isotope analysis of phosphorus cycling and mitigation d4.2_final.pdfisotope analysis of phosphorus...

Isotope analysis of phosphorus cycling and

mitigation

Incl. of Review of state of the science

Work package 4: Mitigation of soil and groundwater impacts from agriculture using mixed waste media

Domiziana Cristini

Helmholtz Centre for Environmental Research – UFZ

September 2019

This report has been prepared within the INSPIRATION (Managing soil and groundwater impacts from agriculture for sustainable intensification) Marie Skłodowska-Curie Innovative Training Network (Grant agreement no. 675120)

INSPIRATION Innovative Training Network

Marie Skłodowska-Curie Actions

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Introduction

Phosphorus (P) is an essential nutrient for all living forms. It plays a key role in fundamental biochemical reactions

involving genetic material (DNA, RNA) and energy transfer (ATP) and in structural support of organisms provided

by membranes (phospholipids) and bone (hydroxyapatite) (Ruttenberg, 2003).

Phosphorus is present in water ecosystems in a wide variety of chemical forms, both dissolved and particulate

forms. These fractions are determined by filtration through 0.45 µm filters. The dissolved fraction (which passes

through the filter) includes inorganic P, generally orthophosphate (H2PO4-, HPO4

2-, PO43-), organic phosphorus

compounds (e.g. ATP) and macromolecular colloidal phosphorus. Particulate P (retained on the filter) includes

living and dead plankton, precipitates of phosphorus minerals (e.g. fluoroapatite, hydroxyapatite) and phosphorus

adsorbed to mixed phases (e.g. clays, clay-organic complexes and metal oxides and hydroxides) (Maher and Woo,

1998; Jarvie et al., 2002; Paytan & McLaughlin, 2007). Dissolved Inorganic Phosphorus (DIP) is the most

bioavailable form of P and is quickly taken up by microbial cells, plants and algae, and altered to organic

phosphorus compounds (Blake et al., 2005, Paytan & McLaughlin, 2007; Li et al., 2011). The largest dissolved

inorganic phosphorus pool is SRP (Soluble Reactive Phosphorus), a measure of monomeric inorganic phosphorus

(orthophosphate) in solution. Typically, inorganic P exists in very small concentrations in natural waters, limiting

primary biological productivity in many freshwater systems and in oligotrophic regions of the ocean (Blake et al.,

2005). Recent increases in bioavailable P fluxes and concentrations induced undesirable ecosystem changes, such

as increases in primary production, shifts in community composition, reduced biodiversity, algal blooms and

hypoxia (Hilton et al., 2006, Sondergaard & Jeppesen, 2007). Unlike carbon and nitrogen, phosphorus added to an

aquatic ecosystem or released during decomposition of organic matter usually stays within the system, resulting

in an enrichment of P in detritus and surface soil/sediment (Reddy et al., 1999). This enrichment of nutrients,

which is termed eutrophication, has limited effect on human health (with the exception of occasional toxic blue-

green algal blooms) but has major ecological, social and economic consequences (Hilton et al., 2006). Most

scientists agree that it is largely caused by an inefficient treatment of human sewage and a high application of

organic and synthetic fertilizers on agricultural lands (Schindler, 2012). However, identifying point and nonpoint

nutrient sources is critical to understanding ecosystem health and management practices and the oxygen isotope

ratio of dissolved inorganic phosphate (δ18Op) represents a potentially powerful stable isotope tracer for

biogeochemical research (Young, 2009; Davies et al., 2014).



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This study investigates phosphorus biogeochemistry and the methodologies available for analysing the oxygen

isotope composition of the dissolved inorganic phosphate (δ18Op). In fact, according to the objectives of the

research programme INSPIRATION, a multidisciplinary European Training Network (ETN) facing the topic of

Sustainable Intensification of Agriculture (SIA) in Europe, the Early-stage Researcher (ESR) 13 sets the goal to

develop and refine isotope analytical tools for tracing phosphate in agricultural catchments, to identify and

quantify P-transformation processes and pathways and to investigate δ18Op together with the fluxes and dynamics

of a catchment.

Phosphorus biogeochemistry

Phosphorus has only one stable isotope, 31P, and stable isotope analysis requires an element to have at least two

naturally-occurring stable isotopes, with changes in the ratio of individual isotopes of an element in a sample,

against a known ratio in a reference (Davies et al., 2014). Biogeochemical cycling of P in aquatic ecosystems has

previously been examined using the radioactive isotopes 32P and 33P (e.g. Benitez-Nelson, 2000; Benitez-Nelson &

Karl, 2002). However, the use of radioisotopes is constrained by short isotope half-lives, perturbation of

experimental systems associated with labelling, or the use of incubations which omit irregular events in natural

ecosystems, such as seasonal algal blooms (Levine et al., 1986; Thingstad et al., 1993; Benitez-Nelson, 2000).

P is often bound strongly to oxygen (O) in the dissolved inorganic phosphate ion (Blake et al., 1997), Pi, and

attention has recently focused on whether the stable isotope composition of O in Pi (δ18O-PO4) can provide new

insights into cycle of P. Although extensive data are available on the oxygen isotopic composition of phosphate

found in mineral forms such as apatite in teeth, bones, and rocks within both the archaeological and

environmental studies on paleoclimate conditions (Longinelli & Nuti, 1973; Kolodny et al., 1983), information

regarding the oxygen isotopic signatures of dissolved phosphate to water bodies are limited.

The value of the oxygen fractionation (16O and 18O) within a sample is expressed as δ18O, relative to Vienna

Standard Mean Ocean Water (0‰ VSMOW on the δ18O scale):

Equation: δ18Osample =1000 $%&'(&)(*+,-./0

%&'(&)(*+1,23,43− 16

P-O bonds in Pi are resistant to inorganic hydrolysis under typical temperature, pressure and pH conditions in

surface water and groundwater ecosystems (O'Neil et al., 2003), negligible O exchange occurs within these



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ecosystems without biological mediation (Tudge, 1960; Blake et al., 1997). However, biologically mediated

processes involving Pi cleave the P-O bonds and exchange O isotopes with ambient water, leading to a change in

the O isotope composition of the Pi (Blake et al., 2005). Pi is the species preferentially utilized by organisms because

it diffuses across cytoplasmic cell membranes and the major enzyme that catalyses intracellular P cycling is

inorganic pyrophosphatase, due to its ability to mediate reversible reactions such as the cleavage of adenosine-

5’-triphosphate to adenosine-5’-diphosphate (Blake et al., 2005). Intracellular reactions catalysed by inorganic

pyrophosphatase (PPase) cause complete O isotope exchange between Pi and oxygen in the surrounding water

molecules (Blake et al., 1997).

Reaction: H2P2O72- + H2O ↔ 2HPO4

2- + 2H+

This reversible reaction is necessary to control the level of dissolved inorganic pyrophosphate (PPi) in cells,

because PPi is toxic at high concentrations (Chen et al., 1990), but on the other hand, low PPi levels make it difficult

to synthesize important biomolecules such as RNA or DNA (Klemme, 1976). Blake (1997) targeted PPase due to

its highly reversible character which suggested the possibility of exchange of all 4 O atoms in DIP with O in ambient

water (i.e. breaking and reforming of covalent P–O bonds), which is required to achieve O-isotopic equilibrium

(Springs et al., 1981), in fact the thermodynamic fractionation that characterizes the exchange results in

temperature-dependent equilibrium between O in Pi and O within intracellular water (Fricke et al., 1998). Hence,

within environments in which biological activity is high, the initial δ18O composition in the extracellular

environment, reflecting sources of P, could be overprinted by equilibrium fractionation (Davies et al., 2014).

The δ18O value of a Pi molecule in equilibrium with surrounding water has been predicted by many authors using

the empirically-derived equation from Longinelli & Nuti (1973):

Equation: T(°C) = 111.4 – 4.3 (δ18Op- δ18Ow)

T is the average growth temperature during shell formation for sampled organisms and δ18Op and δ18Ow refer to

the isotopic composition of Pi and environmental water respectively. Comparing the theoretical value for δ18Op at

equilibrium with δ18Op observed in a sample can provide insight into the extent to which Pi has been recycled

through intracellular metabolic reactions (Davies et al., 2014). This equation has been largely used; however,

analytical techniques have evolved since the work of Longinelli & Nuti (1973) and a recent paper by Chang & Blake

(2015) presented a new experimentally-determined equation:

Equation: 1000 ln α (PO4-H2O) = 14.43(±0.39) 1000/T (K) – 26.54(±1.33) r2 = 0.99

Since: α (PO4-H2O) = (δ18OPO4+1000) / (δ18OH2O+1000)

https://www.sciencedirect.com/science/article/pii/S0016703714006541#b0045

https://www.sciencedirect.com/topics/earth-and-planetary-sciences/rna

https://www.sciencedirect.com/topics/earth-and-planetary-sciences/dna






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Expected equilibrium: δ18OPO4= (δ18OH2O+1000) x e[14.43x(1000/T)-26.54]/1000 - 1000

The new equation is offset by +0.5 to +0.7‰ from recent empirically-determined fractionations based on biogenic

apatite, with both based on modern CF-IRMS TC/EA analysis of Ag3PO4, and by +0.9 to +2.3‰ from the earlier

empirical fractionation determined using fluorination of BiPO4 (Chang & Blake, 2014).

Biogenic apatite forms in or near oxygen isotopic equilibrium with cellular water as a result of multiple enzyme-

catalysed hydrolytic cleavage, condensation, and phosphoryl-group transfer reactions inside of cells (Longinelli &

Nuti, 1973). In contrast to biogenic phosphates where the exchange of Pi occurs within a tightly closed system

(inside the organism), the metabolism of Pi in natural waters and sediments is carried out largely by

microorganisms, algae and plants in relatively open systems, and involves intra/extracellular exchange of Pi across

cellular membranes (Figure 1; Maloney, 1992; van Veen, 1997; Blake et al., 1998a).

Figure 1. Biogeochemical cycling of P in aquatic systems. (1) direct uptake of free Pi by diffusion, enzymes not required; (2) extracellular enzymatic hydrolysis of Porg to release Pi-Corg (2a) subsequent uptake of Pi derived from Porg facilitated by membrane-bound transport proteins; (3) intracellular Pi-water O isotope exchange catalysed by various enzymes; (4) incorporation of Pi into Porg compounds in biomass (for example, RNA phospholipids); (5) release of intracellular Pi, Porg and enzymes* from cells during growth or following death/lysis; (6) Pi recycling via re-uptake of intracellularly cycled Pi; (7) recycling of Porg. Many of the pathways for P cycling involve enzyme catalysis (Blake et al., 2005).

These intra/extracellular processes are usually kinetically controlled. Laboratory experiments conducted on

biological processes and phosphoenzyme reaction pathways showed process-specific results. For example, it came

out that P16O4 is taken up preferentially from inorganic phosphate (Pi) by Escherichia coli, causing the δ18O value



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of the growth medium to increase (Blake et al., 2005). If this process operates more widely in freshwaters, the

effect would be observed given unidirectional reactions in which the reactants are not fully consumed and the

initial value of δ18Op overprinted. However, to date only a small number of enzyme-substrate combinations has

been investigated.

Current isotope methods for the determination of δ18Op

Determining the oxygen isotope ratios of dissolved inorganic phosphate in freshwater as well as in seawater

environment is a difficult task. The first method of isolating dissolved phosphate was published by Longinelli et al.

(1976). This is a three-step method which uses natural sponges impregnated with iron hydroxide to quantitatively

remove phosphate ions from sample solutions. The phosphate ions are then leached by nitric acid, purified, and

finally precipitated as BiPO4. This is a relatively laborious method which suffers from the great disadvantage of

ending up by precipitating a hygroscopic form of BiPO4 crystals (Gruau et al., 2005). Historically δ18Op has been

determined through fluorination or bromination of bismuth(III)phosphate (BiPO4), currently the pure phosphate

salt Ag3PO4 is preferred (Lécuyer, 2004). Fluorination and bromination suffer from the use of hazardous materials,

poor O2 yields, tedious and lengthy chemical procedure and requirement of large size samples (Vennemann et al.,

2002; Lécuyer, 2004). Silver(I)phosphate has been identified as a suitable alternative, due to its weakly

hygroscopic nature, stability, low solubility and relatively short preparation time (Lécuyer, 2004; McLaughlin et

al., 2004). Phosphate salt Ag3PO4 is analysed using a High Temperature Conversion Analyzer (TC/EA) interfaced to

a stable isotope ratio mass spectrometer (IRMS). The TC/EA converts the oxygen in phosphate to CO through

pyrolysis at 1270 to 1450°C in the presence of a carbon source to aid full conversion, typically nickelised graphite

and/or glassy carbon. Water vapour is removed through a water trap and CO is separated from other gaseous

impurities through gas chromatography and carried in He to the IRMS for oxygen isotope measurements

(Vennemann et al., 2002; Li, 2009). The sample mass required for analysis is typically 400-500 µg Ag3PO4

introduced in silver capsules and the precision for isotopic analysis is generally quoted as better than

±0.3‰VSMOW (1 standard deviation) (Lécuyer, 2004; Davies et al., 2014).

The major protocol for the extraction of Pi via precipitation of Ag3PO4 has been developed by McLaughlin et al.

(2004). Although it has been successfully used to determine the DIP-δ18O values of seawater and estuary waters,

it is not directly applicable to freshwaters with high dissolved organic C (DOC). However, some research has



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applied it to freshwater matrices without modification, whilst other has included clean-up stages for samples

containing high concentrations of dissolved organic matter (DOM). Inefficient removal of DOM could significantly

influence measured δ18Op, because it can consist of up to 45% O by weight and has been shown to persist until

the precipitation of Ag3PO4 (Lécuyer, 2004; McLaughlin et al. 2004). In his study, Li (2009) reported that using the

method developed by McLaughlin et al. (2004), the Ag3PO4 precipitates contain very high levels of organic matter

with >20% C and >3% N. In another study, Davies (2016) evaluated the potential extent of co-precipitation of six

Porg compounds with brucite and the results showed a significant co-precipitation of all Porg compounds, ranging

from 34.5% (2-aminoethylphosphonic acid) to 97.5% (sodium pyrophosphate decahydrate). High contamination

of Porg persists in the sample also when the brucite pellet is re-dissolved in a strongly acidic matrix in the

subsequent step of the protocol. Therefore, many Porg compounds can undergo acid hydrolysis, yielding Pi that is

derived from the original Porg compound and altering the final δ18Op determined for a sample. Consequently, all

O-containing contaminants must be removed from the Ag3PO4 precipitate to ensure an accurate determination of

δ18Op value.

Different attempts have been applied for improving the extraction of Pi via precipitation of Ag3PO4 so far. Gruau

et al. (2005) attempted to remove DOM from samples through adsorption of organic compounds to activated

carbon; Li (2009) filtered the dissolved DIP through a 0.2 micron filters and increased the rinse times of cerium

phosphate with a potassium acetate solution; Tamburini et al. (2010) introduced an ammonium

phosphomolybdate and a magnesium ammonium phosphate precipitation to purify and separate phosphate from

other inorganic and organic compounds; Goldhammer et al. (2011) added a repetition of precipitation steps such

as MagIC in order to isolate Pi from a matrix of potential contaminants; Lapworth et al. (2014) uses resins (DAX-8

and Dowex1x8) to specifically remove DOM and a washing step through H2O2 to oxidise organic compounds in the

Ag3PO4 precipitate (Figure 2). Despite all these attempts, no definitive approach exists for all aquatic matrices.



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Figure 2. Categorisation of published protocols for the precipitation of Ag3PO4 (Cristini & Knoeller, 2019).

Analytical methods are still evolving. A recent paper (Tcaci at al., 2019) has just been published and reports an

innovative method to analyse the δ18Op value in samples with a P concentration <0.016 mg P L-1. The protocol is

referred to as twist spinning mode (TSM) because of the pumping of the sample into a column with an angle of

30°, causing the sample to rotate during the passage. Furthermore, the method introduces a new silver phosphate

precipitation step at low temperatures (Freeze-drying Ag3PO4 precipitation). The TSM could be an interesting

alternative to the two purification steps (DAX-8 and Dowex1x8) developed by Lapworth et al. (2014). Even though

these steps have been introduced specifically for freshwaters, the purification may be time consuming and

problematic when large samples (≥20L) are pumped into the resin columns, i.e. swelling of the resin bed and

keeping samples at room temperature for several hours to days. These problems have been encountered by ESR13

already with 10L samples. A possibility to avoid the two column steps could be the pre-concentration of the

samples directly on field through the MagIC protocol (co-precipitation of PO4 with brucite). This procedure allows

to reduce ten times the volume of the samples. The MagIC protocol is combined with the protocol of Tamburini

et al. (2010) that allows the purification of the samples with two precipitation steps: Ammonium Phospho-



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molybdate mineral Precipitation (APM) and Magnesium Ammonium Phosphate mineral Precipitation (MAP). The

fellow will apply the combination of the two protocols as next step (Figure 3).

A main analytical challenge remains the lack of internationally certified Ag3PO4 standards, even though Ag3PO4

has been used for a long time in the determination of δ18Op in archaeological and palaeoclimate studies (Stephan,

2000; Vennemann et al., 2002; Halas et al., 2011). Using materials other than Ag3PO4 as calibration standards (i.e.

benzoic acids) can influence the analysis, for example through differing temperatures at which efficient pyrolysis

is achieved (Lécuyer et al., 2007). Therefore, some laboratories have produced internal synthetic Ag3PO4 standards

from either internationally recognised δ18O standards such as NBS120c (phosphate rock distributed by NIST), or

KH2PO4 solutions equilibrated with 18O-enriched water (Lécuyer et al., 2007; Fourel et al., 2011; Halas et al., 2011).

These synthetic Ag3PO4 materials have been shown to be stable over long periods (at least 8 years), a key

requirement for any international reference material (Lécuyer et al., 2007). This and other requirements, such as

sample homogeneity and purity and easy of handling, must be rigorously satisfied before an internationally

distributed reference material can be released (Brand et al., 2009).

At the moment an inter-laboratory calibration of a synthetic Ag3PO4 is going on among several international

laboratories, including the Helmholtz Centre for Environmental Research (UFZ-Halle) and the University of Natural

Resources and Life Sciences (BOKU) in Vienna. To obtain the most precise isotope data across the different

Figure 3. Comparison between Lapworth et al. (2014) protocol and the combination of MagIC and Tamburini et al. (2010) protocol.



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laboratories, same sample preparation and normalization of data have been discussed as well as the use of the

same reference materials. The choice of reference materials is particularly important to avoid differences

associated with the standard selection. Two benzoic acids (IAEA 601 and IAEA 602) and a barium sulphate (NBS

127) were selected, since they appeared to behave well without many problems, even though IAEA 602 is reported

to show often large deviations (Brand et al., 2009). This inter-laboratory comparison highlights the need to

develop an internationally certified Ag3PO4 standard to have a reference material that has a chemical nature as

similar as possible to the samples under investigation.

δ18Op values from different sources and aquatic ecosystems

The use of δ18Op value as both a tracer of P sources and as a dynamic tracer of metabolic processes has been

highlighted in recent studies. A restricted dataset is currently available across a range of sources (Figure 4) and

aquatic ecosystems.

Young et al. (2009) published the first collection of δ18Op source values reviewed from the literature and from their

own studies. Davies et al. (2014) expanded the global library of δ18Op data synthetizing values derived from

different sources and aquatic ecosystems (marine, freshwater, estuarine and sediments). Gooddy et al. (2015)

investigated δ18Op values in tap water and two recent papers (Granger et al., 2017; Tonderski et al., 2017)

published the first δ18Op data regarding septic systems and provided new data of freshwater ecosystems. At

present, Figure 4 suggests that the δ18Op values of the various groups of samples tend to overlap. Nevertheless,

Figure 4. δ18Op for potential phosphate sources (Gooddy et al., 2015).



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recent studies demonstrated that constraining geographical and temporal variations of δ18Op would be useful in

understanding phosphate biogeochemical cycling. Site-specific parameters (i.e. DIP concentrations) and dynamics

(i.e. seasonality, residence time, streamflow) seem to influence the actual isotopic Pi-oxygen values (Gooddy et

al., 2016; Granger et al., 2017; Tonderski et al., 2017). Therefore, a good characterization of local sources and flow

dynamics may provide a more precise interpretation of the δ18Op values.

Conclusions

δ18Op represents a novel stable isotope tracer that could be useful as a source apportionment tool, to identify

point and nonpoint nutrient sources, and to investigate flow dynamics of water bodies. The fate of reactive

phosphorus is critical to understanding ecosystem health and, through the use of isotopic phosphate signature,

future policies and practices could be designed to reach the goal of Sustainable Intensification in Agriculture.

Nevertheless the research is still at an early stage and more study is needed to:

1. Develop a robust protocol to analyse δ18Op that ensures the purification of the samples from any O-

containing contaminants and the accuracy of results;

2. Expand the global dataset of δ18Op between sources of P and aquatic ecosystems to guarantee the stable

isotope of oxygen phosphate as a strong apportionment tool;

3. Better understand the way and the extent the biological activity controls P speciation.

4. Combine stable phosphate isotope analysis with other stable isotope signatures and with the knowledge

about hydrological pathways.

The project of ESR13 focuses mainly on developing points 1, 2 and 4, trying to assess isotope techniques for

evaluating the fate and mitigation of reactive phosphorous in agricultural catchments and to provide new data

that will contribute in expanding the existent dataset of δ18Op.



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