The oxygen isotopic composition of phosphate in river water and its potential sources in the Upper River Taw catchment, UK

The need to reduce both point and diffuse phosphorus pollution to aquatic ecosystems is widely recognised and in order to achieve this, identification of the different pollutant sources is essential. Recently, a stable isotope approach using oxygen isotopes within phosphate (δ18OPO4) has been used in phosphorus source tracing studies. This approach was applied in a one-off survey in September 2013 to the River Taw catchment in south-west England where elevated levels of phosphate have been reported. River water δ18OPO4 along the main channel varied little, ranging from + 17.1 to + 18.8‰. This was no > 0.3‰ different to that of the isotopic equilibrium with water (Eδ18OPO4). The δ18OPO4 in the tributaries was more variable (+ 17.1 to + 18.8‰), but only deviated from Eδ18OPO4 by between 0.4 and 0.9‰. Several potential phosphate sources within the catchment were sampled and most had a narrow range of δ18OPO4 values similar to that of river Eδ18OPO4. Discharge from two waste water treatment plants had different and distinct δ18OPO4 from one another ranging between + 16.4 and + 19.6‰ and similar values to that of a dairy factory final effluent (+ 16.5 to + 17.8‰), mains tap water (+ 17.8 to + 18.4‰), and that of the phosphate extracted from river channel bed sediment (+ 16.7 to + 17.6‰). Inorganic fertilizers had a wide range of values (+ 13.3 to + 25.9‰) while stored animal wastes were consistently lower (+ 12.0 to + 15.0‰) than most other sources and Eδ18OPO4. The distinct signals from the waste water treatment plants were lost within the river over a short distance suggesting that rapid microbial cycling of phosphate was occurring, because microbial cycling shifts the isotopic signal towards Eδ18OPO4. This study has added to the global inventory of phosphate source δ18OPO4 values, but also demonstrated the limitations of this approach to identifying phosphate sources, especially at times when microbial cycling is high

Changes of oxygen isotope values of soil P pools associated with changes in soil pH

Field data about the effect of soil pH on phosphorus (P) cycling is limited. A promising tool to study P cycling under field conditions is the 18O:16O ratio of phosphate (δ18OP). In this study we investigate whether the δ18OP can be used to elucidate the effect of soil pH on P cycling in grasslands. Soils and plants were sampled from different fertilisation and lime treatments of the Park Grass long term experiment at Rothamsted Research, UK. The soils were sequentially extracted to isolate different soil P pools, including available P and corresponding δ18OP values were determined. We did not observe changes in plant δ18OP value, but soil P δ18OP values changed, and lower δ18OP values were associated with higher soil pH values. At sites where P was not limiting, available P δ18OP increased by up to 3‰ when lime was applied. We show that the δ18OP method is a useful tool to investigate the effect of pH on soil P cycling under field conditions as it highlights that different soil processes must govern P availability as pH shifts. The next challenge is now to identify these underlying processes, enabling better management of soil P at different pH

The stable oxygen isotope ratio of resin extractable phosphate derived from fresh cattle faeces

Phosphorus losses from agriculture pose an environmental threat to watercourses. A new approach using the stable oxygen isotope ratio of oxygen in phosphate (δ18OPO4 value) may help elucidate some phosphorus sources and cycling. Accurately determined and isotopically distinct source values are essential for this process. The δ18OPO4 values of animal wastes have, up to now, received little attention. Methods Phosphate (PO4) was extracted from cattle faeces using anion resins and the contribution of microbial PO4 was assessed. The δ18OPO4 value of the extracted PO4 was measured by precipitating silver phosphate and subsequent analysis on a thermal conversion elemental analyser at 1400°C, with the resultant carbon monoxide being mixed with a helium carrier gas passed through a GC column into a mass spectrometer. Faecal water oxygen isotope ratios (δ18OH2O values) were determined on a dual-inlet mass spectrometer through a process of headspace carbon dioxide equilibration with water samples. Results Microbiological results indicated that much of extracted PO4 was not derived directly from the gut fauna lysed during the extraction of PO4 from the faeces. Assuming that the faecal δ18OH2O values represented cattle body water, the predicted pyrophosphatase equilibrium δ18OPO4 (Eδ18OPO4) values ranged between +17.9 and +19.9‰, while using groundwater δ18OH2O values gave a range of +13.1 to +14.0‰. The faecal δ18OPO4 values ranged between +13.2 and +15.3‰. Conclusions The fresh faecal δ18OPO4 values were equivalent to those reported elsewhere for agricultural animal slurry. However, they were different from the Eδ18OPO4 value calculated from the faecal δ18OH2O value. Our results indicate that slurry PO4 is, in the main, derived from animal faeces although an explanation for the observed value range could not be determined

A rapid ammonium fluoride method to determine the oxygen isotope ratio of available phosphorus in tropical soils

Rationale The isotopic composition of oxygen bound to phosphorus (δ18OP value) offers an opportunity to gain insight into P cycling mechanisms. However, there is little information for tropical forest soils, which presents a challenge for δ18OP measurements due to low available P concentrations. Here we report the use of a rapid ammonium fluoride extraction method (Bray‐1) as an alternative to the widely used anion‐exchange membrane (AEM) method for quantification of δ18OP values of available P in tropical forest soils. Methods We compared P concentrations and δ18OP values of available and microbial P determined by AEM and Bray‐1 extraction for a series of tropical forest soils from Panama spanning a steep P gradient. This involved an assessment of the influence of extraction conditions, including temperature, extraction time, fumigation time and solution‐to‐soil ratio, on P concentrations and isotope ratios. Results Depending on the extraction conditions, Bray‐1 P concentrations ranged from 0.2 to 66.3 mg P kg−1 across the soils. Extraction time and temperature had only minor effects on Bray‐1 P, but concentrations increased markedly as the solution‐to‐soil ratio increased. In contrast, extraction conditions did not affect Bray‐1 δ18OP values, indicating that Bray‐1 provides a robust measure of the isotopic composition of available soil P. For a relatively high P soil, available and fumigation‐released (microbial) δ18OP values determined by Bray‐1 extraction (20‰ and 16‰, respectively) were higher than those determined by the AEM method (18‰ and 12‰, respectively), which we attribute to slightly different P pools extracted by the two methods and/or differences resulting from the longer extraction time needed for the AEM method. Conclusions The short extraction time, insensitivity to extraction conditions and smaller mass of soil required to extract sufficient P for isotopic analysis make Bray‐1extraction a suitable alternative to the AEM method for the determination of δ18OP values of available P in tropical soils

How to Design a Study Including the Analysis of δ18OP

To plan a research study, one needs to (1) establish a research question, (2) make a set of observations, (3) form a hypothesis in an attempt to explain the observations and (4) test the hypothesis based on the data collected. The following questions should be addressed when designing a study including the analysis of δ18OP: (i) what is the research hypothesis? (ii) what is the main objective of the study? (iii) what are the aims to address these objectives? and (iv) which techniques are appropriate to address such research question. In addition, one needs to consider (1) which kind of samples needs to be collected, e.g. soil, vegetation or water? (2) in case of soil and sediment samples, which sampling depths and increments need to be sampled? (3) which P pools need to be extracted and analysed for the corresponding δ18OP values? (4) when and how often should samples be taken and (5) how many samples can be processed per week

Conclusions and Way Forward

With an increasing number of researchers using the δ18OP method to investigate P cycling in the environment, it is necessary to conduct an inter-laboratory comparison study for the purification protocol as well as the measurement of silver phosphate with the TC/EA-IRMS like Watzinger et al. (2021) did. For the δ18OP method to progress, further fundamental research as well as field and laboratory studies need to be conducted. To the best of our knowledge, the effect of synthesizing enzymes on the δ18OP has not been investigated yet, despite the importance of those enzymes in the P cycle

Modifications and Issues During Purification

Depending on the extract, it is necessary to modify the purification protocol slightly. Each sample is different and despite a thorough testing of the purification protocol, issues might occur. The three modifications suggested include (1) adjustments in pH, (2) magnesium ammonium phosphate (MAP) precipitation and (3) reductions, prior to A1, of cations like iron (Fe), silica (Si) and calcium (Ca) which could cause interferences during the purification process. Some of the major issues often encountered are (1) no APM precipitation due to the presence of high carbonate concentrations, (2) the presence of high organic matter that requires additional steps in the protocol, (3) crystals not dissolving and (4) discoloration of solution

Purification Protocol

The five stepwise purification of extracts and final precipitation of silver phosphate (A1–A5) are described. The first two steps (A1 and A2) are removing organic matter and are concentrating the phosphate in the extract by reducing the volume. Certain cations could interfere with the precipitation of silver phosphate and are removed in step A3. Silver chloride, which, if not removed, could co-precipitate with silver phosphate, is removed in step A4. The final analyte is then precipitated in step A5. The filtration steps can be quite tedious, using vacuum filtration equipment is therefore recommended. Following step A5, the silver phosphate samples need to be weighed in for the measurement with a thermal conversion elemental analyser (TC/EA) coupled to a continuous-flow isotope-ratio mass spectrometer (IRMS)

Extraction Protocol

Studies showed that the δ18OP is a useful tool to study P in the environment. Adequate extraction protocols for the targeted P pools of the study are a prerequisite for a successful study. Likewise, for most environmental samples, including water, soil, sediment and plant samples, it is crucial that the samples are processed as soon as possible after they have been taken to avoid any alterations of the original δ18OP signature. This is especially true when more bioavailable P pools, like soluble reactive P (SRP) in water samples, are extracted and analysed. Brucite precipitation of water samples should be directly done in the field, fresh soil and sediment samples have to be extracted within 7 days (if microbial P is targeted, on the day of sampling), and plant samples have to be extracted within a few hours of sampling or be frozen. The chapter briefly describes the P cycle in aquatic and terrestrial ecosystems and give an overview about extracting the most common P pools for δ18OP analysis: soluble reactive P in water samples, sequentially extracted P pools of soil, sediment, fertilizer and plant samples