Macroalgae use energy-dependent carbon concentrating mechanisms (CCMs) to take up HCO3−, the dominant inorganic carbon for marine photosynthesis, but carbon-use strategies may depend on the pCO2, pH and irradiance. We examined photosynthesis in eight tropical macroalgae across a range of irradiances (0–1200 μmol photon m−2 s−1), pH levels (7.5–8.5) and CO2 concentrations (3–43 μmol kg−1). Species-specific CCM strategies were assessed using inhibitors and δ13C isotope signatures. Our results indicate that the log of irradiance is a predictor of the photosynthetic response to elevated pCO2 (R2 > 0.95). All species utilized HCO3−, exhibited diverse C-use pathways and demonstrated facultative HCO3− use. All fleshy species had positive photosynthetic responses to OA, in contrast to a split amongst calcifiers. We suggest that shifts in photosynthetically-driven tropical macroalgal changes due to OA will most likely occur in moderate to high-irradiance environments when CCMs are ineffective at meeting the C-demands of photosynthesis. Further, facultative use of HCO3− allows greater access to CO2 for photosynthesis under OA conditions, particularly amongst fleshy macroalgae, which could contribute to enhance fleshy species dominance over calcifiers.Fleshy macroalgae may increase photosynthesis with greater CO The oceans sequester this excess atmospheric CO2 with the resulting consequence of lowering ocean pH, referred to as ocean acidification (OA). Ocean acidification is resulting in a wide-range of impacts on marine organisms and ecosystems sensitive to shifts in the carbonate chemistry7 (an increase in pCO2 and HCO3− and a decline in CO32−). While the effects of elevated atmospheric CO2 levels on terrestrial plant photosynthesis has been well studied over the last several decades, our understanding of elevated CO2 effects on marine macroalgal photosynthesis has only been emerging over the last decade13. In marine systems, increased pCO2 and HCO3− levels under OA has the potential to provide dissolved inorganic carbon (DIC) for algal photosynthesis when carbon is limiting. A review of OA studies indicate that raising ocean pCO2 and lowering pH have negative effects on growth, calcification and metabolism of many calcifying macroalgae, but a positive effect on the growth of non-calcareous, fleshy species14. This is a disturbing trend as many fleshy macroalgae can readily become nuisance species15 and outcompete calcifiers important for cementing and accreting reefs. While an increase in fleshy over calcifying macroalgae under OA is recognized as a potential problem17, the mechanisms accounting for OA-induced growth responses in macroalgal species are elusive. OA-induced growth responses may be related to species-specific photophysiology, as has been found near naturally acidified volcanic seeps or vents18, which are considered a useful proxy for long-term exposure to elevated pCO2 in the field.The oceans have been absorbing ~30% of the total global anthropogenic COpCO2, because individual species use different mechanisms of inorganic carbon uptake. HCO3− use can be advantageous in marine macroalgae because the ocean pH (8.1) maintains CO2 in seawater at a low concentration (13 μmol kg−1) compared to HCO3− (1867 μmol kg−1). Furthermore, CO2 diffuses 10,000 times slower in water compared to air, thus CO2 availability can limit marine macroalgal photosynthesis5. Many marine macroalgae depend on HCO3− to supplement CO2 as a source of inorganic carbon for photosynthesis to overcome inorganic carbon limitation19. Carbon concentrating mechanisms (CCMs) in macroalgae are characterized by a range of efficiencies in providing CO2 to rubisco for photosynthesis18. One CCM mechanism is the secretion of external carbonic anhydrase (CAext) into the cell wall where it catalyzes the dehydration of HCO3− to CO2 after which it can passively diffuse into the cell21. A second mechanism is the use of ATPase H+ pumps that lower the pH at the algal surface and shift the carbonate equilibrium towards CO2. This mechanism can also generate a proton-motive force for active transport of CO2 or HCO3− 24. So far, evidence for H+ pumps has been presented for only a few temperate marine macroalgal species and microalgae25 and its linkage to photosynthetic OA response is unknown. Another mechanism is an anion exchange (AE) protein that facilitates the active uptake of HCO3− 29.Inorganic carbon uptake strategies may be a factor in determining macroalgal responses to elevated 3− may be less carbon limited under current pCO2 than species that rely solely on CO2, it has been hypothesized that algae which use HCO3− and possess CCMs will be less responsive to increases in pCO2 than species that only use CO2 6. This is supported by studies that have shown temperate macroalgae that rely exclusively on CO2 have increased growth and photosynthesis under elevated pCO2 22, whereas macroalgae that utilize HCO3− elicit no photosynthetic response to elevated pCO2 30. Counter to these results, lower isotopic signatures, an indicator of greater CO2 use, was more prevalent in macroalgae closer to high CO2 seeps in the field, suggesting species that utilize greater CO2, when available, may be more competitively dominant, even when they also possess CCMs. These data suggest potential facultative HCO3− use may be selected for in a high CO2 ocean if CCMs are downregulated30, and may confer competitive dominance. While facultative HCO3− use may be an optimal carbon use strategy, species in low-light environments can be restricted to non-CCM strategies due to the energy requirements of active C-use mechanisms32. Thus, establishing the role of C-use mechanisms and light in modulating responses to OA is required to clarify under what conditions photosynthesis has the potential to increase under greater ocean CO2 availability.Due to the fact that species using HCOpCO2 and lower pH, including those predicted for 210033 (scenario RCP 8.5). We asked if species employing CCMs are obligate HCO3− users or if they respond to greater CO2 availability indicative of a facultative, flexible physiology in regard to inorganic carbon sequestration. Stable isotope signatures were used as an indicator of their inorganic carbon source for photosynthesis based on the assumption that macroalgae with δ13C values > −10 only use HCO3−, those between −11 and −30% are both HCO3− and CO2 users, while those with values < −30% are restricted to CO2 use34. Specific HCO3− use mechanisms were assessed using inhibitors. Further, we examined the interactive role of light by assessing the photo-physiological responses to pH across a broad range in irradiances , comparing photosynthetic efficiency, maximum net/gross photosynthesis, light compensation point and respiration, based on photosynthesis-irradiance curves. We hypothesized that species with the capacity to utilize HCO3− for photosynthesis would show little or no enhancement of photosynthesis in response to lowered pH and elevated pCO2. Based on the dominance of fleshy species at CO2 enriched sites in the field36, particularly those in the brown phyla, we proposed that fleshy macroalgae would preferentially increase photosynthesis compared to calcifying species under low pH.In this study, we examined if species-specific carbon uptake mechanisms would determine the photosynthetic response of five calcareous and three fleshy tropical macroalgae to elevated −2 s−1 bottom) patch reef along the Florida Reef Tract at Looe Key  on five collection trips (May 2016 to January 2017). Field light levels were measured just above the benthos with a 4π spherical PAR quantum sensor . Species included five calcifying and three fleshy species, representing the three macroalgal phyla: calcified green algae , calcified red algae , fleshy brown algae , and a fleshy red alga (Laurencia intricata). With the exception of CCA and Saragassum, algae were collected by removing the whole thallus from the substrate. Thalli branches were kept intact when subsampling to minimize disturbance. CCA were collected on 80 small Plexiglas plates (2 cm × 1 cm).Macroalgae were collected from a shallow (~3 m), high irradiance (700–1200 μmol photon m2 SYS37). Macroalgae were transported to the laboratory in an aerated cooler and transferred to aquaria with carbonate sand and seawater from the study site situ seawater. Aquaria were kept in a water bath at 27 °C, the average seasonal temperature on the Florida Reef Tract; light was maintained on a 12:12 light/dark cycle (150 μmol photon m−2 s−1). Salinity and temperature were measured and maintained at ambient levels (~36 psu and 27 °C) throughout the experiment. All experiments were run within two weeks of collection. Replicates for each experiment were run sequentially to account for any differences in responses for algae immediately taken from the field growing at 700–1200 μmol photon m−2 s−1 and those in the lab maintained at a lower light level; our excellent replication among treatments provides confidence that algal responses were not significantly influenced by short-term exposure to lower irradiance. Further, no photoinhibition was found for any algal species at high experimental irradiance.During macroalgal collections, site pH , temperature, irradiance and salinity were determined in the field. Water samples  were collected and total alkalinity determined within 48 h . Five carboys of seawater were collected to run experiments with seawater from the study site seawater. Alkalinity, temperature, conductivity and pH data were used to calculate DIC speciation (CO33 and low (7.5). Different individuals were used for each run  and runs conducted between 10:00 to 19:00 in filtered (0.45 μm) seawater. To achieve pH treatments, CO2 gas was bubbled into seawater to lower pH (7.8 and 7.5) and 0.1 M NaOH was added to raise pH (8.5). The pH meter (Orion A211) was calibrated daily with a pH standard . Alkalinity, temperature, conductivity and pH were used to calculate CO2 concentrations in each pH treatment (CO2 SYS). Alkalinity was 2,369, 2,378, 2,449, and 2,805 μmol kg−1 for pH treatments 7.5, 7.8, 8.1, and 8.5 respectively. The higher alkalinity in the high pH treatment was due to adjusting pH with NaOH38; however, the change in alkalinity was due to an increase in hydroxyl anions (OH−), because no additional carbon was added to the system. The four pH treatments  resulted in approximately an order of magnitude difference in CO2 levels  based on DIC speciation calculations .Photosynthetic and respiration rates were determined at four pH values: high (8.5), ambient (8.1), projected levels for 2100 ns Table . Before 2 electrode and data acquisition system which recorded O2 concentrations every second . The O2 electrode was calibrated daily. Light was provided by an LED light source , calibrated daily with a 2π PAR quantum sensor  held up to the chamber’s glass portal, and subsequently checked at 3 light levels  with a resulting accuracy of approximately ±5 μmol photon m−2 s−1. The Chlorolab 3 was programmed to increase light every two minutes to preset irradiances ; this resulted in 16 min incubations. A short incubation time of 16 minutes resulted in minimal changes of seawater pH (average ±0.01) during each incubation. The 120 points over two minutes at each light level were linearized and the slopes used to calculate the rate of O2 flux . In the Chlorolab 3 system, the light source is projected from one side of the chamber, thus the respiration:photosynthesis ratio in this system would be expected to be lower than field conditions, resulting in relatively high compensating irradiances; however, all algae were subjected to the same chamber conditions across treatments. Each algal sample was dark acclimated for ~5 minutes prior to experimentation. Water temperature was controlled using a circulating water bath set to 27 °C. Each replicate (n = 6–8) of 0.5 g fresh tissue mass of calcified species or 0.25 g fresh tissue mass of fleshy species was placed into the 20 mL Chlorolab chamber with filtered (0.45 μm) seawater. O2 flux rates were normalized to fresh tissue mass with the exception of CCA, which was normalized to surface area. PI curves were calculated using a hyperbolic regression model  + R) and photosynthetic parameters calculated using Excel’s data solver tool40. Parameters included photosynthetic efficiency (α), maximum net photosynthesis (Pmax), maximum gross photosynthesis (Pgmax), light compensation point (Ic), and respiration (R).Photosynthesis-irradiance (PI) curves were determined using an Olux Fig. . Irradia26. Inhibitors included acetazolamide  that blocks the dehydration of HCO3− into CO2 via external carbonate anhydrase (CAext)26, pyridoxal (5) phosphate  that inhibits active uptake of HCO3−, Tris buffer  that interferes with proton pump acidification of the thalli boundary layer25 and sodium orthovanadate  that obstructs plasmalemma ATPase H+ pumps23. Solutions of AZ (200 μM), PLP (480 μM) and Tris (50 mM) were dissolved in filtered seawater (0.45 μm) followed by pH adjustment to 8.1 (1 M HCl). A 200 mM stock solution of vanadate was prepared by dissolving sodium orthovanadate in deionized water and activated using several cycles of boiling, cooling, and adjusting the pH to 10. For each experimental replicate, vanadate stock solution was added to filtered seawater for a final concentration of 400 μM. The seawater pH was checked after adding the vanadate stock solution. Concentrations of each inhibitor were chosen based on previous studies41 and preliminary dose response curves.Photosynthetic rates were determined in the presence and absence of inhibitors that blocked specific inorganic C uptake mechanisms. Each experimental run used different individuals resulting in ~320 total runs (8 sp × 8 replicates × 5 [1 control and 4 inhibitors]). Inhibitors were chosen based on previous studies which identified inorganic carbon uptake mechanisms in algae2 evolution in the same Chlorolab 3 system as used in the pH experiments. Irradiance levels, incubation time, normalization to fresh tissue mass or surface area, PI curve construction and parameter determination were also as described above for pH experiments.Photosynthetic rates were measured by Oext-supported photosynthesis, PI curves were established across a range of pH in the presence and absence of AZ. Two species, C. cervicornis and J. adhaerens, were chosen for these experiments based on a significant CAext and pH response in preliminary studies. Photosynthetic rates were measured by O2 evolution as in the pH and inhibitor experiments.To determine the effects of pH on CA13C isotope analyses. Upon returning to the lab, tissues were acidified to remove carbonates, dried at 60 °C to constant weight, and ground with a mortar and pestle for analysis. Tissue δ13C was determined using a mass spectrometer (Thermo Electron DeltaV Advantage) coupled with a CNS Elemental Analyzer (ConFlo II interface linked to a Carlo Erba NA 1500) at the Stable Isotope Mass Spec Lab . All carbon isotope results are expressed in standard delta notation relative to VPDB.Fresh tissue samples (n = 5) of each species were collected at the Looe Key patch reef site for δ42 and SigmaPlot (v13 Syststat Software Inc.). Assumptions of normality of residuals and homogeneity of variance were examined using a Shapiro Wilkes and Levene’s test, respectively. For parameters where assumptions were not met, data was transformed with a log or square root transformation. In the case that homogeneity of variance was not met after transformations, a non-parametric Kruskal-Wallis rank sum test was used in place of an ANOVA. The effects of pH treatments and inhibitors on PI parameters  were tested using ANOVA. A post hoc Tukey pairwise comparison test was used to determine which pH levels had significant differences in photosynthetic parameters. A post hoc Dunnett test was used for the inhibitor experiment to compare each inhibitor group to the control. For the pH x AZ experiment significant Pgmax was determined for C. cervicornis and J. adhaerens using a two-way ANOVA followed by a post hoc Tukey pairwise comparison test. Data was transformed using a square root transformation. Differences in species δ13C signatures were determined using a Kruskal Wallis test on ranks followed by Dunn’s test. ANOVA tables for pH and inhibitor experiments are presented in the supplement.Statistical tests  were conducted using R2 flux used to calculate photosynthetic rates at each light level and the average results from pH and inhibitor experiments as a function of irradiance. Statistical tables and average values generated from the study presented in graphs are also included in the supplement. All other datasets are available from the corresponding author upon request.Examples of raw data generated from this study are graphed in the supplement, including an example of the linearity of OpCO2 on tropical macroalgal photosynthesis was on maximum photosynthetic rates, as illustrated by the significantly higher Pgmax  and calcified algal  species. While comparison among mean Pgmax was primarily significant between pH 7.5 and 8.5 using ANOVA, regression analysis indicated significant trends across the four pH treatments. There was a significant linear increase in Pgmax from pH 8.5 to 7.5 for all fleshy  and two calcified species  that exhibited pH end member differences in Pgmax, suggesting that the trend in elevated photosynthesis has the potential to track pH declines with OA. Two red calcifiers showed no significant trends  using regression analysis across the four pH levels and no significant differences among means based on ANOVA.The greatest effect of lower pH and higher max Fig.  parametemax Fig.  and S3. gmax also showed similar pH effects on Pmax with the exception of S. fluitans. Sargassum fluitans respiration rates were lower at 8.5 compared to 7.8 pH, although were similar  across pH treatments  and without a linear trend  at lower pH . In the two fleshy species, L. intricata and S. fluitans, α significantly increased as a function of decreasing pH . Halimeda opuntia, L. intricata, and U. luna also had lower irradiance compensation points at lower pH  and two fleshy  species , and the fleshy species, S. fluitans, showed linearity at low irradiance  and then a modest decline above saturation . These data indicate that irradiance is critical to the enhanced photosynthetic response under OA conditions in both fleshy and calcified macroalgae that respond to elevated pCO2.The PI curves showed the difference in photosynthetic rates between pH treatments increased with irradiance Fig.  in the snts Fig. . Compariies Fig. . The calgmax and/or Pmax rates were significantly lowered with the inhibition of CAext by AZ  in Pgmax and/or Pmax , with an approximately 30% decline without CAext. In contrast, J. adhaerens showed no significant decline in photosynthesis with CAext  as pH increased, compared to a decline without CAext . These data, together with those in Fig. 3−, and specifically CAext, to sequester DIC may decline as OA provides greater CO2 availability under a lower pH.The relative importance of CAudy Fig. . Signifiudy Fig. . Pgmax i13C signatures for all species examined fell within a relatively narrow range between −14 and −20  with H. opuntia exhibiting a more depleted δ13C signature than S. fluitans, N. strictum, CCA and U. luna. There were also significant differences between C. cervicornis and U. luna. Further, the two red species  that responded significantly to pH treatments had 20% lighter signatures than the two red species that did not .The organic tissue δ−20 Fig. . Statist3− use, all the fleshy (100%) and a high proportion of the calcified (40%) species had the ability to increase photosynthesis under OA conditions, especially at high irradiance. Our results support the supposition that inorganic carbon physiology may underlie macroalgal responses to elevated pCO2. These data concur with those from field studies at naturally high CO2 sites where flexibility to utilize CO2 for photosynthesis increased a species abundance in the community adjacent to the CO2 seeps18. Several experimental and culture studies have also shown an increase in photosynthesis and growth in temperate macroalgae in response to elevated pCO244. Others suggest that greater CO2 availability only advantages macroalgae that solely depend on CO2 diffusion and lack CCMs46. We suggest that facultative use of HCO3− under greater access to CO2, regardless of CCM mechanism employed, is more likely to predict tropical macroalgal responses to OA, particularly if they are not light limited.Although the tropical macroalgal species examined showed evidence of HCOpCO2 and irradiance is critical to predict how macroalgae are likely to respond to OA. Six of the eight species studied, including both fleshy and calcifying species, increased maximum photosynthetic rates at light saturation (Pgmax or Pmax) as a result of lower pH and higher pCO2, indicating the importance of irradiance for the OA response in these autotrophs. Irradiance influenced the degree to which species responded to lower pH. Maximum photosynthetic rates increased as a log or linear function of irradiance in six species under OA conditions and the photosynthetic efficiency parameter (α) significantly increased in two species as a function of decreasing pH. Thus, the specific photosynthetic response to changes in pH was dependent on the light gradient. Since all species within the patch reef community sampled were growing in a high-light environment (700–1200 μmol photons m−2 s−1), light limitation should not constrain their response to elevated pCO2 and their ability to utilize CCMs in the patch reefs of the Florida Reef Tract. However, under lower light, macroalgae may be saturated with respect to inorganic carbon, where light is the primary limiting factor47, or CCMs may be downregulated due to low-energy availability. Based on a survey of macroalgae on the Great Barrier Reef, the only non-CCM species were from deep (10 m) reef sites9, consistent with the findings of non-CCM macroalgal species from deep temperate rock reefs45. Thus, CCMs likely require adequate irradiance to support energy-dependent C-uptake of HCO3−, but the carbon demand under high irradiance also increases and is likely satiated by greater CO2 uptake when available.Given that most macroalgae reside primarily on the benthos where the light environment can vary substantially, understanding the interactive effects of elevated 13C ratios <−30‰ found at depth on the Great Barrier Reef 9 were fleshy red species, while the calcifying red and green species had stable isotope ratios (−13 to −19‰) indicative of both HCO3− and CO2 use, consistent with those found on patch reefs in this study and the majority (~75% or more) of macroalgae on the Great Barrier Reef. While the literature to date indicate a lower photosynthetic response to OA by calcifying versus fleshy algae in temperate macroalgae8, our results with tropical calcifying species present a diversity of HCO3− use mechanisms and CO2 uptake responses. For example, pH had no effect on photosynthesis in the red calcareous species N. strictum and the CCA assemblage. These two species also presented the highest δ13C signatures amongst the eight species, suggesting more dependency on HCO3− and/or less ability to sequester CO2. However, based on inhibitor studies, no HCO3− uptake mechanism was discerned for N. strictum. In contrast, the red calcareous alga, J. adhaerens, showed a statistically significant difference in Pmax and Pgmax as a function of pH and had a moderately lower δ13C signature than N. strictum and CCA. These data confirm that, while some red calcareous algae lack a photosynthetic OA response49, there are exceptions50. Our results support the contention that ecologically important red calcifiers should be examined for their species-specific OA responses. This is particularly important in the red phyla because of its bimodal distribution in δ13C signatures (peak at −35 and −20) compared to green and brown phyla with a unimodal peak (peak at −20) based on a global meta-analysis of marine macroalgae51. In the two green calcifying species, H. opuntia and U. luna, photosynthetic rates significantly increased at low pH and were highly correlated to the log of irradiance. The results for H. opuntia are in contrast with others that found no or negative effects of pCO2 on photosynthesis54, but isotope signatures from this study, and those from the Great Barrier Reef9, indicate both HCO3− and CO2 uptake. Calcifiers are also unique in that CO2 can become more available via the calcification process, thus more research is needed to understand linkages between photosynthesis and calcification.Interestingly, the three non-CCM species identified by δC. cervicornis, S. fluitans, and red, L. intricata, phyla increased Pgmax and/or Pmax at lower pH. This is consistent with a high percentage of species within the brown phyla show increases in photosynthesis under OA conditions57 and fleshy species in general increasing percent coverage near natural CO2 seeps58 at the expense of calcifiers. The idea that calcifiers do not respond photosynthetically as a group to elevated pCO2 may be confounded by observations that they decline in the field adjacent to CO2 seeps. This observation may be better explained by either direct OA effects on their carbonate thalli structure, due to changes in calcification and dissolution processes, or dominance by fleshy species that are more successful competitors for resources under OA conditions59.In contrast to calcifiers, all three-fleshy species studied, including those from brown, pCO2, they all showed evidence of HCO3− use based on stable isotope ratios. The δ13C range between −14 and −20 imply HCO3− use in photosynthesis for the tropical macroalgae studied. The most prevalent mechanism for HCO3− uptake was the use of CAext shown by the reduction in photosynthesis in the presence of AZ. Two fleshy and one calcifying species also showed evidence for active uptake and proton pumping to facilitate HCO3− exchange. Evidence for an AE protein role was found for the first time in J. adhaerens and L. intricata. To date, the evidence for AE proteins has only been found in temperate brown29 (Macrocystis pyrifera), green and red species26. Acidification of the boundary layer was important for S. fluitans and L. intricata based on Tris inhibition. One mechanism for acidification of the boundary layer is the use of an active ATPase H+ pump, but only L. intricata showed evidence for proton pumping in the inhibitor experiments. Based on inhibitor studies, it appears that several mechanisms exist in tropical macroalgae to moderate CO2 limitation at current seawater CO2 concentrations by gaining access to the more abundant HCO3− ion. A meta-analysis of 613 species world-wide showed δ13C values increase with decreasing latitude for both brown and red phyla, indicating the significance of CCMs in tropical macroalgal species51. However, δ13C values still ranged between −10 and −30 in these two phyla globally, demonstrating the diversity of C-uptake strategies in tropical macroalgae.Even though the majority of calcifiers and all the fleshy species’ photosynthetic rates increased in response to elevated 2 becomes more available to HCO3− users under low pH, the dependency on HCO3− use via CAext appears to be dampened, as was shown for C. cervicornis and J. adhaerens. This finding is consistent with recent volcanic CO2 seep studies that found lowered δ13C values in macroalgae closest to natural CO2 seeps18. At this site, HCO3− users were hypothesized to have decreased HCO3− uptake and acquired more CO2. The greater CO2 use by macroalage under long-term elevated CO2 exposure at vents support our conclusion, using highly controlled short-term experiments, that macroalgae utilizing HCO3−, and constrained by low pCO2 in seawater, are likely to take advantage of the greater CO2 availability as the oceans become more acidic. Because marine macroalgae developed HCO3– use mechanisms to facilitate CO2 acquisition in seawater with low CO2 availability, it is reasonable to assume they would take advantage of increased seawater pCO2, supported by trends in greater CO2 use in macroalgae with increasing latitude51. In the field, CO2 availability can be enhanced from short diurnal cycles of community metabolism where respiration rates exceed photosynthesis and from longer trends controlled by cultural eutrophication and OA. The potential for some fleshy macroalgal species to decrease HCO3− uptake or increase gross photosynthetic production with greater CO2 availability can result in elevated growth and abundance, as has been shown experimentally14 and reported from naturally acidified volcanic seeps18.As CO3− uptake can increase their photosynthetic rates under low pH and greater CO2 availability. The consistent positive photosynthetic response to OA by fleshy species, in contrast to a split amongst calcifiers, support the possibility that fleshy forms could outcompete calcifiers. While we present data that tropical macroalgae utilize a diverse suite of HCO3− use pathways, some species are likely to become less dependent on HCO3− as an inorganic C source for photosynthesis under OA. This study also clearly indicates the importance of irradiance in controlling the photosynthetic response of tropical marine macroalgae to OA. Our results show that at low irradiance, light limitation, rather than carbon limitation, probably controls photosynthetic responses to OA. This presents two important implications for OA research: (1) studies must take careful measurements and control light levels in order to compare amongst OA studies, (2) in low-light environments, such as on deep reefs and under ledges, elevated pCO2 and a lower pH may not affect photosynthesis of macroalgae unless CO2 is limiting and HCO3− use is energetically constrained, and (3) the major shifts in photosynthetically-driven macroalgal changes due to OA will most likely occur in moderate to high-irradiance environments when CCMs are inefficient at meeting the C-demands of photosynthesis and greater access to CO2 enhances direct and/or indirect competitive interactions. While we identify diverse C-use strategies for photosynthesis in tropical macroalgae using inhibitors, this approach is complicated by species-specific responses due to thalli structure and potential effects on non-target metabolic functions, thus more work is needed to further support C-use mechanisms presented herein. Further, studies that identify the importance of elevated photosynthesis to growth for these species are warranted.We conclude that tropical macroalgae that utilize multiple mechanisms for HCOSupplementary Information


Alpha-synuclein is an intrinsically disordered, highly dynamic protein that pathogenically aggregates into inclusion structures called Lewy bodies, in several neurogenerative diseases termed synucleinopathies. Despite its importance for understanding disease, the oligomerization status of alpha-synuclein in healthy cells remains unclear. Alpha-synuclein may exist predominantly as either a monomer or a variety of oligomers of different molecular weights. There is solid evidence to support both theories. Detection of apparent endogenous oligomers are intimately dependent on vesicle and lipid interactions. Here we consider the possibility that apparent endogenous alpha-synuclein oligomers are in fact conformations of membrane-bound alpha-synuclein and not a bona fide stable soluble species. This perspective posits that the formation of any alpha-synuclein oligomers within the cell is likely toxic and interconversion between monomer and oligomer is tightly controlled. This differs from the hypothesis that there is a continuum of endogenous non-toxic oligomers and they convert, through unclear mechanisms, to toxic oligomers. The distinction is important, because it clarifies the biological origin of synucleinopathy. We suggest that a monomer-only, lipid-centric view of endogenous alpha-synuclein aggregation can explain how alpha-synuclein pathology is triggered, and that the interactions between alpha-synuclein and lipids can represent a target for therapeutic intervention. This discussion is well-timed due to recent studies that show lipids are a significant component of Lewy pathology. In different synucleinopathies, neurons and/or glia bare the hallmark intracellular deposits of filamentous αsyn21 and oligomerization23 are available; hence, for the sake of clarity, we will not comprehensively discuss the literature.Are there benign αsyn oligomers that have normal cellular functions, and if so, how do they transition to toxic αsynO-β? Here, in this short review, we discuss how lipid–αsyn interactions might help explain the observation of apparent endogenous non-toxic oligomers and highlight alternative models that are monomer-centric. Furthermore, we will extend this viewpoint to consider its implications for synucleinopathy pathogenesis. There has been substantial work done in this area and several exhaustive reviews on lipid–αsyn interactions24 Lipids have diverse cellular functions in biology including cell signaling, energy storage, and structural partitioning.25 Phospholipids contain amphipathic characteristics with a charged hydrophilic phosphate group and a carbon chain of varying lengths.25 Phospholipids spontaneously form bilayer structures in aqueous solutions that are the basis of cellular membranes. Lipids have not been as extensively studied as proteins in vivo, possibly because of their hydrophobicity, chemical complexity, and the fact that they are not gene products.25 However, lipids are crucial for cellular function and are implicated in several neurodegenerative diseases including synulceinopathies.26 Recent technological advances with lipodomic analysis have furthered the study of lipids.30 Current lipodomic analyses, however, are focused on whole-cell lysates and therefore insensitive to cellular spatial and temporal dimensions, which are crucial for understanding lipid function.25The brain is ~60% lipids by weight.31 the lipid-binding properties of asyn were documented and the significance of disease-causing point mutations in lipid-binding domains was recognized.32 Indeed, one of the earliest histochemical descriptions of Lewy bodies noted that they stain positively for phospholipids, particularly sphingomyelin.33 Since then, interactions between αsyn and vesicle lipids have been implicated in the initial generation of synucleinopathy.36 The hypothesis that lipids control pathological αsyn aggregation primarily stems from observations that lipids/vesicles influence aggregation kinetics in vitro,40 and that disease-related missense mutations of SNCA alter residues within the N-terminal lipid-binding domain of αsyn.46 αSyn may redistribute to lipid compartments early in disease pathogenesis.36 Several disease-causing αsyn mutants abnormally associate with intracellular vesicles and lipid droplets,48 and dyshomeostasis of intracellular lipids are likely an early molecular event preceding pathology formation.49 The key to pathogenesis lies within the lipid-binding domain of αsyn.Shortly following the discovery of αsyn in Lewy pathology,55 and regulation of the presynaptic vesicle pool.57 αSyn–lipid interactions may have a vesicle tethering function. It has been proposed that the broken α-helical N-terminus can function to tether intracellular vesicles via a “double-anchor” mechanism.61 The biological significance of αsyn-mediated clustering is unknown; however, it could serve to promote the exchange of lipids between adjacent vesicles62 and possibly promote vesicle fusion.64 αSyn preferentially binds to membranes with lipid-packing defects68 and high curvature.72 In the neurons, αsyn is densely clustered around intracellular vesicles and vesicular tubule structures, most prominently at the nerve terminal.73 When incubated with small (~10–30 nm) unilamaller vesicles, the N-terminal of αsyn adopts an extended α-helical conformation as it coats the vesicular surface and a broken α-helical conformation when interacting with micelles.80 The interaction between the N-terminus of αsyn with lipid membranes is driven by electrostatic interactions between positively charged residues and lipid phosphate head group.81 When membrane bound, the N-terminus residues (1–26) of αsyn rigidly bind to the membrane and the internal segment (residues 26–97) acts to sense lipid properties and regulates binding affinity.82 Interestingly, the hydrophobic stretch of residues 71–82 are required for pathological aggregation of αsyn83 and, therefore, lipid-sensing properties of αsyn and pathological aggregation occur through the same functional domain. It is not clear whether mutation of the N-terminus results in a toxic gain of function or loss of function.αSyn binding to vesicular membranes is important, because it influences oligomerization and pathological aggregation. Evidence suggests that non-pathological αsyn is involved with vesicular dynamics in cells84 αSyn interacts with SNARE proteins at the vesicle surface.88 αSyn binding to membranes promotes SNARE complex formation and may function as a SNARE chaperone protein.89 Vesicular membrane-binding promotes the oligomerization of αsyn.9 In vitro phospholipids can also increase the rate of pathological aggregation  by decreasing lag time of primary nucleation.90 The effect of lipids on αsyn aggregation is dependent on lipid to protein ratio, with a low ratio promoting aggregation and higher ratio being inhibitory.91 This bimodal phenomenon probably results from a lack of monomer available for oligomer elongation when the lipid ratio is too high. Interestingly, increasing αsyn expression, presumably shifting the intracellular lipid to protein ratio, promotes aggregation of αsyn in cells. Notably, it has been hypothesized that reducing monomeric αsyn is an important therapeutic target.94Many cellular functions have been attributed to αsyn and membrane interactions, including soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex assembly and exocytosis; however, the exact cellular function of αsyn remains unclear.97 and to promote αsyn aggregation.47 This may be due to differences in assay conditions between studies, such as membrane lipid composition and αsyn concentration. Indeed, recent studies using lipodomics implicated specific fatty acid oleic acid in the pathogenesis of Parkinson’s disease.49 In the model proposed by Fanning and colleagues49, soluble αsyn binds to oleic acid, effectively sequestering the monomer to lipid membranes and ultimately culminating in pathological aggregate formation. As they also observed an increase in oleic acid in response to αsyn overexpression, there may be a toxic lipid dyshomeostatis that preceeds aggregate formation. Their results suggest a complex origin of synucleinopathy where both lipid metabolism and αsyn are central players.A confusing aspect to the literature is that binding of αsyn to membranes has been reported to both inhibit98 αSyn is highly expressed in erythrocytes under the control of transcription factor GATA1.100 During the terminal step of erythropoiesis, α-syn expression dramatically increases and remains elevated in the mature erythrocyte.98 αSyn is then found associated with phospholipids and vesicle membranes in the mature erythrocyte.101 Thus, which of the proposed cellular functions does αsyn perform during erythropoiesis? One likely explanation is that αsyn plays a role in the dramatic intracellular lipid organization, analogous to asymmetric cytokinesis, which occurs prior to the phenomenon of enucleation. Enucleation is the process by which organelles are condensed and extruded from the cell to form a mature erythrocyte. Indeed, just prior to enucleation αsyn can be found associated with lipids of the cell, particularly the nucleus and ER, which are key players in enucleation. αSyn accumulates at the site of nuclear extrusion,102 suggesting it is directly involved with enucleation lipid dynamics. Concurrently, SNARE machinery is decreased in the erythrocyte, suggesting that the potential involvement of αsyn in lipid dynamics during enucleation is independent of hypothesized SNARE functions.103 However, if αsyn is involved in this cellular phenomenon, it is non-essential or interchangeable with beta or gamma synucleins, as only minor phenotypic abnormalities of erythrocytes are observed in α-syn-knockout models.99αSyn may have a more generalized cellular function as an effector of lipid dynamics, and not as a factor of a specific subprocess or pathway. To highlight this concept, consider the curious relationship between αsyn and erythropoiesis  for which others have hypothesized αsyn that may have an underlying redundant mechanism in the two cells of different linage.8 However, the existence of a soluble αsyn tetramer is based mostly on results from crosslinking experiments.106 The interpretation of crosslinking experiments is non-trivial. αSyn tetramers are captured when using a permissive chemical cross-linker with spacer arm length (DSG spacer arm length 7.7 Å) and perhaps not with a shorter spacer arm .107 A milieu of progressively larger oligomers are formed and captured even when purified recombinant αsyn is incubated with glutaraldehyde.108 The successful detection of an αsyn tetramer in tissues and cells is dependent on sample preparation conditions and can be detected when cells remain intact prior to crosslinking or when tissue lysates are kept highly concentrated.107 Indeed, purification of αsyn prevents the detection of a soluble tetramer further suggesting a cofactor is required and this factor is likely of lipid origin.109 Although the question remains which lipid cofactor might be responsible for the tetramer formation, the tetramer and αsyn–lipid interactions are inextricably linked. This is highlighted when recently a transgenic mouse model  of tetramer deficiency was generated by introducing 3E– > K mutations in αsyn’s lipid-binding N-terminus.48 These 3K mice exhibit aggressive αsyn aggregation, loss of an apparent tetramer, and a motor phenotype that has some semblance to Parkinson’s disease. The lipid-binding domain was mutated in the 3K mice and correspondingly lipid interactions were enhanced48 and similar to what was observed with similar mutations in cells.110 In both mice and cells, mutated 3K αsyn clustered around vesicles and intact tissue crosslinking captured less soluble tetramer.110 Was this due to less tetramer or alternatively less soluble tetramer? The results could be explained either way, but if the captured αsyn species is truly an insoluble tetramer, one would expect less detection in the soluble fraction, as the authors observed. Indeed, the results appear to fit a scenario where folding on the vesicular membrane is driving αsyn pathology without the need for a soluble tetramer.  that is devoid of vesicle/lipid binding.111 Interestingly, the same crosslinking chemicals used to capture tetramers chemically modify lysine side chains of αsyn and neutralize their charge.112 Chemically modifying the lysine side chains during tissue crosslinking would presumably disrupt membrane binding, because it neutralizes the required electrostatic charge of lysine residues. Therefore, captured oligomers would dissociate from the membrane and would be detected in the soluble fraction, producing the characteristic gel-shift of the αsyn tetramer.106 To highlight this concept, the neutralization of αsyn charge by chemical cross-linkers is routinely used to enhance the retention of αsyn on polyvinylidene difluoride membranes during western blotting protocols.112 Together, adjacent αsyn molecules bound to intracellular vesicle surfaces  might disassociate into the soluble fraction once chemically modified by the crosslinking reagent. This phenomenon would also help explain why the soluble tetramer has similar intermolecular n-terminal structure as the membrane-bound form.Detection of endogenous oligomers, including the tetramer, could be explained by compartmentalized αsyn residing on the vesical membrane Fig. . Membran8 Erythrocytes have millimolar concentrations of hemoglobin. Hemoglobin oxidatively catalyzes the formation of intramolecular dityrosine bonds resulting in a mixture of αsyn dimers and tetramers.113 Dityrosine crosslink formation occurs rapidly114 and would likely occur to some extent during αsyn purification from erythrocytes. αSyn in erythrocytes associates with vesicles102 and stable dityrosine αsyn occurs in clinical blood samples.115 Together, it is probable that stable αsyn oligomers isolated from erythrocytes are due to oxidative crosslinking of adjacent αsyn molecules bound to vesicle membranes. Heat denaturation irreversibly abolished the tetramers α-helix structure, indicating that the captured configuration was not in equilibrium but instead was a stabilized structure originating from the tissue . Lipid binding of the tetramer was enhanced when compared with the monomer, further suggesting it retained a lipid-binding confirmation.8 Removal of lipids with Lipodex 1000 did not affect the tetramer detection, suggesting the tetramer was not associated with any stabilizing lipid structure. Stabilization of small oligomers via oxidative crosslinking can prevent progressive aggregation and might explain why the stable tetramer appears to resist aggregation.116 Together, it is likely to be that the stable soluble αsyn tetramer purified from erythrocyte is a covalently stabilized membrane-bound confirmation similar to that captured using exogenous crosslinking agents.The apparent soluble tetramer may be stabilized by covalent bonds formed in the oxidative environment of the erythrocyte . Biochemical characterization of a putative αsyn tetramer was mostly done using erythrocyte derived αsyn.117 FRET has been employed to study various aspects of αsyn oligomerization mostly in vitro120 but also ex vivo121 and in vivo.122 In vitro, purified αsyn forms distinct oligomer conformers, which then can spontaneously convert to protease resistant and toxic αsynO-β.12 Biomolecular fluorescence complementation (BiFC) technique uses fluorescent constructs to determine protein–protein interaction. BiFC constructs have been used to study αsyn aggregation in vivo;124 however, the resolution of this technique cannot differentiate between small oligomers and complex formation . The method detects diffuse staining in neurons lacking pathology, suggesting either small aggregates or close association of αsyn molecules normally within the cytosol.124 αSyn comes into close proximity around synaptic vesicles and possibly forms multimers on the membrane.15 Other BiFC techniques employing photoactivatable fluorescent molecules can increase the spatial resolution to several nanometers,125 but this type of imaging has yet to be done with αsyn.Soluble αsyn oligomers have been detected using several imaging techniques. Förster resonance energy transfer (FRET) is a powerful technique used to determine intermolecular distances between molecules with 1–2 nm spatial resolution.130 αSyn purified from Escherichia coli behaves as an intrinsically disordered protein with a large stokes radius,129 which may be why monomeric αsyn appears to have greater mass in some assays.126 Non-denaturing purification procedures from several tissue sources also produce a disordered monomeric αsyn.126 A disordered soluble monomer has been observed directly using in-cell nuclear magnetic resonance (NMR) imaging techniques.14 Specifically, investigators transfected cells with recombinant αsyn labeled with 15N isotope to monitor individual αsyn molecules within the living cells. Results showed that the majority of monomeric αsyn maintained a disordered confirmation in the cell, while becoming slightly more compact than in free solution, probably due to molecular crowding.14 The compact structure observed in vivo likely prevents spontaneous aggregation in the cytosol.131 Although this is compelling evidence that the majority of αsyn in the cell occurs as a disordered monomer, the result does not rule out the existence of a tetramer. A tetramer that existed at low concentration would not be detected and it is possible that the recombinant αsyn behaved dissimilarly to endogenous αsyn. Importantly, this study demonstrated that the majority of αsyn in the cell is cytosolic and monomeric, and suggests that membrane interactions are likely transient and highly dependent on local environment . Correspondingly, it would be interesting if αsyn persists as a monomer at axon terminals where it’s vesicle interactions are more prominent than in the cell body.132 The α-helix conformation αsyn was recently described in HELA cells using FRET, where it was demonstrated that αsyn assumes several confirmations when interacting with vesicle surfaces.133 Considered together, the majority of αsyn in the cell exists as a relatively compact disordered monomer and adopts an α-helix structure when interacting with vesicle membranes. The native state of αsyn may not include an oligomer, whether free and soluble, or vesicle bound.There is also good evidence that αsyn exists predominantly as an intrinsically disordered monomer in the cytosol.136 In the cystosol, αsyn remains monomeric and in a slightly compact configuration. Transient interactions with vesicle surfaces induce a conformational shift, but not necessarily oligomerization, and concentrate αsyn molecules on the vesicle surface. This focal point on the vesicle surface is where opposing αsyn molecules bind and might serve as the molecular origins for Lewy pathology. Numerous cellular and genetic factors converge at this focal point in such a way that creates an environment conducive for the initiation of pathogenic αsyn aggregation. Studies using sonicated αsyn PFFs suggest that once the αsynO-β is present, progressive aggregation and toxicity follow.137 Yet, studies that utilize PFF’s to assess pathology are bypassing pathology generation and may be recapitulating downstream pathological events. Therefore, the use of PFF’s to study synucleinopathy is likely to give valuable insight into the progression of these diseases and perhaps are not suitable to study the initiation of the disease.Assuming monomeric αsyn is interacting with vesicle membranes, and remains monomeric at the vesicle surface under normal circumstances, how might pathology begin? Fig. . One pos138 Together with lipid/vesicle interactions at the center of αsyn function, a route to pathophysiology might be the collapse of clustered lipids/vesicles into a pathological inclusion. Similar to a massive star transitioning to a black hole, at some point pathological αsyn and lipids form a critically dense structure, and compact to form a Lewy body. Interestingly, the architecture of the Lewy pathology supports this interpretation.144 Neuropathological examination of patient brains shows a mixture of αsyn staining in neurons that consist of a pale diffuse, punctate irregular shape , discrete body , and a massive dense structure with a pale core .139 These structures have been hypothesized to be snapshots of a pathological process with Lewy bodies being the result. Indeed, pale bodies contain a mixture of granular and vesicular structures and are often found near mature LB.145 Using a lipid centric view, punctate irregular “early” pathology might represent the initial vesicle clustering48 or lipid droplet formation47 before the characteristic pathology develops. Overexpression of αsyn in yeast models produces lipid only inclusions, lacking the filamentous αsyn that is the hallmark of synucleinopathies.146 Lipids have been identified as a core component of Lewy pathology148 but this has largely been ignored and the pathology is often considered “proteinaceous.”At the membrane, αsynO-β might act similar to a “molecular shovel” inserting itself into the membrane with destructive, toxic, consequences.149 provided substantial evidence that Lewy pathology consists of compacted lipid components from a variety of organelles with αsyn oligomers interspersed. Their work strongly suggests that Lewy pathology is actually an inclusion of fragmented lipids, for which αsyn–lipid interactions play a causative role. Ultrastructural characterization of Lewy pathology showed tubule vesicular, fragmented membranous, and mixed lipid–protein structures, all of which can be formed from αsyn interactions with vesicle membranes. Electron dense structures, consistent with lysosomes, were also observed throughout Lewy pathology.149 Lysosomes are central mediators of lipid metabolism150 and the conspicuous presence of lysosomes surrounded by fragmented membrane structures strongly suggests a deficit in lysosomal/autophagic pathways, specifically the removal of lipid membranes. Large-scale genome-wide association studies have implicated lysosomal/autophagy pathway in several neurodegerative diseases, including synucleinopathies.152Recent work by Shahmoradian and colleagues153 The presence of lipid inclusions in the absence of αsyn are not generally considered when examining patient tissues.A lipid-centric view of Lewy pathology is transformative in that it helps unify and identify disease-causing pathology of several molecular origins. Several neurodegenerative diseases are currently characterized by protein aggregation, when instead we may be missing the lipid components that are the core of the pathology. For example, clinical cases resembling synucleinopathies are documented without the presence of Lewy pathology , as measured by αsyn staining.155 This strategy offers flexibility with analysis and offers unambiguous evidence of direct αsyn–lipid interactions in vivo. Captured lipid–αsyn molecules can be subsequently labeled or purified for downstream analysis. Labeling the structures will help determine where αsyn–lipid interactions are most relevant in the cell. Purification of the structures with subsequent analysis by liquid chromatography–mass spectrometry could determine specific αsyn proteoforms involved with pathological lipid interactions, as well as global analysis of other proteins that are involved. However, the drawback to this strategy is the investigator can only assess one specific lipid species at a time and a synthetic bifunctional lipid must be available or developed for application. Recently, a bifunctional analog of glucosylceramide, a lipid implicated in synucleinopathy,156 has become commercially available and could aid in these studies.αSyn interactions with lipids and vesicles has been investigated mostly in vitro and needs to be characterized in living cells. There are several promising strategies to start understanding lipid–αsyn interactions. The first strategy uses synthetic bifunctional lipids to directly determine lipid–protein interactions.49 However, it is difficult to draw distinct conclusions or find drug targets based on the description of a total lipid species. The resulting information is most useful in implicating lipid metabolism or catabolism pathways, and not the characterization of the specific localized lipids that may be involved with initiating pathology. To find a disease-relevant target, a focused lipodomic approach looking at specific organelles, or better yet, early Lewy pathology, will be the most illuminating. Lipodomic arrays can also be used to screen many lipid–protein interactions; however, they have the disadvantage of not representing in vivo binding conditions.A shotgun lipidomic analysis may also be useful, but because of the complexity of whole-cell lipid determination, the data may not give insight into the localized αsyn–lipid interactions that precede pathology formation. Recently, a shotgun lipodomic analysis was conducted on various αsyn mutant models and αsyn was found to have an effect on lipid metabolism.134 This is the consequence that restrains monomeric or low-molecular-weight oligomeric αsyn diffusion from a three-dimensional to a 2D space upon interaction with the plasma membrane or extracellular vesicles plane.A key question remains: at what point during its interaction with cytoplasmic membranes and extracellular vesicles does αsyn adopt a pathological confirmation? To answer this question, one needs to consider the membrane as a chemical reactor favoring molecular encounters.Evidence for a soluble αsyn oligomer might be best explained by folding intermediates on the plasma or vesicle membranes that remain soluble for subsequent extraction and detection. This interpretation does not require a soluble functional oligomer and seems to fit much of the experimental data. The distinction between a soluble native oligomer and vesicle-bound oligomers/folding intermediates is important, because it clarifies the origins of pathological aggregation of αsyn. With this perspective, determining the molecular details of αsyn–vesicle/lipid interactions is important for understanding the endogenous origins of synucleinopathy. Although there is consensus that aggregation of αsyn is associated with neurological disease, the precise molecular origin of the aggregate pathology remains a mystery.


The effect of a sporadic nucleation was therefore minimized in the experimental crystallization temperature range and a good proportionality between overall crystallization rate (k) and crystal growth rate (G) was inferred. Similar bell-shaped curves were postulated to express the temperature dependence of both k and G rates, corresponding to the maximum of these curves close to a crystallization temperature of 14–15 °C.Thermal properties and crystallization kinetics of poly(4-hydroxybutyrate) (P4HB) have been studied. The polymer shows the typical complex melting behavior associated to different lamellar populations. Annealing processes had great repercussions on properties and the morphology of constitutive lamellae as verified by X-ray scattering data. Kinetics of isothermal crystallization was evaluated by both polarizing optical microscopy (POM) and calorimetric (DSC) measurements, which indicated a single crystallization regime. P4HB rendered banded spherulites with a negative birefringence when crystallized from the melt. Infrared microspectroscopy was applied to determine differences on the molecular orientation inside a specific ring according to the spherulite sectorization or between different rings along a determined spherulitic radius. Primary nucleation was increased during crystallization and when temperature decreased. Similar crystallization parameters were deduced from DSC and POM analyses (e.g., secondary nucleation parameters of 1.69 × 10 Escherichia coli K12 } against log(t − t0) ((t − t0) a. The vaZ and k values evaluated from both kinds of crystallizations are also summarized in τ1/2), calculated as the inverse of the difference between crystallization start time  and the time required to achieve a relative crystallinity of 0.5, are also given in τ1/2 = (Z/ln2)n1/). k, was very similar to that found for the experimental crystallization data.The values of the corresponding reciprocal crystallization half-times (1/dv) of the melted polymer and the crystallization rate (cv) [cT is the crystallization temperature, t is the whole diffusion time, G0 is a preexponential factor, generally assumed to be constant, U* is the transport activation energy, T∞ is the temperature at which all motions are associated with viscous flow cease, f is a factor which corrects for variation in the heat of fusion with temperature below the equilibrium melt point and taken as 2T/(T∞), ΔT is the supercooling degree, and gK is the secondary nucleation parameter which is related to the growth regime. Banded morphologies are usually observed when dv becomes lower than cv [Crystallization of P4HB from the melt state rendered spherulitic morphologies with a banded texture a at all ate (vc) ,31. Thes than vc ,31,32. NThe observed spherulitic morphologies clearly revealed an increase of the zigzag irregularities of each band when temperature was increased, as well as, the differences between the width of birefringent and non-birefringent bands  . SpacingKinetics of crystallization of P4HB from the melt state could be studied by optical microscopy but not for the glassy state since a high crystallinity was developed during the previous cooling run. The study of the development of spherulites was therefore precluded despite some cold crystallization taking place when samples were heated at the appropriate temperatures.2 to 3200 nuclei/mm2 when crystallization temperature was reduced from 49 °C to 36 °C. Crystal grown measurements were not possible at lower temperatures than 36 °C due to the small size of the derived spherulites.The number of spherulites increased during the isothermal crystallization process  and ester groups  can be detected.Microspectroscopic experiments were undertaken in order to get insight on the hypothesis concerning the structure of ringed P4HB spherulites. −1 CH2 band. Interpretation of the peak is in progress, but the result points out the different orientation of the chemical bond that gives rise to a typical cross with two sectors with higher intensity (red) and two with the lower intensity (blue).Solvent casting of chloroform diluted solutions gave rise to films with a thickness lower than 5 μm that could be analyzed by transmission Fourier transform infrared microspectroscopy. Polarized optical microscopy images showed the development of spherulites b with weThe indicated peak was also considered to discern, if some differences can be detected, between dark and bright rings. G, was analyzed by means of the Lauritzen-Hoffman (LH) equation in order to deduce the corresponding kinetic parameters [The crystal growth rate, rameters : G = G0 A similar equation can also be derived by considering the overall crystallization rate as deduced from DSC data:k and G values, which in general is accepted for athermal crystallizations and obviously becomes progressively wrong as primary nucleation varied during the crystallization process [The validity of this last approach implies a proportionality between  process ,36,37,38G + U*/R(cT − T∞) versus 1/(cT(ΔT)f) (k + U*/R(cT − T∞) versus 1/(cT(ΔT)f) (2r = 0.99) when the “universal” values reported by Suzuki and Kovacs [U* = 1500 cal/mol and T∞ = gT − 30 K) were used in the calculation. Kinetic features at low supercoolings are basically governed by the nucleation term, and consequently crystallization rates could become relatively insensitive to both U* and T∞ parameters. Plot allowed estimating secondary nucleation constants of 1.58 × 105 K2  measurements) and 1.69 × 105 K2 (from DSC data). The good agreement that is found allows to infer that the applied approximation  seems correct and consequently the impact caused by the thermal nucleation is scarce. Probably, all primary nuclei became active when the exothermic crystallization peak starts to appear  and consequently a minimum discrepancy between the derived gK values is found.Following LH equation, the experimental spherulitic growth rates and overall growth rates of P4HB were plotted as ln Tc(ΔT)f) a and ln Tc(ΔT)f) b. The pld Kovacs   and (8), and the estimated P4HB shows a complex melting peak associated to different lamellar populations and that is strongly dependent on the processing conditions. Thus, the temperature associated to the predominant melting peak can be increased by more than 12 °C after an annealing process. SAXS experiments demonstrated clear differences on the lamellar supramolecular order between annealed and melt crystallized samples. Lamellar thickness and contrast, between amorphous and crystalline regions, become higher for the annealed samples. These samples showed also a higher macroscopic crystallinity, although the order within lamellar stacks decreases due to the high increase of the amorphous lamellar thickness. An equilibrium melting temperature of 79.9 °C was deduced from the typical Hoffman-Weeks analysis.5 K2. P4HB experimented a cold crystallization process from the glassy state that occurred under high geometrical constrictions and lead to a decrease of the exponent to a value of 1.93.DSC analysis of the isothermal crystallization of P4HB from the melt state allowed determining an average Avrami exponent of 2.56 and a single secondary nucleation constant of 1.69 × 105 K2) that is in full agreement with that determined from DSC experiments. Similar bell-shaped curves defined the dependence of the crystal growth and the overall crystallization rate, with 14–15 °C being the inferred temperatures for those associated to the maximum rates.Optical microscopy observations indicate that P4HB crystallized from the melt, gives rise to banded spherulites with clear differences between the widths of black and white rings and an interring spacing. Infrared microspectroscopy analysis indicated that chemical bonds had the same orientation in the dark and bright rings supporting a model based on a rhythmic growth. Primary nucleation increased by lowering the crystallization temperature, and even during an isothermal experiment. Nevertheless, the impact of the sporadic nucleation was reduced as could be deduced from the Avrami exponent and also by the value of the secondary nucleation constant (1.58 × 10


Immune checkpoint inhibitors (CPIs) have shown promising results in many solid tumors. There are limited data on the safety and efficacy of these drugs in HIV infected patients as they have traditionally been excluded from CPIs clinical trials.We present a case of an HIV-positive patient with extensive extrapulmonary high-grade small cell carcinoma who was treated with dual CPIs (nivolumab and ipilimumab) with a complete response to therapy and with a manageable safety profile. We performed a comprehensive literature review identifying 62 total HIV positive cases treated with CPIs showing this to be a potentially safe option in HIV-positive patients.HIV infection is not an absolute contraindication to CPI therapy. Our case and others provide justification for ongoing trials of CPI therapy in patients with HIV infection, a group that has traditionally been excluded from clinical trials. Immune checkpoint inhibitors (CPIs) including anti-programmed cell death 1 (PD-1), anti-PD ligand 1 (PD-L1) and anti-cytotoxic lymphocyte associated protein 4 (CTLA-4) antibodies have shown promising results in several solid cancers including small cell lung cancers. Small cell lung cancer (SCLC) is a poorly differentiated high-grade neuroendocrine carcinoma representing 15% of all lung cancers with very poor prognosis , 3. ChecTP53, MLL3, MEN1, FAT1, CDKN2A, BCORL1, BCOR, ATRX and TSC2 genes. There is currently no approved targeted therapy for any of these mutations. He was started on chemotherapy with carboplatin and etoposide. He had a partial response (PR) after 2 cycles of chemotherapy. He had disease progression after 6 cycles of chemotherapy with carboplatin and etoposide. He was then started on chemotherapy with FOLFIRINOX  as second line therapy. He received four cycles but continued to have disease progression on imaging. He was then treated with carboplatin and paclitaxel but his disease continued to progress with clinical deterioration and significant abdominal pain. At that point, treatment with dual CPI therapy (nivolumab and ipilimumab) was pursued given the available data in refractory SCLC. Before the start of this therapy, his CD 4 count was 294 cells/uL with an undetectable HIV viral load (less than 20 copies/ml). He received Nivolumab 1 mg/Kg along with Ipilimumab 3 mg/kg every 3 weeks. After 2 doses of the combination, he developed acute kidney injury with creatinine of 4.2 mg/dl from a baseline of 1.0. The therapy was suspended and he was admitted to the hospital and a renal biopsy was performed which showed severe drug-induced acute interstitial nephritis (AIN). He was treated with high dose steroids; 500 mg of IV methylprednisolone for 3 days followed by a steroid taper. His renal function improved after 4 weeks with return of creatinine to baseline. He was then re-started on single agent Nivolumab at 1 mg/Kg and later ipilimumab at 1 mg/kg was added. The patient had significant clinical improvement soon after starting the dual CPI therapy with resolution of abdominal pain which previously required high-dose opioids. Repeat scans at 12 weeks (including MRI of the head and PET/CT scan) showed complete response (CR) as per PERCIST criteria with disappearance of all metabolically active lesions  and the CD4 count of 850 cells/uL at the time of presentation. In December 2016, he presented to the emergency department with chief complaint of diplopia. A magnetic resonance imaging (MRI) of the orbits revealed a mass in the left orbit with involvement of the optic nerve. He was referred to ophthalmology and underwent a lateral orbitotomy and removal of the orbital mass. Pathology showed metastatic small cell carcinoma. A Computed Tomography (CT) scan of the chest, abdomen and pelvis and a Positron Emission Tomography (PET) scans were negative for any intrathoracic mass; however, there were multiple liver lesions and a large pancreatic tail mass. Given these findings his final diagnosis was extrapulmonary high-grade small cell carcinoma of the pancreas. Next Generation Sequencing of his tumor showed an intermediate tumor mutation burden with 9 mutations/megabases and deleterious alterations in Many cases of exceptional and durable responses to CPIs have been reported. However, this case is unique in that it highlights the impressive efficacy of dual CPIs in a HIV positive patient. The only drug-related adverse effect was acute interstitial nephritis (AIN), successfully treated with high dose steroids. The patient achieved a complete clinical and PET response. Patient continued on antiviral therapy during the entire course of therapy. There was no significant adverse effect on the HIV viral load and CD4 cell counts. Interestingly, his viral load which was undetectable before the start of the CPI therapy increased to 106 copies/ml at week 10 followed by a drop in HIV viral load to 80 copies/ml at week 12. At the same time his CD4+ cell count increased to 593 cells/Ul at week 10 and dropping down to 507 cells/Ul at week12. Day et al. have shown that in untreated HIV infection PD-1 was significantly upregulated on HIV specific CD4 and CD8 T cells leading to T cell exhaustion and PD-1 blockade resulted in proliferation of HIV specific CD4 and CD 8 T cells and decrease in viral load [We performed a comprehensive literature review for use of CPIs in HIV positive patients. Search terms “immunotherapy”, “PD-1”, “CTLA-4”, “PD-L1”, “Checkpoint inhibitors”, “nivolumab”, “ipilimumab”, “pembrolizumab”, “HIV”, “AIDS” were used to search PubMed, congress abstracts from the annual meetings of the American Society of Clinical Oncology, European Cancer Congress, Society of Immunotherapy of cancer, American Association of Cancer Research and Google Scholar and for ongoing trials in ClinicalTrials.gov..... reported a series of 44 patients with malignant tumors and concurrent solid organ transplant, HIV, HBV or HCV infection treated with CPIs. Among these, 11 were HIV-positive and there were no adverse effects on their HIV viral load and CD 4+ cell counts [Our search identified 12 case reports , 9–18, 3l counts . Similarl counts , 19. SevIn summary, it appears that HIV infection is not an absolute contraindication to CPI therapy. Although the safety is not fully defined, we present a patient who had an exceptional response to therapy with a manageable safety profile. Apart from the therapeutic effect against the malignancy, there are emerging data that the HIV disease itself may be favorably affected by CPI therapy and studies for this indication are also ongoing. Our case and others provide justification for ongoing trials of CPI therapy in patients with HIV infection, a group that has traditionally been excluded from clinical trials.


To investigate the development of peak oxygen uptake (Multiplicative multilevel modelling, which enables the effects of variables to be partitioned concurrently within an allometric framework, was used to analyze the peak Mean peak Data should not be pooled for analysis but data from either ergometer can be used independently to interpret the development of peak  Peak oxygen uptake  and peak − 1·min− 1). Collectively longitudinal studies indicate boys’ peak There is a plethora of both treadmill- and cycle ergometer-based cross-sectional studies of youth aerobic fitness with the vast majority attempting to control for growth by focusing on a single anthropometric variable and interpreting peak Allometric scaling has challenged the ‘convenient and traditional’  to adopt a multiplicative allometric approach to investigate the influence of mode of exercise on peak 136 (72 boys) students aged 11–14 years volunteered to participate in a series of studies of aerobic and anaerobic fitness. Individual ages were computed from date of birth at each test session. The studies received ethical approval from the District Health Authority Ethical Committee and all participants and their guardians provided written informed consent to participate in the studies. The treadmill-determined data have been integrated into a review of over 1000 treadmill determinations of youth peak Participants visited the Research Centre on three annual occasions and were well-habituated to the laboratory environment, to the laboratory personnel who were unchanged throughout the study, and to the experimental procedures. The annual exercise tests took place in randomized order with a 1 day rest interval between tests. The same equipment and procedures were used throughout the study. Body mass was assessed using Avery balance scales , and skinfold thicknesses over the triceps and subscapular regions were measured using Holtain skinfold calipers . Apparatus was calibrated according to the manufacturers’ instructions. Maturity status was visually assessed by the Research Centre nurse using the indices for pubic hair (PH) development described by Tanner . FFM was− 1 (7 km·h− 1) or 2.22 m·s− 1 (8 km·h− 1) and increased by 0.28 m·s− 1 (1 km·h− 1) every 2 min until a speed of 2.78 m·s− 1 (10 km·h− 1) was reached. Subsequently, belt speed was held constant and the gradient was incrementally increased by 2.5% every 2 min until voluntary exhaustion. Cycle ergometer tests commenced at an exercise intensity of either 60 or 80 W depending on age. The pedal cadence was fixed at 60 rpm and the exercise intensity increased by 20 W every 2 min until the children were exhausted and could no longer maintain the pedal cadence. The highest 30 s Following a standardized warm-up, peak Data were analysed using SPSS v25 (IBM SPSS Statistics). To describe age, body mass, and estimated FFM relationships with peak Factors associated with the development of treadmill- and cycle ergometer-determined peak l et al.  and impll et al.  was adopmentclass2pt{minimLog transformation linearized the model as in Eq. k) (intercept term) which was allowed to vary randomly at level 2  and the multiplicative error ratio (ε) which also varied randomly at level 1  as denoted by the subscripts i (level 1 variation) and j (level 2 variation). Age was centred on the group mean. From the baseline model of age, age2, and body mass, additional explanatory variables were explored. In the initial models sex differences were investigated using the indicator variable boys = 0, girls = 1 which sets the boys’ constant as the baseline from which the girls’ parameter is allowed to deviate. The interaction term age by sex investigates differential development of peak 2 term indicates changes in the size of the age effect as the rate of change in growth decreases. In the sex-specific analyses, from the baseline model of age, age2, and body mass (or estimated FFM), additional explanatory variables including indicator variables for maturity status  which set stage 1 as the baseline from which effects due to maturation can be explored. With age and body mass (but not estimated FFM) models, sum of triceps and subscapular skinfold thicknesses was also entered.All parameters were fixed with the exception of the constant (p < 0.05) when their value exceeded 2 × the standard error (SE). A comparison of the goodness of fit of the different models was obtained from the change in the deviance statistic (− 2 × log-likelihood) with reference to the number of fitted parameters. In a comparison of two models with the same number of fitted parameters, the model with the smallest − 2 × log-likelihood reflects that with the best fit. Additional parameters contribute to improved fit from the change in − 2 × log-likelihood according to a Chi-squared statistic for additional degrees of freedom added.Parameter estimates were considered significant (p < 0.001) correlated with age , body mass , and estimated FFM . Similarly, girls’ peak p < 0.001) correlated with age , body mass , and estimated FFM . The data are illustrated in Figs. Mean age-related treadmill-determined peak mentclass2pt{minimp > 0.05) between those who were unable to attend an annual test occasion and the rest of their age- and sex-specific group in body mass, skinfold thicknesses, or peak Both the number of observations per individual and the temporal spacing of the observations may vary within a multilevel analysis enabling data representing 5 years to be collected over a 3 year period. In the present study, there were no significant differences  between peak r = ~ 0.73–0.86). The fallacy of the ratio-scaled peak Mean treadmill-determined peak mentclass2pt{minims Tanner  which is2 diff max)  changes in maturity status-driven FFM exert a powerful influence on the development of peak


Out of the 526 MPs targeted, a total of 123 different MPs were detected above the limit of quantification at the three different RBF sites. Of the 75–96 MPs detected in each river, 43–59% were attenuated during RBF. The remaining total concentrations of the MPs in the raw drinking water accounted to 0.6–1.6 μgL−1 with only a few compounds exceeding 0.1 μgL−1, an often used threshold value. The attenuation was most pronounced in the first meters of infiltration with a full elimination of 17 compounds at all three sites. However, a mixing with groundwater related to regional groundwater flow complicated the characterisation of natural attenuation potentials along the transects. Additional non-target screening at one site revealed similar trends for further non-target components. Overall, a risk assessment of the target and estimated non-target compound concentrations finally indicated during the sampling period no health risk of the drinking water according to current guidelines. Our results demonstrate that monitoring of contamination sources within a catchment and the affected water quality remains important in such vulnerable systems with partially short residence times.Riverbank filtration (RBF) is used worldwide to produce high quality drinking water. With river water often contaminated by micropollutants (MPs) from various sources, this study addresses the occurrence and fate of such MPs at three different RBF sites with oxic alluvial sediments and short travel times to the drinking water well down to hours. A broad range of MPs with various physico-chemical properties were analysed with detection limits in the low ng L  •123 of 500 micropollutants were detected with LC-ESI-HRMS/MS in surface and groundwater samples.•About 50% of the micropollutants were attenuated during riverbank filtration with short residence times.•17 micropollutants were attenuated >50% at all 3 sites within <24 h and 13 persisted >50%.•Target and non-target screening of raw water indicate no significant human health risk. Due to Varying MP loads discharged with WWTP effluents due to differences in regional usage and prescription, as well as varying WWTP efficiencies implicate observable stream-to-stream and within-stream spatio-temporal variability of MP concentrations. Processes including biotransformation, photolysis, sorption, dispersion, and volatilization influence the fate and attenuation of MPs in aquatic systems . All theKOW (octanol water distribution coefficient) values which are negatively charged or neutral move rapidly with the infiltrating river water into the groundwater and can only be eliminated by microbial degradation  < 4.5 between pH 4–10) to be transported through natural and urban barriers and reach sources of drinking water have been defined as PMOCs  and have received more attention . Hence, oxic, redox-independent conditions predominate RBF processes. As a consequence of the predominance of mainly alluvial gravel aquifers, RBF is often highly dynamic with short groundwater residence times. The growing number of river restoration projects that are conducted along Swiss rivers as modern flood protection measures and to achieve a “good ecological status” of rivers as required by the European Water Framework Directive  might fuTherefore, an improved understanding of the environmental fate and transport of MPs during RBF is essential to provide a basis on which to decide whether drinking water sources are sufficiently protected or additional measures are needed. As thousands of MPs in daily use enter rivers directly or indirectly after incomplete treatment from their various sources such as agriculture, households and industry, it is extremely important to study not only a few selected compounds herein, but to get a comprehensive overview on MPs in surface waters that either attenuate or persist during RBF.−1 range using solid phase extraction followed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS). The concentrations of MPs in each river sample and the respective fate during RBF was evaluated, correlated with physico-chemical data and compared with literature data. Additionally, a profiling of peak pattern of non-target compounds was conducted at one site. Finally, the implications with regard to drinking water consumption of the resulting raw water was assessed. Due to the breadth of compounds it was not possible to investigate the time-variable nature of contaminant distribution in the river and groundwater samples in more detail and constrain fate processes.Thus, in the presented study we aimed to determine the occurrence of several hundreds of MPs from various sources at three different RBF sites with varying residence times from hours to months to get a comprehensive picture on the contamination. More than 500 MPs with a broad range of physico-chemical properties were analysed with detection limits in the low ng L22.1−5 to 10−2 m s−1. The rivers were once canalized at the end of the nineteenth century, and river bed levels have subsequently eroded by several meters into the former floodplains, from which they were eventually disconnected. Whereas the rivers at the study sites Birs and Frenke are connected to the groundwater systems, at the location of the study site Ergolz the river is partly disconnected from the groundwater system and infiltration occurs via the unsaturated zone  from 0.1 to 146 m3 s−1, respectively. Discharge at the Frenke  was 0.04–27.3 m3 s−1 between 1978 and 2013  ranged from 0.83 to 383 mand 2013 .95%: 3.1 m3/s), Ergolz (Q95%: 0.5 m3 s−1), and Frenke (Q95%: 0.2 m3 s−1), respectively  and forestal use (41%), and a smaller portion by urban areas (18%), historically most pronounced close to the rivers. In total, 30 WWTPs with a population equivalent of over 220000 are connected to the different streams in the Canton, resulting in a proportion of up to 13%, 23%, and 7% treated wastewater during base flow conditions in the rivers Birs (Qectively .3 s−1 during sampling). Both transects were chosen according to previous investigations from At the three sampling sites in total four RBF transects were investigated . Transec3 s−1, mainly during night time. Whilst the observation wells 35.L.1 and 35.L.2 are close to the river bank , the distance between 41.J.1 and the river is around 280 m and the extraction well 41.A.4 is located around 1500 m downgradient of the river sampling site . With a typical particle size contribution of the material <0.2 mm of 2–5% determined for the Frenke sample, the fraction of organic carbon ftzerland .2.2All samples were taken during low discharge conditions in December 2013 when the concentrations in rivers are expected to be relatively stable and wastewater-derived compounds usually show the highest concentrations see .−1. In the well houses of the extraction wells the samples were taken directly from the tap. In all cases, the physico-chemical parameters temperature, pH, specific electrical conductivity (EC), the oxidation-reduction-potential (ORP) as well as dissolved oxygen concentrations were measured . Sample collection was conducted after stabilization of the physico-chemical parameters which was achieved after around 10–15 min.The samples at the observation wells were taken by submersible electric pumps  with an average pumping rate of 10 L minEven though air temperatures were below 5 °C during the sampling campaign, all samples obtained in the field were immediately placed in a cool box equipped with thermal packs. All samples obtained during a day were transported to the laboratory and stored at −4 °C in the dark until sample preparation and analyses.2.3® LC/MS 99.9%) was purchased from Thermo Scientific, and ethyl acetate  from Sigma Aldrich. Ultra purity water was obtained using a NANOpure®21 Barnstead from Thermo Fisher Scientific. High-purity N2 (99.99995%) was purchased from Carbagas.Ammonia (25%), ammonium acetate, formic acid (98–100%), and ethanol (HPLC purity) were obtained from Merck AG. Methanol  by vacuum filtration to remove any particulate matter. Before solid phase extraction (SPE), ammonium-acetate buffer was added  and the pH was adjusted to 6.5 using formic acid and ammonia. The exact volume of 1 L was spiked with 163 isotope labelled internal standards see SI  before p2O. Water samples were passed through the cartridges with a consistent speed of 3–5  mL min−1. Afterwards the loaded cartridges were dried completely on the vacuum manifest by pulling air through the cartridge and stored overnight at 4 °C until elution. The cartridges were eluted in back-flush mode using ethylacetate/methanol  containing ammonium (0.5%), and subsequently ethylacetate/methanol  containing formic acid (1.7%). Afterwards the cartridges were rinsed with pure methanol (2 mL) resulting in a final combined neutral extracts of 11 mL. Sample aliquots were reduced to 100 μL under a gentle nitrogen gas stream at 35 °C. The extracts were reconstituted with 900 μL of Milli-Q water and centrifuged for 30 min at 35′000 rpm using a Heraeus Megafuge 1.0R (Thermo Scientific).For the enrichment a SPE-LC-HRMS/MS method as described by 2.5−1.Sample analysis was performed on a high performance liquid chromatograph (HPLC) coupled to a tandem high-resolution mass spectrometer (HR-MS/MS). The HPLC consisted of a CTC Pal auto sampler , a RHEOS 2200 pump with degasser (Flux Instruments) and a column oven. The analytes were separated using a reversed phase column  coupled with a pre-column . The mobile phase consisted of water and methanol both acidified with formic acid (0.1%) and a gradient from 10 to 95% methanol was applied. The injection volume was 20 μL, and the mobile phase flow was 0.2 mL min−1 using Tracefinder 3.1. (ThermoFisher Scientific). For substances without own internal standard the closest-matching internal standard according to retention time and structure was used. These substances were corrected for relative recovery using spiked control samples. Limits of quantification (LOQs) were determined based on the external calibration curve  and corrected for matrix effects using river water (Birs) and groundwater (Ergolz).Detection of analytes was performed using a Q-Exactive™ Hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific), with an electrospray ionization (ESI) probe performed in two separate runs for the positive and negative ionization modes each. Full scan accurate mass spectra were acquired from 100 to 1000 m/z with a nominal resolving power of 70′000 referenced at m/z 200. The automated gain control (AGC) was set to 500′000 and the maximal injection time was 200 ms with a mass accuracy of ±5 ppm. Data-independent high-resolution product ion spectra (HR-MS/MS) were recorded at a resolving power of 17′500 at m/z 200, AGC set to 200′000 and maximal injection time to 100 ms. Quantification was carried out with a calibration row of extracted standards between 0.5 and 750 ng L2.64, M + Na, M + K]  were re-measured in triplicate in the positive ionization mode on a Q Exactive Plus Orbitrap with an increased nominal resolution of 280′000 at m/z 200 (Thermo Fisher Scientific). The raw files were converted with ProteoWizard v 3.0.7162 from raw to mzXML formats . Further, M + K]  to focus, M + K] . Subsequgrouping  was carrgrouping . Profile33.1KOW: 3–9, pH dependent octanol-water distribution coefficient log Dow: −5.7–6.9 (at pH 6.6–6.9), molecular size: 101–1447 Da, different speciation, and molecular structures containing various functional groups (526 target compounds were selected based on consumption data and occurrence in former monitoring campaigns of surface and groundwater in Switzerland  and covel groups ).−1 and for 24% even below 1 ng L−1 in the surface and groundwater samples. The determined LOQs varied only slightly between the surface and groundwater . Even though the highest number of compounds detected was in the river Ergolz, where also the highest proportion of treated wastewater within the three different sites was reported (up to 23%), the highest median concentrations were determined for the river Birs . According to −1 in the first observation well and afterwards remained almost constant.In all observation well samples collected a few meters (≤20 m) from the river with low residence times in the aquifer the number of quantified MPs > LOQ decreased to 43% and 56% , 59% (Ergolz), and 51% (Frenke), respectively, showing overall a good RBF potential. At the Birs transect A the number of quantified MPs decreased from 86 in the river to 37 at the observation well 24.J.28 within 10–14 h flow time, which could also be observed in the significant decline of the median concentration from 9.0 to 4.4 ng L−1 . However−1 but the differences between the median concentrations are rather marginal.A comparable situation could also be documented for all the samples obtained at the Ergolz site. Again the number of detected MPs decreased markedly from 96 to 57 in the first observation well and then remained constant within the measurement uncertainty - taking into account that quite some compounds were quantified close to the LOQ . Similar−1. According to . Additionally, the fraction of organic carbon foc of the aquifer material is estimated to be quite low (0.002%–0.05%). For the positively charged atenolol and metoprolol cation exchange was reported to be relevant  and behaviour. This is complicated by the fact that sorption and degradation cannot be distinguished. With regard to biodegradability the prediction program 3.6−1, and for perfluorooctanic acid at 0.5 μg L−1 . −1 and for all other organic chemicals at 0.1 μg L−1 based on the TTC and further considerations. They addressed also the occurrence of mixtures and proposed target values for the total sum of genotoxic chemicals, the total sum of steroid hormones and the total sum of all other organic compounds at 0.01, 0.01 and 1 μg L−1, respectively.The −1 in drinking water was defined.Furthermore, for several individual chemicals the German Federal Office for the Environment has proposed threshold and guiding values . For 1H-−1 for anthropogenic non-natural compounds such as pesticides and pharmaceuticals was also proposed by the International Associations of Water Works of the large rivers in Europe  in their memorandum to protect the provision of drinking water  a threshold value of 0.1 μg Lng water . For all−1 was exceeded in the raw drinking water analysed in this study under dry weather conditions for only four out of the more than 500 compounds analysed. Acesulfam exceeded the threshold value significantly at all sites, showing the high usage of this artificial sweetener. The other sweetener sucralose exceeded the threshold value only slightly at one site (0.12 μg L−1). Otherwise, benzotriazole (0.1 μg L−1) and tris showed each an exceedance at one of the sites. The total concentration of all compounds is at two of the sites above the proposed threshold of 1 μg L−1 by The widely suggested threshold value of 0.1 μg L4•From the 461 MPs analysed with sufficient sensitivity, 75–96 MPs were detected in the rivers and therefrom 43–59% eliminated during RBF.•No correlation of natural attenuation of MPs with physico-chemical properties was revealed.•−1.The remaining total concentrations of the MPs in the raw drinking water accounted to 0.6–1.6 μg L•−1, a threshold value often used to indicate the absence of health risks.During the sampling period only a few of the targeted compounds exceeded 0.1 μg L•Non-target screening at one site indicates no further high contamination with organic contaminants during the sampling period.Multi-target and non-target screening of MPs at three RBF sites revealed that RBF is an effective natural process to reduce or even remove MPs from river water, and even for the short residence times observed.The authors report no conflict of interest.