For the coarse-textured soil, arsenic concentrations in sampled pinnae ranged up to 1890 mg/kg after 11 weeks and increased 2–3-fold after 21 weeks . Pinnae arsenic concentrations were con siderably lower for the medium-textured soil, never reaching the hyper accumulation threshold . The interaction of soil by time indicated that pinnae arsenic concentrations were lower at 21 weeks in ferns growing in the medium-textured soil com pared to coarse-textured soil. The mass of arsenic accumulated in sampled pinnae increased over time, increasing 4–5 times up to 1.1 mg at 21 weeks in the coarse-textured soil and 2–3 times up to 0.67 mg at 21 weeks in the medium-textured soil . At final harvest, soil type similarly affected fern frond arsenic concentrations and mass of accumulated arsenic per fern yet had the reverse effect on whole plant biomass . Fern arsenic concentrations in coarse-textured soil ferns ranged between 2666 and 3570 mg/kg for the whole plant, up to 10 times higher than the values in medium-textured soil ferns. The total mass of accumulated arsenic in coarse-textured soil ferns ranged from 15.2 to 20.2 mg/fern, about two times higher than in the medium textured soil . However, the fern dry biomass was 3–4 times higher in the medium-textured soil than in the coarse-textured soil, with values between 20.1 and 23.5 g for the whole plant . Soil treatment did not affect whole plant arsenic concentrations , mass of accumulated arsenic , or biomass . Arsenic concentrations were greater by up to 2 orders of magnitude in fern above ground biomass compared to the rhizome and roots . Mass of accumulated arsenic was greater by an order of magnitude in above ground biomass compared to rhizomes . Interactions of plant part biomass by soil and by treatment showed that senescent fronds were larger in the medium textured soil ferns compared to coarse-textured soil ferns, yet were smaller in phosphorus-treated ferns across both soils compared to in other treatments.
Within above ground biomass alone, arsenic concentrations were up to 8 times lower in mature and senescent fronds,hydroponic gutter compared to young fronds. However, total mass of accumulated arsenic was greater in senesced fronds , compared to young fronds across both soils. Here, the interaction of frond age and treatment indicated phosphorus-treated senesced fronds accumulated less total arsenic than young fronds. Whole plant phosphorus concentrations were 20% lower in ferns grown in the medium-textured soil , compared to in the coarse-textured soil . In contrast, mean iron concentrations were two to six times higher in ferns grown in the medium textured soil , compared to in the coarse textured soil . Phosphorus concentrations were higher in phosphorus-treated ferns .Across soils, soil , time , and depth affected pore water total arsenic concentrations . In the coarse textured soil, arsenic concentrations decreased after 3 to 7 weeks, with con centrations higher in the 27 cm depth than surface depths for the remainder of the experiments. In the medium-textured soil, pore water could be extracted from the phosphorus-treated columns till 17 weeks, longer than the control and F. mosseae-inoculated columns, where pore water extraction was not possible as early as 5 weeks into the study. Interactions of soil and time , soil and depth , and soil and treatment showed that in the medium-textured soil, pore water arsenic concentrations slightly increased with time, decreased with depth, and in creased with phosphorus treatment. Arsenic concentrations were very low in the coarse-textured soil pore water, less than 4.4 μg/L, but were higher in the medium-textured soil, with a mean of 13.6 μg/L increasing up to a peak of 109 μg/L in weeks 5 to 9 in the phosphorus treated columns . Concentrations of DOC were less than 32 mg/L in the coarse-textured soil pore water but higher in the medium-textured soil pore water, where they reached 165 mg/L . Depth affected pore water DOC concentrations. Interactions of soil by week and soil by depth showed that DOC concentrations increased with time and depth in the medium-textured soil but not coarse textured soil. Pore water iron concentrations were less than 66 μg/L in the coarse textured soil but higher in the medium-textured soil, up to 164 μg/L, and decreased with time .
A significant soil by depth interaction showed that iron concentrations decreased with depth in the medium-textured soil pore water. Phosphorus concentrations were less than 0.60 mg/L in the coarse textured soil pore water but were higher , up to 3 times those values, in the medium-textured soil pore water, and decreased with time in both soils . Phosphorus and total arsenic concentrations were moderately correlated . Soil , time , depth , and treatment affected pore water pH, which ranged from 6.0 to 8.9 in both soils and increased significantly with depth across both soils . Interactions of soil by time , by depth , and by treatment showed in the medium-textured soil that pH increased over time, decreased with depth, and increased with phosphorus treatment.Effluent elemental concentrations, volume, and cumulative leaching A soil by presence/absence of ferns interaction indicated that effluent arsenic concentrations were higher in the presence of ferns in the medium-textured soil , but that presence of ferns did not affect effluent arsenic concentrations in the coarse-textured soil . Across both soils, effluent arsenic concentrations increased with time . Effluent arsenic concentrations were lower in the medium-textured soil than in the coarse-textured soil . Phosphorus treatment lowered arsenic concentrations in effluent of both soils , regard less of whether ferns were present. Effluent volume and cumulative arsenic loss were greater in the absence of ferns, with up to 67 mL/day effluent leading to cumulative arsenic loss of up to 12.6 μg/day by leaching . Regardless of whether ferns were present, effluent volumes were greater in the coarse-textured soil where effluent flow lasted 22 weeks in the presence of ferns , leading to cumulative arsenic loss of up to 5.9 μg/day by leaching. This cumulative arsenic loss was more than from the medium-textured soil in the presence of ferns, where effluent flow ceased at 7 weeks with 50% less loss by leaching. Although in the presence of ferns effluent volumes were greater in phosphorus-treated columns , this did not lead to greater cumulative loss of arsenic from phosphorus-treated soil .
In the absence of ferns, cumulative arsenic lost from soil was lower in the phosphorus-treated soil .Bulk arsenic K-edge XANES spectra of the coarse-textured soil samples indicated greater abundance of arsenic in rhizosphere soil, compared to whole roots or bulk soil . In contrast,raspberry plant container in medium-textured soils, bulk spectroscopy showed rhizosphere soil arsenic fractions lesser than or equal to those in roots, with even lower abundance in bulk soil . In both soils, a similar or higher fraction of arsenic was found in phosphorus-treated soils compared to control samples . Micro-focused arsenic K-edge XANES spectra from coarse- and medium-textured soil showed that across all sample types, a higher fraction of arsenic was present in medium- than coarse-textured soil . In both textures we found very little evidence of arsenic reduction in aggregates from control soil . Compared to control aggregates, a higher fraction of arsenic was found in aggregates of phosphorus-treated soil in coarse- , Fig. 6C, Table SI-4 and medium- , Fig. SI-6C, Table SI-5 textured soil. Similarly, in medium-textured rhizo sphere soil, μXANES spectra showed a lower fraction of arsenic in control soils, but a higher fraction in phosphorus-treated soil . In coarse-textured soil, the fraction of arsenic on/within whole roots ranged up to 28.5% on control roots, 80.9% on F. mosseae-inoculated roots, and 20.0% on phosphorus-treated roots . In the medium-textured soil, the fraction of arsenic on/within whole roots ranged up to 99.8% on control roots, 106.0% on F. mosseae-inoculated roots, and at least 13.8% on phosphorus-treated roots , but good fits could not be obtained. In contrast, on particles adjacent to roots in medium textured soil the fraction of arsenic was only 18.2% in control soil and 3.5% in F. mosseae-inoculated soil . Bulk iron K-edge XANES spectra indicated iron oxyhydroxides were the most abundant species in coarse textured bulk soil, rhizo sphere soil, and roots . In medium-textured bulk soil, rhizosphere soil, and roots, iron oxyhydroxides were less abundant and were not present at all in phosphorus-fertilized roots . Other mineral groups identified through bulk and iron K-edge μXANES spectra in both coarse- and medium-textured soil included iron silicates, iron silicates, and iron silicates .The large increase in frond arsenic concentrations we observed with increasing soil particle size suggests changes in soil texture have a strong effect on arsenic phytoextraction rates, directly through arsenic phyto availability and/or indirectly through nutrient content and availability. Because arsenic strongly associates with the clay particle size fraction including iron oxides , arsenic phyto availability is lower in soils with higher clay contents . Our findings build on previous work showing P. vittata frond arsenic concentrations decrease as clay content in creases in medium to fine-textured soils and across wider clay content intervals . Even in the presence of apparently highly plant-available arsenic, P. vittata did not use the rhizome as a secondary storage organ, in contrast to previous observations . We showed that under high phytoavailability conditions, arsenic tolerance and hyper accumulation are simultaneous functional traits in P. vittata, if genetically independent . However, effective hyper accumulation—and/or tolerance— appears to exact a metabolic cost. We found lower biomass coupled with higher arsenic concentrations in ferns growing in the coarse textured soil, which suggests that at higher levels of phytoavailable arsenic , biomass decreases as resources are allocated to tolerance and hyper accumulation mechanisms.
In arsenic hyper accumulation, en ergy is used for active transport of arsenic via phosphate transporters, glutathione production, arsenic reduction, transport within xylem, and sequestration . The lower fern biomass could also be a response to the lower nutrient content in the coarse textured soil. Like arsenic, nutrient retention can be greater in soils with higher clay and organic matter contents . Extensive nutrient scavenging in the lower-nutrient coarse textured soil could expend metabolic energy, release arsenic from soil, and increase plant uptake of arsenic, requiring more resource allocation away from bio mass production toward sequestration.The greater fern arsenic accumulation coupled to greater loss of arsenic by leaching observed in the coarse- compared to the medium-textured soil suggests that plant-available arsenic is also available to leach. In the coarse textured soil, characterized by a lower iron content and adsorption capacity, arsenic appeared to leach from soil at all depths, not resorb to soil, and accumulate in pore water, leading to higher effluent arsenic concentrations. The peak in effluent arsenic concentrations in the coarse-textured soil suggests rapid, linear leaching of the most available arsenic fraction, similarly to what was observed in soils with 8% clay , followed by a decrease in concentrations as the most available arsenic fractions are depleted. Moreover, we attribute the greater effluent flow rates and duration in the coarse-textured soil to lower transpiration from the smaller above ground fern biomass. Lower biomass leads to lower transpiration, greater infiltration, and greater leaching of available arsenic, compared to the medium-textured soil which better supported plant growth. Yet even though the most leachable fraction was depleted early on from the coarse-textured soil, the highest fern arsenic concentrations were found in young fronds produced later in the study. This could suggest that arsenic continued to be plant available even after leaching decreased. Even in a soil with low adsorption capacity, the pools of arsenic available for leaching and plant uptake are overlapping but not identical.The lower arsenic leaching observed in the medium-textured soil is consistent with the greater clay and iron content and adsorption capacity, lower leachate volume, and more diffusive transport in this soil. Arsenic sorption and desorption processes appeared to occur at all depths, leading to constant pore water arsenic concentrations with depth, stable effluent arsenic concentrations lower than in the coarse-textured soil, and soil arsenic concentrations that increased with depth, indicating retention of arsenic re leased from the surface depth . Nonetheless, pore water and effluent concentrations in our study were still 4 to 40 times higher than in soils with similar or higher clay content , likely due to influent water pH and application rate simulating maximum daily rainfall conditions for an extended period.