**2. Materials and Methods**

Suspended sediment samples were collected from two agricultural catchments located in the east and southeast of Ireland, namely, Ballyboughal (BB) and Tintern Abbey (TA), as shown in Figure 1. Both catchment streams are medium- to high-level eutrophic according to the 2015–2017 water quality map released by the Irish Environment Protection Agency [30], which makes them suitable locations to study the influences of agricultural catchment sediments to fluvial waters. The BB site has an area of 23 km2, with soils primarily composed of river alluvium, fine loamy drift with limestones, and siliceous stones. TA has an area of 10 km2, with soils composed of primary river alluvium and fine loamy drift with siliceous stones.

**Figure 1.** Location of the Ballyboughal (BB) and Tintern Abbey (TA) catchments in Ireland.

Fluvial sediment was gathered using time-integrated sediment traps, which were deployed to collect in-stream suspended solids over a 10-week period. One time period of samples was collected for this study, and taking into account the significant period of time (10 weeks) the traps were left in place, it is reasonable to consider the samples are representative of the catchment areas. Such sediment traps

are known as Philips samplers [31], and their ability to collect suspended soils is based on slowing down the water velocity, resulting in sediment accumulation within the sediment trap tube. These samplers were installed horizontally in the middle of the channel and installed securely at approximately 60% of the average water depth using steel uprights and plastic cable ties to steel rebar embedded deep into the river channel [28]. The sediment traps were carefully removed from streams and emptied into clean 10 L containers which are kept as near as possible to the stream temperature while being transported back to the laboratory. The sediments were stored in a refrigerator for a few hours and processed by wet sieving the fluvial sediment through 63 μm steel stainless sieves, then centrifuged, and the supernatant decanted. The sediments were then freeze-dried for further analyses.

The sediment samples were sequentially extracted using a modified chemical extraction procedure [16,32], as shown in the flow diagram (Figure 2). Briefly, 1 M NH4Cl was used to extract P adsorbed loosely to surfaces (PNH4Cl); bicarbonate-dithionate (BD) extraction represents redox-sensitive P that is mainly bound to oxidized Fe and Mn compounds (PCBD); 0.1 M NaOH was used to extract inorganic P compounds, such as Al and Fe oxyhydroxides (PNaOH) and poly-P, P in detritus or complexes (PNRP); humic P complexes (Phum), acid-soluble P mainly bound to Ca (especially apatite), and Mg were differentiated using 0.5 M HCl (PDetr); and, lastly, the residual P (PRes) represents refractory organic P and non-extractable mineral P extracted using 1 M HCl at 120 ◦C. Phosphorus assumed to be associated with humic matter was precipitated by adding 2 M H2SO4 to a subsample of the NaOH extract [17]. Organic P (Figure 2) is calculated as the difference in TP between the first NaOH step and the second digested NaOH step [18].

**Figure 2.** Modified Psenner sequential chemical P extraction procedure flow diagram [16,32].

The bulk and μ-XANES at P, Ca, and Fe K-edges were recorded using the Soft X-ray Microcharacterization Beamline (SXRMB) beamline at the Canadian Light Source. At SXRMB, a DCM monochromator using Si (111) crystals is used to cover an energy range of 2–10 keV. At the bulk station, the powder samples were mounted on the double-sided carbon tape and loaded into the vacuum chamber. The bulk spectra were recorded using a 7-element Si drift detector for sediment samples with low P concentration, and in TEY mode for reference samples. A pair of KB mirrors was used to focus the beam to a spot size of 10 <sup>μ</sup>m <sup>×</sup> 10 <sup>μ</sup>m with 109 photons/100mA/s flux [33]. A high-resolution and large area CCD camera is equipped to obtain the optical image of sample. A 4-element Si drift detector is used for μ-XANES analysis. A thin layer of sediment was spread on the carbon tape, and a large area of sample (3 <sup>×</sup> 3 mm2) was first mapped with coarse resolution. Fine-resolution <sup>μ</sup>-XRF maps were acquired by selecting areas based on the elemental distribution and correlation. P, Ca, and Fe μ-XANES

were acquired for selected hotspots. A photon energy of 7200 eV was used to record the XRF maps so that the distribution of P and other relevant elements can be tracked.

#### **3. Results and Discussion**

#### *3.1. Total P and P Fractionation in Suspended Sediments*

In this study, the overall median TP concentrations in the fluvial suspended sediments from the sum of all sedimentary P fractions at the Ballyboughal (BB) outflow was 3.4 mg·g−1, and for the Tintern Abbey (TA) outflow, it was 0.9 mg·g−<sup>1</sup> (Figure 3a). Such TP concentrations are comparable with previous studies on fluvial suspended and streambed sediments [5]. There is a relatively high TP concentration observed for the BB fluvial suspended sediment, likely due to additional sedimentary P contributions from domestic septic tanks and village wastewater treatment plant (WWTP) outflows [34] as the catchment is a relatively highly populated agricultural catchment in north county Dublin, Ireland.

**Figure 3.** (**a**) Sequential chemical P fraction pools including total P (mg g−<sup>1</sup> DW) for (**a**) Ballyboughal outflow (BB) and (**b**) Tintern Abbey outflow (TA); (**b**) sequential chemical P fractionations in relative percent (%) for (**i**) Ballyboughal (BB) outflow and (**ii**) Tintern Abbey (TA) outflow.

Within the BB suspended sediments, the most dominant P fractions included PCBD, POrg, and PNH4Cl with concentrations of 1.18, 0.3, and 0.75 mg·g−1, respectively. By contrast, the most prevalent P fractions in the TA sediments were the PCBD, PNaOH, and POrg with concentrations of 0.29, 0.18, and 0.16 mg·g−<sup>1</sup> (Figure 3a). Figure 3b presents the relative proportional percentage chemical P fractionation results of representative fluvial suspended sediments from (i) BB and (ii) TA sites. The sequential chemical P fractionations separated the fluvial sediment TP into pools with diverse bioavailability. The distribution of P fractions differs in the suspended solids of the two geologically contrasting agricultural catchments. Loosely sorbed (PNH4Cl) was elevated at 22% in BB while there was a lower percentage at 7.5% in TA. The BB agricultural catchment is typical of many Irish agricultural catchments in that additional P contributions, including PNH4Cl, may come from domestic septic systems or from village WWTP outflows [34]. The relative PCBD fraction which constitutes P bound to reducible species of Fe and Mn was almost the same in the suspended solids of both catchments being slightly elevated at the TA outflow at ~32% in comparison to ~35% at the BB outflow. Within the BB suspended solids, there is a relatively elevated percentage composition of POrg, PDetr (P bound to Ca and Mg), and PRes at ~9% for all three. The relative percentage composition of organic P for TA is ~17%. A previous study on fluvial suspended solids from the mixed land-use Bras d'Henri River watershed in Quebec City, Canada, reported POrg comprising up to 20% of TP using sequential chemical P fractionations, which was an order of magnitude greater than streambed sediments [5]. Previous studies have shown that mineralization of organic P (POrg) to inorganic P (iP) through enzymatic hydrolysis has a direct effect on P bioavailability in freshwater systems [35,36]. In addition, the TA outflow suspended sediment contains relatively elevated PHum and PNaOH at ~7% and ~20.5%, which may be associated with the higher Fe and Al concentrations reflected in the catchment soils [34]. The PHum fraction may include Fe(III)-bearing colloids or organic matter–Fe(III)–P ternary complexes which can play an important role in P transport [8,12,17]. BB sediment samples clearly showed a higher relative fraction of P extracted by HCl (PDetr) ~15%, indicating the presence of relatively elevated Ca-bound P, in agreement with the calcareous soils of the catchment [37]. The BB catchment does contain some areas rich in sandstone with elevated Fe, which may influence the relatively elevated percent composition redox-sensitive P (PCBD) fraction. The relative percentage of redox-sensitive P (PCBD) and NaOH-extracted P (PNaOH) are both elevated in the TA sediment, consistent with the Fe-rich composition of the local mineralogy in the TA catchment [34]. The origin of such redox-sensitive P (PCBD) and Al and Fe oxyhydroxide P (PNaOH) fractions is associated with redox fluctuations impacting Fe(III)-oxides in hyporheic and riparian environments [38]. Organic matter degradation creates reducing anoxic conditions in such environments, particularly in summer, which can lead to reductive dissolution of Fe(III)-oxides to release Fe(II) and sorbed P [38,39].
