Additionally, plant–microbe rhizosphere interactions that regulate plant growth and productivity, bio-geochemical cycling, and ecosystem functioning occur over the long time scales of agricultural production.Such interactions could affect CNM-exposed plants under realistic conditions, since soil microbial communities and microbe–plant interactions appear sensitive to CNM exposures. Thus, researching CNM effects on plants in soil over long time periods usefully allows for assessing plants through their developmental, including reproductive, stages; life cycle studies in soil also allow for CNMs to interact with fast- or slow-growing rhizosphere microorganisms and symbioses. In this study, we investigated the relative effects of multi-walled carbon nanotubes , graphene nanoplatelets , and carbon black on the growth, nodulation, and dinitrogen fixation potential of soybean grown to maturity in soil. Soybean is an important food, animal feed, and bio-fuel crop species, accounting for 68% of global legume production.Soybean has a critical role in the global nitrogen cycle by forming symbioses with N2 fixing bacteria in root nodules, fixing up to 16.4 million metric tons nitrogen annually in global agricultural systems .N2 fixation by soybean decreases the need for fossil fuel-intensive synthetic nitrogenous fertilizers, whose extensive use leads to environmental impacts.These impacts include nitrous oxide emissions, degraded ground and surface water quality, eutrophication, and harmful algal blooms.Yet, soybean production and its associated N2 fixation apparatuses are potentially vulnerable to soil pollutants,precio de macetas de plastico including nanomaterials. For example, cerium dioxide nanoparticles inhibited soybean growth and yield, caused foliar stress and damage, and diminished N2 fixation potential.
However, less known are the potential effects of CNMs in soils on soybean growth and symbiotic N2 fixation. Here, we evaluated three concentrations of each CNM to represent a range of possible environmental exposures and explored how effects on soil grown soybean varied with CNM type and concentration. Owing to our observations of plant effects, we performed separate studies of CNM concentration-dependent agglomeration in soil water extracts to determine how CNM concentration affected CNM dispersal and thus CNM bio-availability in soils. Our results demonstrate how the soil matrix can affect CNM bio-availability and thus dose-dependent effects of CNMs on soil-grown soybean and root nodule symbioses. CB, MWCNTs, and GNPs were characterized by transmission electron microscopy , thermogravimetric analysis , and inductively coupled plasma optical emission spectroscopy before use. The three CNMs showed distinct morphologies and sizes . CB particles were spheroidal with an average diameter of 36.6 ± 8.3 nm.MWCNTs had an outer diameter of 18.8 ± 4.1 nm, which was slightly thinner than the manufacturer’s reported value . GNPs were two dimensional flakes with an average size of 350 ± 320 nm , which was much smaller than manufacturer’s reported value . The primary oxidation temperatures for CB, MWCNTs, and GNPs differed and were approximately 620, 585, and 623 °C, respectively . Consistent with the high nanomaterial purities reported by the manufacturers , the noncarbon impurities for CB, MWCNTs, and GNPs were 1.3%, 2.2%, and 1.0%, with an overall low metal content . An exception was that MWCNTs had a nickel content of approximately 0.9%, which indicated the likely use of Ni catalyst for MWCNT synthesis.
In all treatments, plant stem length initially increased exponentially, but reached a maximum at approximately 22 d post-transplantation, which just followed the intermediate harvest . Mostly, the CNMs had no significant effect on stem length . The exception was the low MWCNT treated plants, which were significantly shorter than the control plants at the final harvest . Without including the control, the final stem length increased with MWCNT concentration, with plants in the low MWCNT treatment being significantly shorter than the medium and high MWCNT treatments . Hereafter, we refer to such patterns as “inverse dose–response relationships”. The inverse dose–response relationship here between MWCNT concentration and the final stem length was well described by a power function . Further, plants from the low MWCNT treatment showed slower leaf cover expansion and had reduced final total leaf area than the control plants . Both the leaf cover expansion rate constant and the final total leaf area increased with MWCNT concentration . Without including the control, the inverse dose– response relationship was significant for the leaf cover expansion rate constant when modeled by a power function and for the final total leaf area when modeled by either a power or linear function. These trends suggest that the inhibitory effects of MWCNTs on soybean leaf development were reduced at the medium and high MWCNT concentrations . There were no significant differences in either final leaf area or leaf cover expansion rate when comparing either the CB or GNP treatments with the control . Further, the CB, MWCNT, or GNP treatments did not affect trifoliate leaf count per plant . Previous hydroponic studies reported that MWCNTs reduced shoot lengths of red spinach, lettuce, and cucumber but not soybean,while graphene inhibited shoot lengths, leaf counts, and leaf areas of tomato, cabbage, and red spinach.
In our study, only the low MWCNT treatment significantly decreased soybean final stem length, leaf cover expansion rate constant, and final leaf area. In another study with soil-grown soybean, the low concentration of CeO2 nanoparticles most strongly reduced soybean stem and leaf growth, compared to the medium and high concentrations.24 In contrast to the trends in vegetative growth, CNMs appeared to stimulate soybean reproductive development. Relative to the controls, plants in all CNM treatments had more flowers at the beginning of their reproductive stage . The pod number per plant mostly appeared unaffected by CNM treatments, although plants in the high CB treatment had more total pods and all CB treatments had more mature pods , relative to the control . There was no significant difference in either the average seed count per pod or the pod size across all treatments . Prior studies have reported that CNMs could either inhibit or stimulate plant reproduction. For example, MWCNTs decreased the flower count of red clover20 and delayed the flowering of rice plants;in contrast, tomato produced twice as many flowers and fruits when watered with MWCNTs relative to unamended water.These mixed effects could be attributed to the differences in exposure conditions, CNM characteristics and concentrations, and plant species. In our study, all CNM treated soybean produced more flowers, and the vegetative growth was significantly inhibited in the low MWCNT treatment. One mechanism that could explain the earlier flowering is that plants may accelerate their flowering when exposed to environmental stressors in order to maximize the chance of reproduction.Thus, soybean may have responded to the low MWCNT inhibition by switching to reproductive growth and flowering early, at a cost to the final plant height and leaf area. The above ground tissue dry biomass and moisture content were measured at both harvests. From the intermediate to final harvest, each tissue type and the total above ground dry biomass all increased substantially but did not differ significantly from the control or across CNM treatments . The above ground plant tissue moisture contents at both harvests were similar across treatments . In summary,macetas para viveros above ground effects varied with CNM type and concentration. All CNMs accelerated soybean flowering, but only the low MWCNT treatment reduced plant height and final leaf area and slowed leaf canopy expansion.Soybean below ground dry biomass and root nodule N2 fixation potential were measured at both harvests. Although dry root biomass did not vary across treatments at the intermediate harvest , plants in the low MWCNT treatment had lower dry root biomass than the controls at the final harvest . Additionally, the final dry root biomass was significantly lower in the low MWCNT treatment than in the medium and high MWCNT treatments . Without including the control, this inverse dose–response trend followed a power model . The final dry root biomass was not affected by the CB and GNP treatments .
The nodule count per plant is an indicator of the overall N2 fixation capacity.24 At the intermediate harvest, plants from both the low and medium CB treatments as well as from the low MWCNT treatment formed fewer nodules than the controls . At the final harvest, the nodule count was significantly lower in the medium CB and low MWCNT treatments than in the control . Further, the nodule count increased from the low MWCNT treatment to the medium and high MWCNT treatments, although the medium and high treatments did not differ from each other . At the final harvest, the apparent inverse dose–response trend was significant when fitted by a power model . In the low GNP treatment, the nodule count decreased from the intermediate to the final harvest, when it appeared to be lower than the control level . This would result if some of the early nodules decayed and failed to mature due to GNP exposure. Within the GNP treatments, there was an apparent inverse dose–response relationship: the final nodule count increased linearly with GNP concentration . In the period between the intermediate and final harvests , root nodule dry biomass per plant increased substantially in all treatments, but the average 9.6-fold increase significantly varied across treatments . At the intermediate harvest, the dry nodule biomass was reduced for both the low and medium CB treatments as well as for the low MWCNT treatment . The reduction in nodule biomass was more extensive at the final harvest, since all MWCNT and most CB treatments as well as the low GNP treatment resulted in less dry nodule biomass than the control . At the intermediate harvest, the dry nodule biomass in the medium CB treatment was similar to that in the low CB treatment, but significantly lower than in the high CB treatment ; at the final harvest, both the low and medium CB treatments had less dry nodule biomass than the high CB treatment . Within the MWCNT treatments, the final dry nodule biomass was significantly less in the low MWCNT treatment than in the medium and high MWCNT treatments . Also, there was an inverse dose–response relationship within the GNP treatments without including the control: the final dry nodule biomass increased linearly with GNP concentration . Dividing total dry nodule biomass by nodule count for each individual plant yielded the average dry biomass per nodule . The dry biomass per nodule is indicative of how many bacteroids are contained in the nodule central tissue, since the thickness of nodule cortex tissue is relatively constant regardless of nodule size.At the intermediate harvest, plants from the medium CB and low MWCNT treatments formed nodules with lower average dry biomass than the controls . At the final harvest, for the low and medium CB treatments and for all MWCNT treatments, the dry biomass per nodule was approximately half or less than half of the control level . Within the CB treatments, the final dry biomass per nodule was lower in the low and medium CB treatments as compared to the high CB treatment . The final dry biomass per nodule was significantly lower in the low MWCNT treatment than in the medium and high MWCNT treatments . Additionally, the dry biomass per nodule at the intermediate harvest appeared to increase with GNP concentration toward control levels; without including the control, this inverse dose–response trend was significant when modeled by a power function . The decrease in the dry biomass per nodule arose from nodule sizes decreasing, since nodule moisture contents were mostly invariant across treatments . In previous hydroponic studies, nanoscale zinc oxide and titanium dioxide were found to reduce the nodule size in the symbioses between garden pea and Rhizobium leguminosarum bv. viciae 3841. It may be noteworthy that, at both harvests, the low MWCNT treatment had the lowest total dry nodule biomass, nodule count, and dry biomass per nodule . These results were unlikely to have arisen from the Ni impurity of MWCNTs. Assuming that all of the Ni contained in the low MWCNT treatment was bio-available, the Ni concentration would be only 0.0009 mg kg−1. This concentration is far below the Ni dose reported to inhibit soybean nodulation .Measured as the specific activity of nitrogenase by the acetylene reduction assay, the N2 fixation potential is expressed as ethylene production rate normalized to dry nodule biomass. CNMs reduced soybean N2 fixation potential differentially according to CNM dose, type, and harvest stage .