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

Rationale 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 gas chromatography (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 the 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.

this reason, it is essential we understand better P chemistry, biochemistry and emissions from key sources in the landscape.
Stable isotope ratios have been used to track elements during transfers between different pools and to understand the respective roles of abiotic and biotic processes during these transfers. [9][10][11] However, P has only one stable isotope and therefore the stable isotope ratio approach is not directly applicable. Despite this, a stable isotope approach has been developed which may shed more light on P cycling. This is because in the environment most P is bound to oxygen (O), forming anions such as orthophosphate (PO 4 3− ), hydrogen phosphate (HPO 4 2− ) and dihydrogen phosphate (H 2 PO 4 − ) which can collectively be termed 'phosphate' (subsequently referred to as PO 4 in the manuscript). This new approach uses the ratio between the 18 O and 16   Samples were collected from seven animals whose ages ranged between 359 and 490 days old; six were male and one female, and five were Charolais crosses, one a Limousin cross, and one a Stabilizer.
Animals were not preselected for the study; simply, the first animal to defecate was selected. The animal ID number was noted and about 150 g of faeces was collected from the ground using sterile containers.
Samples of fresh faeces were collected directly after being voided onto the soil surface in clean aluminium containers and returned immediately to the laboratory for sub-sampling and preparation. First, a sub-sample of 2-3 g faeces was placed into a 12-mL glass exetainer, sealed and frozen at −20°C, ready for determination of its δ 18 O H2O value.

| Statistical analysis
All statistical analyses were conducted in R. 32 3 | RESULTS

| Faecal properties
The fresh faeces were found to have a DM ranging from 9.3 to 16.6% with a mean of 11.4% (±2.5) while the δ 18 O H2O values ranged between −1.19 and +0.41‰ with a mean of −0.73‰ (±0.65) ( Table 2). The amounts of PO 4 collected from faeces when using Ringer's solution ranged from 67 to 93 μg PO 4 -P g −1 DM with a mean of 78 (±9.1) μg PO 4 -P g −1 DM. This was found to be significantly less (t 6 = −8.03; p <0.001) than that collected using deionised water which ranged from 3885 to 8635 μg PO 4 -P g −1 DM with a mean of 5713 (±1856) μg PO 4 -P g −1 DM.

| Faecal microbiological content
Fresh cattle faeces had E. coli concentrations ranging from 6.1 to 7.85 CFU g −1 DM (Table 3). The concentrations of E. coli in the two  26,27 to effectively protect bacterial cells from the osmotic shock that they would experience when being suspended in sterile water.
However, the new data from this study (Table 3) indicate that there was no difference between Ringer's solution and deionised water and that the microbial cells were thus not lysed in water and that the extracted PO 4 in both cases does not represent 'microbial' PO 4 released through cellular breakdown during the extraction process but, instead, 'free' PO 4 .

| Resin-extractable PO 4
The amounts of PO 4 extracted in deionised water were significantly higher than in Ringer's solution. This finding is at odds with the initial recovery test undertaken on PO 4 in a pure Ringer's solution matrix.
However, it would seem that the combination of organic material, isotopologues. 36 The microbiological analysis showed that cell lysis and rupture did not occur in either extraction (Table 3). Therefore, the results derived from the Ringer's solution extraction are not considered further in this discussion, as it apparent that the method for distinguishing microbial PO 4 from inorganic PO 4 (as defined earlier) requires further development.

| Faecal water
The fresh faeces %DM values are consistent with those reported elsewhere for cattle grazing pasture. 37 The cattle's main source of water is via drinking troughs supplied using ground water originating from a local borehole. The δ 18 O H2O value of the groundwater is relatively stable and will represent an integrated value of the annual precipitation supplying it. At this location, the δ 18 O H2O value is predicted to be between −5.5 and −6.0‰. 38 The drinking troughs are refilled with fresh water every time that an animal drinks from them and therefore we do not consider deviations from the groundwater water, water that should be far more representative of the body water of the animal. 40 It is unclear why this is the case without further work being carried out to investigate animal P food sources and metabolic processes within the animal.

| CONCLUSIONS
• The extractable PO 4 from fresh cattle faeces was lower using Ringer's solution than deionised water. However, this did not appear to be because of microbial cellular lysis in the deionised water extraction. It would appear to be due to some form of interference between the Ringer's solution ions, compounds in the faeces and the anion resin sheets. Because of this it was not possible to differentiate 'microbial' PO 4  • There were no apparent relationships between the animal variables and the δ 18 O PO4 value. However, to examine these, a more detailed study is required which should also include other animals for which few data exist in the literature.