It was found that dry ice pretreatment of WAS can be an intriguing alternative for the conventional methods used.The freezing process consists of dissipating heat from the product until the final temperature is lower than the temperature of crystallisation of that product. Freezing can be used for numerous applications, including for disruption of waste-activated sludge (WAS). The aim of this study was to calculate the estimated amount of heat conveyed between the solidified carbon dioxide and the WAS, in the following ratios: 0.25:1; 0.5:1; 0.75:1 and 1:1. In heat of phase transformations, dry ice sublimation, water solidification, the amount of heat transferred by other substances and heat transferred from the sludge (dry sludge) were taken into account during the process of WAS freezing. Heat changes on the surface of WAS were registered using a thermovision camera. The effectiveness of WAS disintegration was confirmed by several biochemical parameters such as soluble chemical oxygen demand (increase over 14 times), degree of disintegration (48%), proteins (increase over 5 times), carbohydrates , RNA (increase by 2.23 mg LThe online version of this article (10.1007/s11356-019-05889-2) contains supplementary material, which is available to authorized users. Technological development, population growth and improvement in the quality of life bring about an increase in the amount of sewage sludge. On the other hand, the tightening of regulations forces treatment plants to search for highly efficient methods of purifying sewage and recycling sewage sludge. With the guidelines of the European Union, it is aimed to maximally reuse the sewage sludges, both on the stage of recovery of products and minimisation of sludges, as well as final management. Agricultural management of sludges is subject to strict regulations and storage requires development of a strategy, which aims to reduce the amount of biodegradable waste ), which increases, for example, for ultrasonic disintegration to 57.9%  bind with a group of factors such as rate of freezing and thawing, chemical composition of the environment of microorganisms, species, time of freezing and temperature. However, mechanical destruction of the cells of microorganisms with the use of ice crystals, which rapture them from the inside or damage them from the outside, is put on the first place. Moreover, the process by dry ice freezing of WAS can cause the following: destruction of sludge flocs structure, increase of the volume of freezing water in the cytoplasm, mechanical damage to the wall and/or cytoplasmic membranes of microorganisms, osmotic shock, decrease of the stability of secondary structures of RNA and DNA, and reduction of the efficiency of transcription, translation, replication of DNA in cells, and ‘cold’ death of microorganisms  8.16 ± 0.62 g L−1, soluble chemical oxygen demand (COD) 61 ± 8 mg L−1, total COD 9.82 ± 0.74 mg L−1, soluble ammonium nitrogen (N-NH4+) 2.4 ± 0.2 mg L−1 and soluble phosphate (P-PO43−) 13.7 ± 0.5 mg L−1.WAS samples were collected in 10-L containers and transported from wastewater treatment plant (WWTP), located in the Silesian voivode ship in Poland, to the laboratory of University of Bielsko-Biala. WWTP operates according to the Enhanced Biological Nutrient Removal (EBNR) processes and WAS samples were taken from outflow of secondary settling tanks. The main characteristics of WAS are as follows: pH 7.1 ± 0.2, total solids (TS) 11.81 ± 0.51 g LDry ice used for disintegration was in the form of prills/pellets and was obtained from the company Cryopoland sp. z o.o. Dry ice to the laboratory was transported in thermostatic containers that prevented the sublimation of dry ice.2  and allowing it to expand into the natural atmosphere. This causes the liquid CO2 to expand into both a gas vapour and solid snow. Liquid CO2 is injected into chambers inside the production machines, creating pressure, and liquid CO2 expands; ~ 1 kg of liquid CO2 is flashed to make 0.45 kg of dry ice. The snow is then either compressed or extruded to form pellets. Generally, it is desirable to have dry ice pellets that are well compacted to minimise the entrapment of gaseous CO2 and/or air which may affect product quality. These dry ice pellets are generally in the order of 0.2 to 0.3 cm in diameter and 0.25 to 1 cm in length.Dry ice was produced in a pelletiser which is manufactured by taking pressurised liquid CODisintegration by freezing of a 1-L WAS sample was done by dry ice. The used volume ratios of the WAS to dry ice and time of disintegration (sublimation) are presented in Table In heat of phase transformations, dry ice sublimation, water solidification and also the amount of heat transferred by other substances and heat transferred from the sludge (dry sludge—without water) should be taken into account when preparing the energy balance for the process of freezing sludge. The heat absorbed from the environment during substance mixing is relatively low  and can be omitted in the overall energy balance. Exact energy balancing is extremely difficult, due to the mixing of sludge and dry ice being impeded by the rapid water solidification. Zones differing in temperature are formed, depending on the chemical composition of the specific area. Exact modelling of phase transformation processes can be found in scientific writing .where Qo = Qs + Qw (2)The amount of heat conveyed by WAS can be calculated using the following equation:Qo is the heat conveyed by sludge; Qs is the heat conveyed by solid sludge components; Qw is the heat conveyed by water found in sludge.where The heat conveyed by solid sludge components which do not undergo phase transformations can be calculated using the following equation:cws is the specific heat of solid sludge components; ms is the weight of solid sludge components; tp is the start sludge temperature.where The amount of heat emitted by water can be calculated based on the weight ratio of the mixed dry ice and WAS. The reason for this is that during the first stage the energy that is absorbed by the dry ice causes the cooling of the sludge. After reaching 273.15 K, the temperature stops decreasing, because the heat absorbed by the dry ice causes the phase transformation of water into ice. Only after all the water freezes does the temperature continue to decrease. Based on this, three ranges may be determined where the heat emitted by the water was calculated:tk > 273.15 Kfor Qw is the heat emitted by water; mw is the water weight; cw is the specific heat of water;where tk = 273.15 Kfor Qk based on the system’s energy balance when the components’ volume fraction is known.In this equation, it is only possible to calculate the amount of heat emitted during the solidification of water tk < 273.15 KFor cwl is the specific heat of ice; ck is the heat of water solidification.where In this experiment, it is essential for the entire sludge to freeze; therefore, the minimal amount of dry ice needed for this purpose can be calculated as:From the equation above, it is also possible to determine the average end temperature, when the mixture composition is known. However, as it was previously mentioned, the above calculations are an approximation due to the phenomenon’s complexity; the errors are minimal. This will be shown in further calculations.The surface of the reactors was registered using a FLIR E50 thermovision camera.In the WAS samples, before and after the process of disintegration by dry ice, the following were determined indicative of the release of organic and inorganic matter: soluble chemical oxygen demand (SCOD) value, concentration of proteins, carbohydrates, RNA acid, ammonia nitrogen, phosphates and turbidity value in the liquid phase.4 to destroy the cell walls of bacteria. Then, WAS was mixed with 3 mL 0.3 M KOH solution and held for 60 min at 37 °C . The post-hydrolysis supernatant was collected and the precipitate was washed twice with 3 mL cold 0.5 M HClO4. Finally, all extracts were made up to 15 mL with 0.5 M HClO4 and centrifuged to remove any remaining solid particles. The obtained supernatant was subjected to the absorbance measurement (absorbance of released purines and pyrimidines) by spectrophotometer HACH DR5000 (wavelength 260 nm). The concentration of RNA was calculated according to the formula:SCOD, ammonia nitrogen, phosphates and turbidity value were determined for samples before and after disintegration by dry ice according to the Standard Methods for Examination of Water and Wastewater (22nd ed.) .where The results were taken from analyses performed at least 5 times; arithmetic average was calculated. The standard deviation was determined according to the estimator of the highest credibility in Statistica 6.0.In order to obtain a quantitative measure of the effects of disintegration, the coefficient called the degree of disintegration (DD) was used for calculation according to methodology given by Müller Müller  and usedTo confirm the release of various chemical compounds from the cells of microorganisms during the destruction of WAS by dry ice, the infrared wave analysis (IR) was applied.−1 resolution in the range from 4000 to 400 cm−1 with 128 scans per spectrum. Data collection and post-processing were performed using OMNIC software  which allows to compare obtained spectra in the scope of absorbance proportionally to the amount of the drawn sample, which allowed for quantitative interpretation of the results.A spectrometer Nicolet Magna IR 860  was used. The analysis of IR spectroscopy was carried out on suspension from evaporated liquid phase WAS samples before and after disintegration. The samples were mixed with KBr (1 mg:200 mg) and compressed in 500 MPa into pellets at 13-mm disc size; the background was a reference of pure KBr. The spectra were registered at a 4-cmThe measurement of the capillary suction time (CST) was carried out according to Baskerville’s and Galle’s methodology based on the measurements of transition of frontal boundary layer of the filtrate as a result of the effect of suction forces in the used paper (Whatman 17).Our investigations on the disintegration of WAS using dry ice concerned two aspects. The first was the dry ice and WAS energy balance analysis, taking into account phase transformations. The aim of this was to determine the mixture’s end temperature (with various WAS-dry ice ratio).The physical properties and thermodynamic parameters used in the calculations are presented in Table 2.Theoretical amount of dry ice needed to freeze the sludge was calculated according to Eq. For the mixture 1:0.25 (ratio of WAS to dry ice), the calculation showed that the amount of absorbed energy during the sublimation of dry ice and its subsequent heating to 273.15 K was approx. Q = 207 kJ, whereas to freeze the sludge, at least 387 kJ of heat emission was required. Therefore, the temperature of 273.15 K for this mixture composition was achieved, because part of the water froze and some remained in liquid state.2O kgp.s−1) and that part of the CO2 is released and leaves the system, only partially absorbing heat form the sludge . The surface of the mixtures ratio (WAS-dry ice) was registered using a FLIR E50 thermovision camera and is shown in Fig. Calculations for the 1:0.5 ratio mixture showed that the solid carbon dioxide absorbs approx. 410 kJ of energy; therefore, the temperature of the mixture should be around 263.15 K. The temperature of 267.15 K Fig.  was dete2 absorbs approx. 558 kJ of energy. The mixture should therefore reach the temperature equal to the temperature of dry ice sublimation. The amount of absorbed heat during the sublimation transformation of such an amount of dry ice causes the freezing of sludge and then its further cooling to approximately 194.65 K. Theoretical calculations assume temperature levelling in the entire mixture volume. This is theoretically possible only after accurate isolation of the mixture and complete sublimation of the dry ice. In practice, it is not possible to reach this state, due to the forming of an ice coat around the fragments of dry ice, which hinders the sublimation process. Despite this, in Fig. Calculations for the 1:0.75 ratio mixture showed that the COEven considerably higher temperature points were also found. These are found on the edges of the container and on larger ice fragments. It can be assumed that if the system was left in isolation longer (not frozen at once), the temperature would be much more uniform in the entire mixture volume.2 absorbs approximately 745 kJ of energy. As was in the previous case, the amount of absorbed energy is enough to reach the sublimation temperature, which is ca. 194.65 K. Similarly as in the previous case, it is impossible to reach this temperature in the entire volume in a short space on time due to the heterogeneity within the mixture’s volume (solid and liquid state of WAS). Large areas going beyond the camera’s range can be observed on the thermovision image  did not cause complete freezing of the entire sample volume and the thawing was fastest . A dose of 1:0.75 and higher resulted in complete freezing of the samples and effective disintegration lasting 3–4.5 h.During the next stage, we tried to evaluate the effect of the freezing phenomenon on the release of chemical compounds.Disintegration of WAS by dry ice caused the release of organic matter (expressed as SCOD) to a supernatant of the sludge. Changes in SCOD were associated with the disruption of the WAS flocs and cells of microorganisms, which led to the release of organic compounds to the sludge liquid.2 L−1 (raw sludge) to 205 mg O2 L−1  increased.As expected, with the increase of the dose of the solid carbon dioxide COAs a result of destructive effects of dry ice to the WAS structure for the volume ratio of WAS to dry ice 1:0.25, the DD increased to 15%  to 99 mg L−1 for the volume ratio of WAS to dry ice 1:0.25  from the microorganisms cells was also shown by other researchers  in the sludge liquid. In the last decade, a number of researchers used Schneider’s method with the use of hot perchlorid acid to determine the RNA or DNA concentration in a medium. Another method used in the studies is a technique which uses cold HClO−1, while for the volume ratio of sludge to dry ice 1:1, the concentration of RNA increased by 2.23 mg L−1 , the concentration of RNA amounted to 10.07 mg LAs a result of destruction of cells structure of microorganisms by dry ice, the release of enzymes contained in the protoplast of microorganisms took place, whose effect of hydrolytic action was decomposition of nitrogen and phosphorous organic compounds and thus, there was also an increase of concentration of ammonium nitrogen and phosphates in liquid phase.4+ concentration amounted to 24 mg L−1  to 133 mg PO43− L−1 (the volume ratio of WAS to dry ice 1:1)  can be used as a good index for the ability to dewater of sludge. The test measures the time for free water to pass through a certain distance using suction paper as the medium .−1), S–H and C–H , C=O (at wavelengths around 1650 cm−1) and C–O, P–O and S–O (at wavelengths around 1100 cm−1)  Fig. . These cThe mentioned changes in absorbance at the specific wavelength are indicative of the release of i.e. amines, amino acids, proteins, phosphates, etc. Revealing these chemical groups in the supernatant attests to a destructive influence of freezing on WAS microorganisms and effective cellular lysis.−1. This can be related to the amine group transformation after the dry ice treatment , a destruction of cellular structures of microorganisms appeared, and thus, there was an increase in SCOD, proteins, RNA, PO43−, N–NH4+ and carbohydrate concentration in the sludge liquid. Furthermore, disintegration of WAS by dry ice caused an increase in the turbidity of supernatant and at the same time CST decreased which can be used as a good index for the ability to dewater WAS. FTIR analysis has confirmed the hypothesis of an efficient disintegration of WAS. That could be concluded from the changes in absorbance at the specific wavelengths, which corresponded to the presence of among others, amines, amino acids, proteins and phosphates. The separation of these substances in the supernatant confirms the destructive action on microorganisms  and effective lysis of microorganisms cells.Calculations showed that the dry ice-WAS mixture, ratio 1:0.25, achieved the temperature 273.15 K, whereas the mixture of solidified carbon dioxide and WAS, ratio 1:0.5, reached 267.15 K. In mixtures, with the ratios of 1:0.75 and 1:1, the COESM 1(DOCX 233 kb)


Vaccinium macrocarpon Aiton) extracts on the basis of their recognized anti-adhesive activities against bacteria, may provide potential compounds able to prevent viral attachment to target cells. Nevertheless, only few studies have so far investigated the possible use of cranberry extracts as an antiviral tool. This study focuses on the suitability of a cranberry extract as a direct-acting anti-influenza compound. We show that the novel cranberry extract Oximacro® inhibits influenza A and B viruses  replication in vitro because of its high content of A-type proanthocyanidins (PAC-A) dimers and trimers. Mechanistic studies revealed that Oximacro® prevents attachment and entry of IAV and IBV into target cells and exerts a virucidal activity. Oximacro® was observed to interact with the ectodomain of viral hemagglutinin (HA) glycoprotein, thus suggesting the interference with HA functions and a consequent loss of infectivity of IV particles. Fluorescence spectroscopy revealed a reduction in the intrinsic fluorescence of HA protein after incubation with purified dimeric PAC-A (PAC-A2), thus confirming a direct interaction between HA and Oximacro® PAC-A2. In silico docking simulations further supported the in vitro results and indicated that among the different components of the Oximacro® chemical profile, PAC-A2 exhibited the best binding propensity with an affinity below 10 nM. The role of PAC-A2 in the anti-IV activity of Oximacro® was eventually confirmed by the observation that it prevented IAV and IVB replication and caused the loss of infectivity of IV particles, thus indicating PAC-A2 as the major active component of Oximacro®. As a whole, these results suggest Oximacro® as a potential candidate to create novel antiviral agents of natural origin for the prevention of IV infections.The defense against influenza virus (IV) infections still poses a series of challenges. The current antiviral arsenal against influenza viruses is in fact limited; therefore, the development of new anti-influenza strategies effective against antigenically different viruses is an urgent priority. Bioactive compounds derived from medicinal plants and fruits may provide a natural source of candidates for such broad-spectrum antivirals. In this regard, cranberry ( The influenza viruses type A and type B  are widespread major pathogens among human populations and are responsible for seasonal epidemics and pandemics . Annual Currently, seasonal vaccines represent the most effective measure to prevent and control IV infections . HoweverVaccinium macrocarpon Aiton (Ericaceae) exert antibacterial activities on the basis of their recognized anti-adhesive activity  functions. Indeed, dimeric PAC-A were identified as the active component of Oximacro® that interacts with and affects HA. Together, these results indicate that Oximacro® is an attractive candidate to create novel antivirals for the prevention of IV infections.Based on these premises, the aim of this study was to evaluate the anti-IV activity of OximacroV. macrocarpon Aiton) extract Oximacro® was obtained by Biosfered S.r.l.  as a reddish powder, and resuspended in Dulbecco’s Modified Eagle Medium  at 10 mg/ml. Purified PAC-A dimer (PAC-A2) was purchased from Extrasynthese (France) and dissolved in 96% v/v ethanol  to generate a final concentration of 100 μg/ml. To determine the total PAC content of Oximacro®, the Brunswick Labs p-dimethylaminocinnamaldehyde (BL-DMAC) assay was performed as previously described by ® was determined by HPLC-ESI-MS/MS as previously described by ® by gel filtration chromatography was performed as described by The cranberry  were propagated in DMEM supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. Infections were performed in the presence of 1 μg/ml of trypsin TPCK treated from bovine pancreas  and 0.14% of Bovine Serum Albumin .Madin Darby Canine Kidney cells  and the influenza B virus strain B/Lee/40 (IBV) were a generous gift from Arianna Loregian . IAV and IBV were cultured and titrated by plaque reduction assay (PRA) on MDCK cells as described below. Experiments were performed under BSL-2 conditions.® or its purified fractions was determined on MDCK cells after a 72 h of treatment. The number of viable cells was measured by MTT as previously described  and after 24 h they were exposed 1 h prior to infection to increasing concentrations of either Oximacro®, its fractions or PAC-A2, and then infected with IAV or IBV (40 PFU/well). After virus adsorption (1 h at 37°C), cultures were incubated in medium containing 0.7% Avicel (FMC BioPolymer) plus Oximacro® or its fractions. At 48 h post-infection (h p.i.), the cells were fixed with a solution of 4% formaldehyde in phosphate-buffered saline 1X (PBS) for 1 h at room temperature (RT) and stained with a solution of 1% crystal violet. The microscopic plaques count then allowed to define the concentration of either Oximacro® or its purified fractions that produced 50 and 90% reductions in plaque formation (IC50 and IC90).The antiviral activity of Oximacro® concentrations were added 1 h before virus adsorption (pre-adsorption treatment), during virus adsorption (adsorption treatment), or after adsorption for 48 h (post-adsorption), as summarized in the scheme depicted in Figure 2. The cells were infected with either IAV or IBV (40 PFU/well) for 1 h at 37°C and then washed with warm medium to remove unbound virions. At 48 h p.i., the plaques were stained as described above.For the time-of-addition assay, a modified PRA was performed. Briefly, MDCK cells were seeded as described above for PRA, and the following day different Oximacro® (20-10-5-2.5 μg/ml). After viral adsorption, the cells were washed twice with warm medium and incubated with medium-containing 0.7% Avicel for 48 h at 37°C. The IV plaques were then stained as described above.For viral attachment and entry assays, MDCK cells were seeded as for PRA and after 24 h cultures were chilled on ice for 20 min and washed three times with cold medium. For the attachment assay, chilled MDCK cell monolayers were infected at 4°C for 2 h with either IAV or IBV (40 PFU/well) in the presence of different concentrations of Oximacro® (20-10-5-2.5 μg/ml) were added for 2 h at 37°C to allow viral entry. Then, cell monolayers were treated with cold acidic glycine buffer  for 30 s to remove adsorbed virions, washed twice with warm medium, and then incubated with medium-containing 0.7% Avicel for 48 h at 37°C. The plaques were then stained as described above.For the entry assay, MDCK cell monolayers were infected with either IAV or IBV (40 PFU/well) for 2 h at 4°C. After adsorption, infected cultures were washed with cold medium, and different concentrations of Oximacro® on IV infectivity, 2 × 104 PFU of either IAV or IBV were incubated with 25 μg of Oximacro® for 0, 60, 120, or 180 min at either 37°C or at 4°C. The, viral suspensions were diluted 100-fold in medium to lower the concentration of Oximacro® below that required for antiviral activity, and residual IV infectivity was measured by PRA as described above.To evaluate the effect of Oximacro® with IAV Hemagglutinin (HA), aliquots of purified recombinant HA subtype H1N1 (A/Puerto Rico/8/1934) ectodomain (1–528 aa)  were incubated at 37°C for different times with Oximacro®. Then, mixtures were treated with SDS sample buffer, fractionated through 10% SDS–PAGE, and analyzed either by staining with Coomassie blue or immunoblotting using the rabbit anti-HA antibody . Immunocomplexes were detected with a goat anti-rabbit Ig Ab conjugated to horseradish peroxidase  and visualized by enhanced chemiluminescence .To investigate the interaction of OximacroPAC-A2 (Extrasynthese) was tested for its ability to bind to HA using fluorescence quenching. In this assay, PAC-A2 quenches the protein’s intrinsic fluorescence produced by the tryptophan fluorophores. Measurements were performed in a quartz cuvette using a fluorescence spectrometer LS55 (PerkinElmer). Fluorescence emission spectra were monitored in the 295–500 nm range using a scan-rate of 200 nm/min upon excitation at 290 nm. The excitation and emission slits were arranged to 8.0 and 10.0 nm, respectively. The protein was dissolved in PBS buffer (pH 7.4) at a concentration of 1.2 μM. Samples were measured in the absence and presence of PAC-A2, following 1 h 25°C. Data were normalized by subtracting the contribution of PAC-A2 fluorescence.Ligand and protein structures were prepared using the YASARA structure package . YASARA,In the docking experiment, the substrate is originally outside the simulation cell and is placed inside the cell by exploiting the Autodock Lamarckian Genetic algorithm, resulting in a series of binding modes classified by the binding energy outputs . Among tThe chemical structures of PAC-A dimers (PAC-A2), PAC-B-2, Kuromaninin, Delphinidin 3-glucoside, Rutin, Quercetin, and Isorhamnetin were obtained from Pubchem. Structures were optimized in terms of molecular geometry optimization using YASARA. The ligands were docked to the crystal structure of IAV HA A/Puerto Rico/8/34 .® was in line with the values declared in the CoA (366.06 mg/g ± 4.96). Furthermore, analysis by HPLC-ESI-MS/MS revealed a high percentage content of total PAC-A (86.72% ± 1.65), of which the majority corresponded to PAC-A dimers, and only a small amount was of PAC-A trimers.The total PAC content of Oximacro® by Sephadex LH-20 chromatography produced five major fractions whose chemical characterization highlighted the presence of different phytochemicals, such as anthocyanins, flavonoids, and PACs. As shown in Supplementary Table 1, fraction 1 contained delphinidin, and cyanidin glycosides; fraction 2 was composed of quercetin and isorhamnetin; fractions 3 and 4 were composed most of PAC-A dimers and of small amounts of PAC trimers; fraction 5 did not show detectable compounds. Oximacro® and its fractions 1–5 were then analyzed for anti-IV activity.Fractionation of Oximacro® inhibited both IAV and IBV replication in MDCK cells in a concentration-dependent manner . The calculated IC50 and IC90 values were 4.5 ± 0.2 μg/ml and 10.4 ± 0.2 μg/ml for IAV, and 4.5 ± 0.5 μg/ml and 9.7 ± 0.5 μg/ml for IBV, respectively. The 50% cytotoxic concentration (CC50) was 141 ± 0.8 μg/ml, thus indicating that the antiviral activity of Oximacro® did not stem from a non-specific cytotoxicity. The selectivity index for IAV and IBV (CC50/IC50) was therefore 31.1 and 31.3, respectively.Treatment with Oximacro50 of 0,017 μg/ml for IAV and of 0.22 μg/ml μg/ml for IBV, respectively, and consistent with a previous report , thus indicating that the components responsible for the antiviral activity of Oximacro® were PAC-A dimers and trimers.To pinpoint the active phytochemical of Oximacro®, time-of-addition assays were carried out in accordance with the scheme depicted in Figure 2. Briefly, Oximacro® was added: prior to IV adsorption ; during infection ; or after viral adsorption (from 0 to 48 h p.i.). Evaluation of the IC50 indicated that Oximacro® was more effective if added either in the pre-adsorption  or adsorption phases  than after infection. In fact, addition of the extract at the end of the adsorption period increased the IC50 to more than 20 μg/ml for both IAV and IBV, thus indicating that Oximacro® was more active against the early stages of IV replication cycle, such as attachment and entry. To investigate this possibility further, selective attachment and entry assays were performed. In the attachment assays, prechilled MDCK cells were infected with either IAV or IBV in the presence of different concentrations of Oximacro® for 2 h at 4°C, so that to enable the attachment of IV particles, but not their entry into target cells. Oximacro® and unattached virions were then washed away, and the remaining IV infectivity was evaluated at 48 h p.i. As shown, in Figure 3A, Oximacro® inhibited the attachment of both IAV and IBV in a concentration-dependent manner. Next, to investigate the inhibitory activity of Oximacro® on IV entry, prechilled MDCK monolayers were infected with IV at 4°C for 2 h to enable viral attachment but not entry. Different concentrations of Oximacro® were then added and infected cultures were incubated at 37°C for 2 h to allow entry of adsorbed IV particles. At the end of this incubation period, IV viruses still bound to the cells were inactivated by briefly treating with acidic glycine buffer, and the remaining IV infectivity was evaluated at 48 h p.i. As depicted in Figure 3B, Oximacro® prevented the entry of both IAV and IBV in a concentration-dependent manner. Together, these results revealed that Oximacro® is able to interfere with both viral attachment and entry, therefore suggesting its suitability as an early-acting inhibitor of IV replication.To understand which phase of the IV replicative cycle is targeted by Oximacro® to IV particles prior to their interactions with host cells. To verify this hypothesis, suspensions of IAV or IBV particles were incubated with Oximacro® (25 g/ml) for 0, 60, 120, and 180 min at either 37°C or at 4°C. After incubation, mixtures were diluted to reduce the concentration of Oximacro® well below that which prevents virus attachment and entry , and then the residual IV infectivity of diluted samples was measured by PRA on MDCK cells. Figure 4A shows that the incubation of IV particles with Oximacro® at 37°C, determined a complete loss in infectivity of IVA and IBV after 180 and 120 min of incubation, respectively. In contrast, incubation at 4°C, even at longer times, led to no detectable loss of IV infectivity , thus suggesting that Oximacro® exhibits time-dependent and temperature-dependent inactivation of infectious IV . Likewise, a virucidal activity against IAV and IBV was observed for fraction 4, the richest in A type PAC dimers and trimers (data not shown). Thus, these results suggest that the anti-IV activity of Oximacro® stems from its ability to affect IV virions, thus impairing their attachment and entry into host cells.The observed inhibition of IV attachment and entry could stem from damages caused by Oximacro® decreased the infectivity of IV particles , we then investigated whether it targets HA, thus impairing its functions in the early stages of the IV replication cycle. To this end, aliquots of purified recombinant HA from the A/Puerto Rico/8/34(H1N1) strain, produced in baculovirus, and corresponding to the ectodomain of the glycoprotein (1–528 aa) , were mixed with increasing concentrations of the extract and incubated at 37°C. Figure 5B shows that the exposure of purified HA to Oximacro® caused a reduction in the amount of HA protein band, and altered its electrophoretic mobility with the occurrence of an evident smear toward high molecular weights at the highest Oximacro® concentrations. Similarly, the exposure of HA to Oximacro® for different times determined a time-dependent reduction of the HA protein band, and the appearance of a protein smear with lower electrophoretic mobility compared with the untreated control. Thus, Oximacro® was able to interact with HA in a concentration- and time-dependent fashion. These findings suggest that interactions between Oximacro® and IV HA may hamper its functions in virus attachment and entry, and underlie the overall antiviral activity of Oximacro®.Influenza virus hemagglutinin (HA) is responsible for both the IV attachment and entry by mediating both the initial interaction with cell receptors containing sialic acid, and the fusion between endosome membranes and the viral envelope. Because Oximacro® to HA is mediated by its PAC-A dimers. The recombinant extracellular domain of our test H1N1 HA (A/Puerto Rico/8/1934) contains nine tryptophans, and the emission maximum of its fluorescence spectrum occurs at 342 nm . The addition of 25 μM of purified PAC-A dimers (PAC-A2) caused a reduction in the intrinsic tryptophan fluorescence of HA, but overall it did not markedly modify the shape of the emission spectrum . Thus, this result sustains the hypothesis that PAC-A2 binds to HA; moreover, since there was no considerable change in the emission maximum of the intrinsic protein fluorescence, it implies that the interaction does not bring about significant conformational change in the overall structure of HA.Fluorescence spectroscopy is widely used to investigate enzyme–ligand interactions, because alteration of the fluorescence emission spectrum of proteins can be used to detect conformational changes, binding to ligands, or unfolding processes. In this regard, it has been reported that PACs can reduce the signature fluorescence of bound proteins, thus fluorescence quenching can be used to demonstrate the binding of PACs to proteins ,b. Here,in silico docking simulations were performed. Since PAC are known to bind protein structures at multiple binding sites , and subsequently to the surface of the HA structure . The best binding pose is shown in Figure 7B, where HA residues  forming hydrogen bonds to PAC-A2 are highlighted.To support the occurrence of interactions between HA and PAC-A2 further, ng sites , all thein silico docking analysis tests to investigate the ability of the different components present in Oximacro® fractions (Supplementary Table 1) to interact in silico with the crystal structure of HA. Of the different Oximacro® components tested, the PAC-A dimer was identified as the molecule with the best docking capability. In fact, when comparing the theoretical dissociation constants for the top 100 scoring poses , clustered to separate complexes with a RMSD higher than 5.0 Å, the simulated PAC-A2-HA complexes formed most favorably with an affinity under 10 nM. Furthermore, the distribution of all the docking data  confirms that even the worst 100 theoretical complexes (out of 999) are still much more likely to bind HA efficiently than any of the other Oximacro® components.We then performed further in silico docking simulations are therefore consistent with the fluorescence analysis  and support the hypothesis that PAC-A2 constitute the chemical component of Oximacro® responsible of binding to and altering HA.These Figure 8A, PAC-A2 produced a concentration-dependent inhibition of both IAV and IBV. However, its IC50 against both IAV and IBV was higher than that of Oximacro®: 14.8 μg/ml for IAV, and 11.2 μg/ml for IBV, respectively. This result may suggest the occurrence of a possible synergistic effect of PAC-A2 with PAC-A trimers, since the IC50 of fraction 4, the richest in PAC-A dimers and trimers was similar to that of Oximacro® (Table 1). Moreover, when tested on IV particles’ infectivity , PAC-A2 determined a complete loss of IAV and IBV infectivity after an incubation of IAV and 180 min at 37°C, which indicated the ability of PAC-A2 to bind to IV particles and thereby impair their infectivity prior to attachment to host cells. Altogether, these results suggest PAC-A dimers as the active components of Oximacro® responsible of the majority of the overall anti-IV activity of the extract.To verify experimentally the role of PAC-A dimers as antiviral molecules, we tested purified PAC-A2 in some antiviral assays against IV. As shown in New anti-influenza molecules need to be continually developed, and this can be done through the adoption of a variety of approaches, such as: (a) improvement of existing drugs; (b) selection and validation of new viral molecular targets; (c) the search for molecule able to inhibit early events in the replication cycle and thus to prevent the establishment of infections; (d) the search for agents able to modulate the host antiviral defense responses; and (e) exploitation of the synergistic effects of compound mixtures acting on different molecular targets . Regardl®, a cranberry extract characterized by a remarkable amount of PAC-A, exerts a potent dose-dependent antiviral activity against IAV and IBV by a virucidal mechanism that involves the interaction with viral particles, and the subsequent impairment of the HA-mediated adsorption and entry into host cells.In this study, we report that OximacroMany phytochemicals of relatively low molecular weight, such as polyphenols, flavonoids, terpenes, glucosides, and alkaloids extracted from different plants have been shown to be endowed with an antiviral activity against IV . In partRumex acetosa polyphenol-enriched extract exerts an inhibitory activity against IAV replication by impairing the attachment of viral particles to target cells  from cranberry juice prevents IV infectivity and attachment  and inte® abolishes both IAV and IBV replication through a virucidal mechanism that involves its direct binding to HA, as indicated by the alteration of electrophoretic mobility of the HA ectodomain upon incubation with Oximacro® , and that such interaction can be mediated by PAC-A2 present in the extract . However, our direct comparison between PAC-A2 and PAC-B2 by in silico docking simulations showed a lower binding affinity to HA of the latter .Likewise, here, we report that OximacroTaken together, these findings support the view that polyphenols derived from different plants are able to bind with a certain degree of propensity to HA and NA on the IV envelope.R. acetosa (V. macrocarpon (this study) to interact with and affect IV HA, could be a consequent to the natural ability of polyphenols to bind and aggregate proteins . Thus, it is possible that the incubation of Oximacro® with the ectodomain of HA may result even in covalent bonding between the two partners. Moreover, our docking simulations  predicted the formation of three hydrogen bonds between the PAC-A2 and Phe99, Asn210, and Trp234 of HA, with a computed binding energy of 14.13 kcal/mol and a dissociation constant of 44.04 pM. Together, the formation of both hydrogen and covalent bonds between the reactive quinone groups of PAC-A dimers and trimers . However, it remains to establish whether these HA’s alterations stem from the binding of PAC-A to specific domains of the IV glycoprotein, or whether PAC-A of Oximacro® simply “upholster” HA surfaces  and, in this manner, they lead to the inhibition of HA functions in the initial stages of the IV replication cycle. In this regard, our docking simulations also suggested that PAC-A2 binds more strongly to the central HA groove, as confirmed by the top 100 possible poses . Based on this observation, a predicted binding mechanism would entail an initial binding of PAC-A2 to the HA groove and then, once all the highest affinity sites have been saturated, the PAC-A2 would bind to other residues on HA surfaces .The observed capability of PACs contained in extracts of  acetosa  or V. maproteins . Dealingproteins . In this trimers  and HA m® against IV advocates its potential application in the prevention of influenza infections. It is therefore possible to envisage that a local application of Oximacro® in the upper respiratory tract, administered in the form of either tablets/chewing gums or through inhaling devices, would allow the active PAC-A to inactivate infecting virus, thus preventing the occurrence of an infection. This proposal may be sustained also by the high safety profile that many formulations of dried cranberry extracts have shown in their widespread use to prevent urinary tract infections (UTIs)  . HoweverAL, MT, GC, MM, and GGri conceived and designed the experiments. AL, MT, and GC performed the experiments. AL, MT, GC, GGil, MM, and GGri analyzed the data. MM and GGri wrote and revised the manuscript. All authors read and approved the final manuscript.® and partly supporting the research. Biosfered did not interfere with the design, analysis, and decision to publish this paper. The remaining 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.MM is also currently a fellow of the Biosfered S.r.l. company. No patents are pending and Biosfered supported the study by providing Oximacro


In situ breakdown of illite provides a previously-unobserved pathway for the release of dissolved Fe2+ to porewaters, thus enhancing clay-rich Antarctic sub-ice shelf sediments as an important source of Fe to Fe-limited surface Southern Ocean waters during ice shelf retreat after the Last Glacial Maximum. When sediments are underneath the ice shelf, Fe2+ from microbial reductive dissolution of illite/Fe-oxides may be exported to the water column. However, the initiation of an oxygenated, bioturbated sediment under receding ice shelves may oxidize Fe within surface porewaters, decreasing dissolved Fe2+ export to the ocean. Thus, we identify another ice-sheet feedback intimately tied to iron biogeochemistry during climate transitions. Further constraints on the geographical extent of this process will impact our understanding of iron-carbon feedbacks during major deglaciations.Recent recession of the Larsen Ice Shelf C has revealed microbial alterations of illite in marine sediments, a process typically thought to occur during low-grade metamorphism.  Recent recession of the Larsen Ice Shelf C has revealed that microbial alteration of illite can occur within marine sediments, a process previously thought to only occur abiotically during low-grade metamorphism. Here, the authors show that such microbial alteration of illite could provide a potential source of Fe release to Southern Ocean waters during Holocene glacial cycles. Relief of this Fe limitation, and sequestration of carbon into the deep ocean, is thought to enhance the Southern Ocean’s ability to act as a mediator of global climate over millennial to ice-age timescales4. However, the source, transport, and fate of Fe in the Southern Ocean has been widely debated, with sources ranging from dust, ice sheets, iceberg-rafted debris (IRD) to sub-ice shelf, and other continental shelf sediments5. With evidence supporting spatially variable contributions of Fe from numerous sources to the Southern Ocean, consideration of the configuration of ice shelves and their dynamics is vital for understanding how changes in Fe supply help to drive carbon uptake in the Southern Ocean.The limited availability of iron (Fe) in the surface Southern Ocean, compared to major nutrients such as nitrate and phosphate, leads to underutilization and thus outgassing of upwelled CO6, acts as a sentinel for changes in Antarctic ice dynamics. Since the short period of satellite observations, rapid disintegration of ice shelves and retreat of continental glaciers has been observed on the Antarctic Peninsula and linked to potential increases in sea level rise7. Larsen ice shelves A and B (LIS-A and LIS-B) disintegrated catastrophically in 1995 and 2002, respectively, whereas the largest of the peninsular ice shelves, LIS-C, has persisted, decreasing in the size, and thinning through the Holocene to the present8  of 25 m/year, particularly between 2010 and 2016.The Antarctic Peninsula, one of the fastest-warming regions on the Earth11. These redox changes in the sediments may cause a variation in clay mineral alteration13. Around 50% of the clay minerals of Antarctic sub-ice shelf sediments are composed of illite14, which contains redox-sensitive Fe in its crystal structure, and is thus potentially sensitive to changes in sediment redox conditions. Traditionally, however, alteration of illite crystallinity (IC) has been assumed to require temperature and pressure, and thus be restricted to low-grade metamorphic settings16. Recently, although, in situ alteration of illite has been identified beyond such metamorphic settings, and used to reconstruct paleoclimate conditions such as the Holocene monsoonal weathering, for example, Yangtze River17, and Southwest Indian continental shelf18. In these studies, chemical weathering in organic-rich sediments is thought to drive the IC alteration, accelerated by wet and warm monsoonal conditions. However, none of these studies have considered the possibility of microbial alteration of illite at low temperatures. Microbially induced geochemical reactions can modify both mineral structure and chemistry13, properties that are sensitive to redox conditions12, and microbes are present in cold but diverse glacio-marine environments19 associated with ice sheets20, introducing the possibility that microbes may play a role in biogeochemical weathering and mineral alteration, even at very low temperatures21. Whereas a range of iron minerals are known to be sources of dissolved Fe upon breakdown by iron-reducing or -oxidizing bacteria23, adding illite to this group, and at low temperatures, provides a potential new broadly distributed mechanism of dissolved Fe2+ to sediment porewaters from the microbial alteration of clay minerals25. This process is distinct from reductive Fe production by microbial respiration of Fe hydroxides26.Environmental change on the Antarctic Peninsula also significantly impacts the redox conditions of surface sediments during the retreat of ice shelves, driven by the overlying bottom waters changing from relatively stagnant to open-ocean oxygenated conditions2+ release to porewaters and the overlying water column and thus productivity and carbon cycling in the Southern Ocean. As such, we present a broadly applicable alternative pathway for dissolved Fe2+ production in sediment porewaters through microbial alteration of clay minerals, in addition to Fe(III) oxides, the strength of which will change corresponding to the depositional conditions during the expansion and retreat of ice shelves.Here we demonstrate that microbe–illite interactions can be significant at low temperatures and pressures, affecting dissolved FeAraon in 2013 (ANA03C Cruise). This core from the continental shelf provides us with information on changes in sedimentary environments during deglaciation, such as the proximity and stability of ice shelves and the influx of meltwater and terrigenous sediments27. The sedimentary sequence unveiled adjacent to LIS-C preserved a depositional record through the Holocene at shallow core depths (<3 m), below the temperatures and pressures of metamorphic modifications28. As such, our core represents an excellent test for the capability of biogeochemical reactions to alter the mineralogical characteristics of the subsurface Antarctic shelf sediments during the Holocene. Core EAP13-GC16B is defined by four distinct lithological units (U1–4)  and rhythmic couplets (~6 couplets/cm) of laminated silt and clay (120–180 cm) are typical of sedimentary facies when an ice shelf retreats and undergoes ice-thinning process during the Holocene climate optimum31. Finally, the presence of IRD and foraminifera in the bioturbated sediment of U1 support oxic and seasonally opened marine conditions10. X-ray diffraction (XRD) profiles show that the major mineral compositions for the clay size sediments throughout the core are smectite, chlorite, kaolinite, and illite and lepidocrocite  on the northwestern part of the LIS-C embayment  Fig. , with th–4) Fig. . These s–4) Fig. , based o15N within U4 16. The average values of IC in Core EAP13-GC15B show variation with depth in the core  – ICgly (median) at each depth (0.14–0.52) correspond to the maximum 3% difference in smectite contents in illite/smectite mixed layers (I/S)35  within U1 compare to U2–4 Fig. . HoweverC34 Fig. . Moreove within U36, far from the conditions observed in sub-ice shelf sediments. Thus, we suggest here that biogeochemical redox-sensitive reactions, possibly by iron-reducing bacteria rather than high temperature and pressure, are the likely cause of the modification of the illite structure observed in U3. Microbial Fe reduction in 2:1 layered phyllosilicate structure results in the alteration of net negative charge, crystal lattice energy, cation exchange capacity, and distribution of Fe(II)–Fe(III) in the octahedral sheet39 that could modify the crystal domain size and structure39 responding in the IC16. Transmission electron microscopy (TEM) measurements on the bioreduced illite confirmed the alteration of illite structure through reductive dissolution and decrease in illite crystalline size24. Indeed, smaller illite domain packet size displayed where reducing condition is favored  patterns of diffused, randomly ordered (U3) to discrete reflections (U1)  Fig. , as illir = 0.90 and 0.69, p < 0.05, Fig. Comamonadaceae sp. (class Betaproteobacteria) and Desulfobacteraceaet sp. (class Deltaproteobacteria)23, comprising only minor components of the microbial community in the top of the core, but becoming abundant below 50 cm (mid-U2) , indicating the occurrence of novel member with not-yet-known functions in those families. Nonetheless, the previous study22 suggested that the family Comamonadaceae is capable of reducing Fe in sediments  may reflect a tight coupling with Fe-reducing bacteria45; however, they are not considered to be a direct driver to change IC  content of illite under anoxic conditions. The relative amount of putative Fe-reducing bacteria diminishes abruptly in the sediments under occasionally open marine conditions (U1), where laminated sediment structures have been destroyed by oxygenation and resultant epibenthic community development. Although such psychrophilic Fe reduction of illite results in similar structural and chemical modification  associated with microbial Fe respiration at low temperature21 in Antarctic regions19.Significant positive correlations between the distribution/abundance of putative Fe-reducing bacteria and IC were observed in the core (U2) Fig. . The gennts Fig. . Furtherion Fig.  may refl IC Fig. . Given t49. This growing consensus of the importance of submarine sediment-derived Fe sources in fueling surface productivity has been driven both by models50 and by observations by the International GEOTRACES program, which have indicated transport of sediment-derived iron over 106 km in the open ocean51, including the Southern Ocean52. In the Antarctic, a number of recent studies have focused on the local importance of Fe from shelf sediments around Antarctica, suggesting that Fe sourced from sediments on the West Antarctic Peninsula is an important Fe source to coastal waters55. Sediment-derived Fe is also thought to be the dominant Fe source fueling blooms in the Ross and Amundsen Seas57, and near Antarctic island chains58.Sediments as an important or even dominant Fe source for the surface oceans has gained traction in recent years, challenging the primacy of atmospheric supply60, consistent with the possibility of long-distance transport of sediment-derived Fe of a reductive origin62 on the Antarctic Peninsula. Such an observation adds weight to the idea that microbial Fe(III) reduction in Fe-rich sediments on the Antarctic margins could play an important role in driving primary productivity in the Southern Ocean, especially near Antarctica. However, dissimilatory reductive dissolution of sediment release isotopically light Fe2+ is typically thought to involve Fe(III) minerals such as goethite, hematite, and ferrihydrite64. Here, we propose that illite may provide an additional substrate for microbial reduction that was previously thought to be largely inaccessible. This study opens the door for iron isotope studies in the future aiming to constrain the proportion of iron release from Fe(III) oxides as well as illite by this process if there is a signature isotope composition for this process. This may be especially important in the Antarctic, because illite  appears so prominently on the continental shelf around Antarctica14, and because redox dynamics are likely to be so intrinsically linked to ice shelf dynamics. Clay minerals comprise 10–30% of bulk Antarctic sediment14, and our experimental data suggest that ~4% of Fe may be released by psychrophilic microbial reduction of the clay mineral smectite  that forms from microbially driven IC downcore  diffusion to the water column, as these sediments are oxygenated and exposed to more vigorous open-ocean exchange  that can be sourced from the microbe–mineral interactions, we have observed that it is important to consider this as a changing source of Fe(II) to the Southern Ocean as ice shelves retreatore Fig. . This prore Fig. , althougore Fig. , will linge Fig. , such acnge Fig.  and prim8. A whole round core sample (238 cm below the seafloor) of marine sediment (EAP13-GC16B) was collected on the northwestern part of LIS-C embayment  along the AP is one of the most dramatic environmental changes directly observed anywhere on Earth68.Sediment cores from the LIS-C embayment are exceedingly rare. The region is often inaccessible to ocean-going and ice-breaking research vessels, and as a result, has not been explored geologically to the extent of the lower latitude embayments of LIS-A and LIS-B. A marine geological expedition (ANA03C Cruise Expedition by the Korea Polar Research Institute (KOPRI)) was conducted in the LIS-A, LIS-B, and LIS-C embayments of northwestern Weddell Sea in 2013  were separated from bulk sediment by wet sieving. For the fine fractions smaller than 62.5 μm (silt and clay), particles were settled down in a 1 L graduated cylinder for 3 days, and then the grain size was determined using Micrometrics Sedigraph 5100. Qualitative ordinal color scale (Munsell color chart) was used to define bedding distinct color variations in the core.A half slab of 238 cm whole round core was examined by X-ray radiograph to investigate the sedimentary structures. The working half of the core was sampled every 4-cm depth interval for measurement of size fractions of clay (<2 μm), silt (>2 to <62.5 μm), and sand (>62.5 μm) following a conventional particle separation procedure13C and δ15N in sediment with depths. Each sample was pretreated with 1 M of hydrochloric acid three times in order to completely remove carbonates, and then isotopic compositions with a precision of 0.2‰ were measured at the Stable Isotope Laboratory, GNS Science, Lower Hutt, New Zealand, using an Isoprime isotope ratio mass spectrometer, interfaced to an EuroEA elemental analyzer in continuous-flow mode (EA-IRMS).Forty-four subsamples (~50 mg of each sample) were collected into tin capsules to analyze isotopic composition of δ70 to minimize the confusion of selecting 10 Å illite packets from the collapsed hydrous clay minerals, such as smectite, displaying the same spacings under the high-energy TEM beam. Illite layers were confirmed by SAED patterns with the strongest Bragg reflections of 1.0, 0.5, and 0.33 nm. The TEM specimens were then sectioned with 700 Å in thickness using a diamond knife microtome . Transmission electron microscopy-electron energy loss spectroscopy (TEM-EELS) was also applied to quantify the oxidation states of Fe in illite structure as a function of the integral ratio of Fe L3/L271 using a TECHNAI F30 ST TEM at the KBSI, Seoul. The operational conditions for EELS acquisition were an energy dispersion of 0.1 eV/channel, entrance aperture of 2.0 mm, and the full-width at half-maximum to 1.0 eV for the zero-loss peak calibration. The statistical optimum signal-window parameters for the integral ratio of Fe-L2,3 edges were calculated using the Gatan Inc.’s Digital MicrographTM software. The backgrounds were removed from EELS spectra by Double arctan functions and Standard power law71.TEM micrographs for the sediment with depths were recorded using JEOL JEM-2100F at Ewha Women’s University, Seoul. The TEM samples were prepared following the impregnation procedure for LR White resin72. IC, also known as Kübler index, comprises of the half-height width of illite 10-Å peak from XRD profiles73 utilizing the Search-Match and OriginPro8 software after the background was removed by Chebyshev polynomial with ≤20 coefficients, and the pseudo-Voigt profile function proposed by Thompson et al. 74. IC was originally referred to as the Weaver index75 that reflects the X-ray scattering domain size and structural distortions16, measuring the crystal alterations76. Three independent sets of XRD profiles were collected in order to reduce the errors in the measurements. Semi-quantitative evaluations of clay minerals were measured after the glycolation treatment. The relative percentage of each clay mineral was calculated using weighting factors77.XRD analyses were performed on air-dried clay sample (<2 μm) with each depth at a scan speed of 1°/min with a Rigaku Miniflex II automated diffractometer utilizing Cu-Kα radiation at Yonsei University, Seoul. Clay size fraction samples were dispersed in distilled water (0.7 mg/mL) and put in an ultrasonic water bath for 30 s to prevent flocculation of particles. Then, air-dried samples were made by pipetting sediment dispersions onto slide glasses for XRD analysis78. We adopted the PCR cycling conditions according to Jorgensen et al. 78, except for applying the nested PCR, in order to minimize PCR bias. PCR amplicon pyrosequencing sequences were processed using the QIIME software package, ver. 1.879. First, raw flowgram data were filtered and denoized by the AmpliconNoise software, version 1.2980, using the platform option for FLX titanium sequence data implemented in QIIME. Sequences were clustered based on operational taxonomic units at 97% similarity using UCLUST81 and classified using the 16S rRNA gene sequence reference database GreenGenes 13_882. To avoid effects of different sample sizes for estimating diversity comparisons among prokaryotic communities, sequences were resampled to the smallest library83.Genomic DNA was extracted from sediment samples every 2 cm from top to 20 cm and every 5 cm below 20 cm (~0.5 g of each sample) using the FastDNA Spin Kit for soil . The quantity of genomic DNAs was measured by Picogreen fluorometry. Polymerase chain reactions (PCRs) for the 16S ribosomal RNA (rRNA) genes of prokaryote (Bacteria and Archaea) were performed using primers Uni787F and Uni1391RShewanella vesiculosa 21939, 21996 and Shewanella frigidimarina 21881) known as cold-tolerant and cold-adapted facultative FeRB84 were isolated from King George Island in western Antarctica by KOPRI. Psychrophilic bacteria were cultured in Luria–Bertani broth (LB) liquid medium aerobically for 7 days at 15 °C to increase cell concentration and activity. The culture was then washed three times with 0.1 mM NaCl solution to remove the residual LB medium, and the washed cells were re-incubated anaerobically for 7 days in liquid M1 medium85 with Fe(III)-citrate (34.5 mM) as an electron acceptor and Na-lactate (20 mM) as an electron donor in order to adapt Fe respiration. The solution was buffered by MOPS (3-(N-morpholino)propanesulfonic acid) to maintain a pH of 7. The incubated cells were finally washed three times with 0.1 mM NaCl solution to remove residual Fe(III)-citrate and M1 medium. Cells were then inoculated in the liquid M1 medium with nontronite (5 g/L) in each set as an electron acceptor and Na-lactate (20 mM) as an electron donor. Control samples were treated identically to experimental ones, except no bacteria to compare the microbial effect on Fe reduction. A sample of 100 mL of this suspension in each serum bottle was placed in the incubator at 4 °C for up to 4 months.Psychrophilic bacteria (Further information on research design is available in the Peer Review FileSupplementary InformationReporting SummarySupplementary data


The purpose of this study was to evaluate the durability and biological response of a new Posterior Dynamic Stabilization system using polyethylene with vitamin E on the sliding surface. The use of polyethylene with vitamin E on the sliding surface in Posterior Dynamic Stabilization has not been reported previously. (1) Fatigue Wear Test. Testing was conducted under a dynamic compressive load of 50N at a speed of 1 Hz for 1 million cycles. We examined the loss of polyethylene due to abrasion in 3 units. (2) Biological Response in Pigs. In two pigs, a new pedicle screw and a conventional pedicle screw were inserted in L2 and L3/4, and L4 and L2/3, respectively. After breeding for 6 months, autopsies were performed. CT imaging was used to evaluate bone union of the facet joint. Abrasive specimens were prepared, and abrasion powder and inflammatory cell infiltration were evaluated microscopically. A developed pedicle screw-based Posterior Dynamic system consists of four parts: a set screw, a rod, a ball, and a pedicle screw. The rod is inserted into the through hole of the ball, and the ball is sandwiched by the set screw. The average loss of polyethylene due to abrasion was -0.01 mg. In all units, polyethylene showed a decrease of 0.1 mm or less at the contact point with the set screw. The facet joints between the conventional screws exhibited bone fusion, but the facet joint between the conventional and the new screw retained mobility with no bony fusion. No abrasion powder was found and inflammatory cell infiltration was only minimally observed. The new Posterior Dynamic Stabilization system exhibited a high level of durability and biological safety. At present, spinal fusion is widely performed. However, spinal fusion surgery increases the load and stress caused by movement in the adjacent segment. The occurrence of adjacent segment disease (ASD) after spinal fusion is reported as 4.8-92.2%  and is cφ 5.5 mm Ti - 6 Al - 4 V) is used for the rod. The pedicle screw has a range of movement of up to 15° on one side around the ball and also allows movement between the rod and the ball. The sliding surface is made of metal - polyethylene, and the two opposing surfaces on which the ball slides feature mirror surface processing. One is a pedicle screw U-shaped groove; another is inside the set screw. Each contact surface has a concave shape because contact is made with a spherical diameter of 8 mm  containing vitamin E . It was achieved by irradiation with a 10MeV electron beam at 300kGy. The ball's diameter is 8 mm and a through hole of 5.5 mm matching the rod diameter exists in the center of the ball. The rod is inserted into the through hole of the ball and fitted to the U-shaped groove of the pedicle screw, and the ball is sandwiched by the set screw. An existing product (Stryker Xia  of 8 mm .The new pedicle screw system was fixed to the testing machine (Mini Bionix: MTS Japan) . TestingThe experiment was conducted on two mini pigs with an age and weight of 3 years, 7 months and 47.4 kg, and 2 years, 8 months and 48.3 kg, respectively. The two mini pigs were anesthetized and screws inserted the pedicles of both sides of L2.3.4. In the first pig, a new pedicle screw was inserted in L2 and a conventional pedicle screw (5.5 × 40 mm Stryker Xia) was inserted in L3,4. In the second pig, a new pedicle screw was inserted in L4 and a conventional pedicle screw was inserted in L2,3. Screws were fixed with a 5.5 mm rod (Stryker Xia) . After bThe loss of polyethylene was 0.02 mg for Unit No. 1, 0.01 mg for Unit No. 2, and -0.06 mg for Unit No. 3. The average loss of polyethylene due to abrasion was -0.01 mg . All uniThe facet joints between the conventional screws exhibited bone fusion, but the facet joint between the conventional and the new screw retained mobility with no bony fusion .No polyethylene or metal abrasion powder was found in the tissue.Abnormal osteolysis was not observed and inflammatory cell infiltration was only minimally observed in the bone and soft tissue surrounding the screw.Aseptic lymphocyte-dominant vasculitis-associated lesion (ALVAL), a reactivity change caused by abrasion powder, was not observed .In order to avoid the complication of adjacent segmental disease after spinal fusion surgery, various systems that preserve intervertebral disc mobility without rigid intervertebral fusion have been developed . HoweverA new Posterior Dynamic Stabilization system using polyethylene on the sliding surface exhibited a high level of durability and biological safety.


Integr Environ Assess Manag 2019;00:000–000. © 2019 The Authors. Integrated Environmental Assessment and Management Published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC)In the United States, there is an absence of federal guidance related to deriving and applying background concentrations at contaminated sediment sites. This absence has resulted in significant variability, uncertainty, and disagreement regarding how representative background concentrations of chemicals of concern should be derived for these sites. The present article discusses important considerations in the derivation of representative background concentrations to be used in the evaluation of contaminated sediment sites. Specifically, a thorough understanding of a site is critical to selecting the background reference areas from which representative background concentrations can be derived, representative background concentrations should account for contributions from those background chemical inputs  that will continue affecting the site even after remediation, perceived outliers should not be eliminated from the background data set just because they are the highest or lowest values, and geochemical evaluation of trace metals is a useful tool for deriving representative background concentrations. On a site‐specific level, representative background concentrations are critical for putting site‐related risk into context, developing a cost‐effective and technically feasible remedial approach, understanding the potential for recontamination, and ensuring long‐term remedy success. In a broader context, clear guidance from the United State Environmental Protection Agency (USEPA) for deriving and applying background concentrations for contaminated sediment sites would help promote national consistency in site assessment and remedy decision making.  Background reference areas should be as similar as possible to the site in terms of surrounding land use and with respect to physical, chemical, geological, and biological characteristics; but importantly, they should not be influenced by site releases.Representative background concentrations should account for input from natural and anthropogenic background sources that will continue to affect the site, allowing for defensible representative background concentrations to be used in remedial investigation, remedy selection, and monitoring.An outlier should not be eliminated just because it is the highest or lowest value in a data set; when the nature of a perceived outlier is unknown or disputed, specific and rigorous statistical analyses should be conducted to evaluate the effect of retaining or eliminating the data point.Once derived, representative background concentrations should remain fixed during remedial investigation, remedial response, and performance monitoring; otherwise, the lack of certainty for stakeholders will be deemed unreasonable and an impediment to the selection and implementation of any remedy. Definitions section) at sediment sites. Workshop attendees included representatives from industry, consulting, academia, and regulatory agencies . The contents of the present article represent the consensus of most of the participants and should not be construed as representing the individual views of all attendees.The United States Environmental Protection Agency (USEPA) has recognized for more than 25 years that establishing a reliable representation of background is a critical issue at Superfund sites USEPA . AlthougClear direction for deriving representative background concentrations at sediment sites is needed because technically defensible, representative background concentrations are critical for putting risk into context; developing an appropriate, cost‐effective, and technically feasible remedial approach; understanding the potential for recontamination; and ensuring long‐term remedy success. Moreover, clear guidance from USEPA would help promote national consistency in site assessment and remedy decision making. The present article has been prepared to present the results of the multistakeholder technical workshop described earlier. As a fundamental premise, representative background concentrations should account for contributions from natural sources and nonsite‐related anthropogenic sources  that will continue affecting the site, even after remediation. Once established, representative background concentrations may be used to develop cleanup goals at sediment sites where background concentrations are greater than risk‐based cleanup levels. This concept is affirmed in USEPA guidance for determining background chemical concentrations in soils as follows: “The reasons for this approach include cost‐effectiveness, technical practicability, and the potential for recontamination of remediated areas by surrounding areas with elevated background concentrations” USEPA . The USEThe following definitions, based on USEPA definitions, are used in the present article:Natural background. Naturally occurring substances present in the environment in forms that have not been influenced by human activity USEPA .Anthropogenic background. Natural and human‐made substances present in the environment as a result of human activities, not specifically related to the site‐related release in question USEPA .Substances or locations that are not influenced by the releases from a site and are usually described as naturally occurring or anthropogenic USEPA , 2002.NThe areas where background samples for chemical concentrations are collected for comparison with samples collected on site. The background reference areas should have similar physical, chemical, geological, and biological characteristics as the site being investigated but should not have been affected by activities on the site USEPA . AlthougA representation of the environmental system and the physical, chemical, biological, and anthropogenic processes that determine the transport of contaminants from sources to receptors. Essential elements of a CSM generally include information about contaminant sources, transport pathways, exposure pathways, and receptors. A good CSM can be a valuable tool in evaluating the potential effectiveness of remedial alternatives USEPA .False outliers. Measurements that are very large or small relative to the rest of the data but represent true extreme values of a distribution and indicate more variability in the population than was expected USEPA .True outliers. Measurements that are very large or small relative to the rest of the data but are a result of transcription errors, data‐coding errors, or measurement system problems USEPA .Measurements that are very large or small relative to the rest of the data and are suspected of misrepresenting the population from which they were collected.Additionally, the term “representative background concentrations” is used frequently throughout the present article. Representative background concentration is defined (for the purpose of this article) as a chemical concentration that is inclusive of naturally occurring and anthropogenic sources affecting the site but is not related to the site release in question. It is derived from sampling within representative background reference areas that may be located on site and/or off site but are not affected by a site release or site activities. For man‐made chemicals, the anthropogenic background concentration and the representative background concentrations are equivalent. For naturally occurring chemicals , representative background concentrations are equivalent to the sum of the anthropogenic and natural background concentrations.At many sediment sites, multiple sources may contribute to the nature and extent of contamination. The largest contribution of contamination at Superfund sites is typically attributed to site releases. However, some contamination can also result from natural and offsite sources.Contamination derived from off site  is considered a component of representative background concentrations and will continue to be a source of contamination to the site, unless all transport pathways are controlled. A primary objective of determining representative background concentrations is to account for any background chemical input  that is expected to continue affecting the site. One of the guiding principles for management of contaminated sediment sites is that sources should be controlled to the greatest extent feasible prior to initiating remediation at the subject site. According to USEPA, “Generally, significant continuing upland sources… should be controlled to the greatest extent possible before sediment cleanup” USEPA . HoweverDetermining a site boundaryDetermining chemicals of concernEstablishing a realistic long‐term monitoring plan, or optimizing existing long‐term monitoring plansAssessing remedy success.When representative background concentrations accurately reflect ongoing chemical inputs to a site from all sources, this results in defensible representative background concentrations for use in the remedial investigation, remedy selection processes, and performance monitoring. In addition to informing or establishing cleanup levels, representative background concentrations can assist in the following:In the absence of representative background concentrations for remedy decision making, risk‐based cleanup levels may be used inappropriately at sites where representative background concentrations are actually greater than risk‐based concentrations. Alternatively, if the representative background concentration has been underestimated or erroneously calculated, inappropriately low cleanup goals could be used in the remedy selection process. Inevitably, in both cases, these sites will eventually return to background conditions after the remedy has been completed, so the remedy would be considered a failure if the site did not meet cleanup goals over the short or long term. This has been demonstrated on a number of sediment sites throughout the United States, under both federal and state lead  has not been issued. The present article has been prepared in the absence of existing USEPA‐issued guidance on the derivation of representative background concentrations for contaminated sediment sites.There are a number of relevant documents with information on the derivation of background concentrations for upland sites, including risk assessment and soil screening guidance USEPA , and chaFurther, USEPA issued a guidance document in 2002 entitled “Role of Background in the CERCLA Cleanup Program” that seeks to clarify the “preferred approach for the consideration of background constituent concentrations of hazardous pollutants, and contaminants in certain steps of the remedy selection process, such as risk assessment and risk management” USEPA . HoweverIn addition to the federal guidance, some states have also issued guidance related to the derivation of representative background and/or the use of background; the present article focuses on representative background as it applies to federally regulated sites. State guidance is typically similar to federal guidance but may use different terminology or may vary in other ways, such as specific statistical procedures recommended for the screening of background data, characterization of background distributions, and calculation of background threshold values (BTVs).Finally, in order to determine representative background concentrations, it is typically necessary to identify background reference areas. A separate, but related, concept that is not addressed here is the use of reference areas in ecological risk assessments. This involves identifying one or more suitable reference areas to facilitate sampling for the comparison of toxicological responses and/or resident biological communities . Note that there are additional documents relevant to the ecological risk evaluation process , polycyclic aromatic hydrocarbons (PAHs), and metals USEPA . ChemicaDirect discharges. Chemical loading from wastewater and/or stormwater discharge is regulated under Clean Water Act programs, which may set limits for chemical concentrations for discharges from these conveyance systems but does not completely eliminate chemical loading from the direct discharge. Therefore, chemical loading to a site from direct discharges should be accounted for in representative background concentrations because they may continue to be a major source of chemical input, even after site remediation ASTSWMO .Sediment transport. Sediment sites are consistently receiving suspended sediments from offsite areas that contain background concentrations of contaminants from anthropogenic sources and may also contain concentrations of naturally occurring chemicals similar to the chemicals of concern for the site. Because sediment resuspension is a transport pathway for contamination, it is important to consider that representative background levels of contamination will inevitably move into a site through this natural process.Atmospheric deposition. Atmospheric deposition from industrial and urban areas, and areas near major transportation corridors, is a recognized pathway of contamination, particularly for those contaminants ubiquitously found in the environment, such as metals, PAHs, PCBs, and pesticides USGS .Source control. Source control should be fully completed, or at least substantially completed, before remediation of a sediment site begins. If source control has not been completed or is not feasible, it is critical that the potential inputs from uncontrolled ongoing sources be included in the determination of representative background concentrations.Sediment physical properties. The physical properties of sediment strongly influence the distribution of naturally occurring and anthropogenic background chemicals in the environment. The abundance of organic material, inorganic material, and porewater within a sediment body; sediment texture; and sorption capacity should be evaluated with geotechnical testing and general chemistry analyses.Hydrodynamic environment and sediment profile. The vertical profile of sediment, rates of sediment deposition, erosion and removal, and mixing vary widely among aquatic environments and should be assessed as part of the CSM because these factors affect chemical distribution in sediments.Geochemistry. Metals concentrations in sediment are controlled by several factors, such as redox, pH, and adsorption–desorption reactions on mineral surfaces. These processes should be evaluated using analytical data for metals (major elements as well as trace elements of concern), anions , and total organic carbon (TOC). When site and background metals concentrations are geochemically evaluated, then mechanistic explanations for elevated concentrations  can be provided.Factors that typically contribute to representative background concentrations are summarized in the list below and are discussed in more detail in the Supplemental Data associated with this article. These factors should be considered when developing the study design for representative background determination.Determination of background conditions at a sediment site almost always requires additional sampling and/or data analyses. To ensure the reliability of these evaluations, appropriate procedures should be considered during each phase of the investigation. Topics that require special attention include those related to the practical aspects of sampling design, selecting the representative background reference areas, using existing site data, choosing appropriate statistical methods for comparison, addressing outliers, and geochemical evaluation of sample data. These topics are discussed further in this section.In general, unless existing contemporaneous data are adequate for extracting site‐specific background data USDON , additioAgency agreement on the scope and scale of the sampling effort to determine representative background concentrations is important and should be captured in a site's data quality objectives, using USEPA's Data Quality Assessment approach USEPA .One of the critical steps in a background analysis is the selection of representative background reference areas. As discussed, representative background reference areas should have “the same physical, chemical, geological, and biological characteristics as the site being investigated, but [have] not been affected by activities at the site,” and they should be informed by the CSM USEPA . FurtherDefining the extent of site influence in urban areas is very difficult and should be considered carefully. Selection of such an analogous area is complicated, due to the fact that sediment background often represents mixtures of naturally occurring and anthropogenic influences. In some cases, these mixtures yield geographically distinct background populations . Under such situations, the parts of the targeted background reference areas (or subareas) that are most analogous to the site should be selected as the background reference areas. Selection of analogous background reference subareas is often supported by multiple lines of evidence, including degrees of urbanization, presence or absence of combined sewer overflows, prevailing sediment TOC content, and grain sizes.In many instances, site data include samples that are free of site influences. Particularly within a larger site data set, there will be samples not affected by site releases that will be reflective of representative background conditions. In these cases, statistical methods, such as probability plot analyses, are recommended for extracting site‐specific background data sets from existing site data sets USDON . This apThe extraction of representative background concentrations from site data not only maximizes the utility of existing data, but also avoids the often complex task of selecting separate background reference areas that adequately represent the site. Even when data from separate offsite background reference areas are available, an extracted site‐specific background data set provides an additional line of evidence for determining representative background concentrations. Therefore, an analysis of existing site data is always recommended.Due to the different types of contamination , USEPA guidance recommends simultaneous use of multiple tests for a valid and complete comparison of background and site distributions USEPA . There aThe point‐by‐point comparison approach is based on comparing individual site measurements to a given BTV, either to delineate the extent of impact or to identify localized (or “hot spot”) contamination. A BTV is a specific value intended to define an upper limit to background concentrations for a given site. Common candidates for BTV include the upper tolerance limit , the upper prediction limit , and the upper simultaneous limit  An outlying observation may be merely an extreme manifestation of the random variability inherent in the data. If this is true, the values should be retained and processed in the same manner as the other observations in the sample. (2) On the other hand, an outlying observation may be the result of gross deviation from prescribed experimental procedure or an error in calculating or recording the numerical value. In such cases, it may be desirable to institute an investigation to ascertain the reason for the aberrant value. The observation may even eventually be rejected as a result of the investigation, though not necessarily so. At any rate, in subsequent data analysis the outlier or outliers will be recognized as probably being from a different population than that of the sample values.”There are well‐established procedures in statistics to identify outliers, including visual inspection of graphs using particular techniques, such as probability and box‐and‐whisker plots, as well as statistical tests, such as Rosner's test and Dixon's test USEPA , 2006. TA careful investigation or review should be conducted for each statistical outlier, with scientific reasoning to ascertain the cause of the aberrant value Grubbs . If therIf the error cannot be corrected, the associated true statistical outliers should be eliminated from the background data set. .If no error can be identified or confirmed, false outliers should not be arbitrarily eliminated.The USEPA has recognized the importance of properly evaluating apparent outliers and not excluding data points simply based on their magnitude. USEPA's “Data Quality Assessment: Statistical Methods for Practitioners” USEPA  divides Thus, an outlier should not be eliminated from the background data set just because it is the highest or lowest value in the data set or based on the perception that the outlying value is too high or too low to fit into the background data set. In this case, the outliers “may be merely an extreme manifestation of the random variability inherent in the data [and] the values should be retained and processed in the same manner as the other observations in the sample” Grubbs . True ouAs noted by USEPA, it is critical that the considerations outlined in this discussion on outliers are followed during the statistical analyses of the background data. Exercising caution to not improperly exclude “false” outliers, which accurately represent conditions at the background reference areas, will ensure technically defensible derivation of representative background concentrations and will also avoid errors in the statistical approach when relying on a preconceived notion that outliers “distort statistics if used in any calculations” USEPA . FinallyGeochemical evaluation is a tool used to evaluate elemental  concentrations in a given data set, which may include exceedances of representative background concentrations  to which the trace element is adsorbed, is plotted on the x‐axis. For further analysis, a ratio plot may also be prepared; like the scatter plot, the concentration of the trace element of interest is plotted on the y‐axis, but the corresponding elemental ratio (trace element concentration divided by reference element concentration) is plotted on the x‐axis. If a metal is found at an elevated concentration and that sample's elemental ratio lies outside the range of background elemental ratios, then that sample should be examined further. For example, the elevated ratio might reflect anthropogenic input of the trace element from the site, or it may indicate that the trace element concentration of that sample is controlled by another geochemical process, such as reducing conditions or trace metal precipitation in the sediment. If the sample's ratio lies within the range of background elemental ratios, then it is considered representative of background conditions.Scatter plots may be prepared in which the concentration of a trace element of interest is plotted on the Clay minerals in the pH range of 6 to 8 have a strongly net negative surface charge and attract positively charged trace metal ions, so these trace metals adsorb to clay mineral surfaces. Aluminum is a primary component of all clay minerals, and detected Al concentrations in sediment serve as proxy indicators of the relative amounts of clay minerals  typically have a net positive surface charge in the pH range of 6 to 8. Detected Fe concentrations serve as proxy indicators of the relative amounts of Fe oxide minerals in sediment samples from oxic environments  can also be used. Grain size and TOC content are additional reference parameters. For example, due to the affinity Hg has to adsorb onto organic matter, covariance of Hg versus TOC concentrations may be observed in the absence of site‐related Hg contamination.The selection of a reference element for the scatter or ratio plot should be based on a careful comparison of the reference element and the trace element of interest, as well as consideration of site‐specific geochemical processes. The following points provide a general overview of a few relevant elemental associations, but the scientific literature provides additional information.x‐axis of a ratio plot.Although quantitative statistical techniques are commonly applied to identify outliers or to develop pass–fail criteria for the presence of contamination, they are not recommended for geochemical evaluations that use scatter plots or ratio plots, for many scientific reasons  to achieve a more complete understanding of historical and ongoing sources to the site. In the absence of detailed guidance, these considerations may be overlooked or discounted when calculating representative background concentrations at sediment sites.Anthropogenic inputs to the site, such as land use, urban runoff, direct discharge, sediment transport, atmospheric deposition, and source control.Natural characteristics of a site, such as sediment physical properties, hydrodynamic environment, sediment profile, and geochemistry.Conceptual site models are critical tools for characterizing the complexity of sources to a site, migration pathways, receptors, and exposure pathways, and they inform the appropriate selection of background reference areas. The CSM developed for a site should include these key considerations (as outlined in the present article):Similarities between the site and the background reference areas are important because they influence the transport and fate of contamination. Anthropogenic sources that cannot be controlled contribute ongoing contamination and should be fully considered and incorporated into the CSM because they represent anthropogenic background chemical concentrations that will persist on site during and after any remedy. It may not be feasible to control all offsite sources of anthropogenic background prior to remediation, which should inform potential cleanup goals.After selecting representative background reference areas and completing a targeted sampling program that uses sampling methods matching those used during site data collection, the data should be closely evaluated. The focus of the data evaluation should be on comparing site data with background data, using appropriate statistical approaches  to derive technically defensible representative background concentrations. During the data evaluation, it is imperative that false outliers are retained and are not arbitrarily removed because natural variability occurs in a data set. A statistically appropriate outlier evaluation should be performed on the background data set, and the evaluation should include the key steps outlined in the discussion on outlier evaluation. Of critical importance, only true outliers should be removed from data sets, and false outliers should not be arbitrarily eliminated.Representative background concentrations should remain fixed for the duration of the remedial investigation and remedial response. Otherwise, the lack of certainty for stakeholders would be an impediment to the implementation of any remedy. Collectively, the considerations and approaches outlined in the present article should increase the ability to derive technically defensible representative background concentrations. These recommendations are offered to help inform, improve, and increase the consistency of sediment site remedy decision making.The peer‐review process for this article was managed by the Editorial Board without the involvement of S Brown.Adapted from Important Considerations in the Derivation of Representative Background Concentrations for the Evaluation of Sediment Sites: Supplemental Material Related to a Conceptual Site ModelThis article includes online‐only Supplemental Data.Supporting InformationClick here for additional data file.