Selection of Method
Dana Zimmer, Rhena Schumann
In nature, phosphorus mainly exists as phosphate or sometimes as phosphonate. Phosphate can form relatively stable (hardly soluble) compounds with numerous ions and organic compounds. For this reason digestion methods have to take into consideration the matrix of sample (Fig. 2.1-1). In some cases, interfering ions, such as arsenates, have to be eliminated before measurement, for example by precipitation. Often it is sufficient to adjust reaction conditions, that interfering ions cannot effect measurement.
With secondary ranking but not negligible is the selection of the analysis method. For example, the photometric P determination by molybdenum blue is much easier and more cost-effective than ICP (Inductively Coupled Plasma) as alternative. However, the measurement range of most photometric method is in the lower µMol-range, but sometimes a higher measurement range is necessary. The ICP has the advantage that almost each sample matrix can be measured and measurement range can, with some limits and in dependency of measurement of other elements, be adjusted. The molybdenum blue method does not tolerate high nitrate concentrations (nitric acid, aqua regia) in solution. Additionally, problems occur if the digestion solution is stored in PE-centrifuge tubes without neutralizing solution.
► Classification of matrix material to be analysed, according to
► concentration of organic matter,
► salinity and
► interfering ions.
► Selection of method of digestion/extraction, according to
► P binding form or sample preparation and
► coordination with P analytical method.
► Coordinate with existing equipment for:
► extraction/digestion,
► P determination method,
► available sample volume,
► measurement range of analytic method and
► potentially, auto analyser for high sample throughput.
All material can be frozen (Fig. 2.1-2). Material with low water content is usually dried and stored by room temperature. High concentrations of organic matter of biomass can be the elimination criterion for persulfate digestion. Under these circumstances, incinerated material can be used for persulfate digestion.
For citation: Zimmer D, Schumann R (year of download) Chapter 2.1 General Approach (Version 1.0) in Zimmer D, Baumann K, Berthold M, Schumann R: Handbook on the Selection of Methods for Digestion and Determination of Total Phosphorus in Environmental Samples. DOI: 10.12754/misc-2020-0001
Dana Zimmer, Rhena Schumann
Important properties affecting selection of the method of digestion or the matrix of measurement solution are: water content, organic substance, carbonate, aluminium and iron, because they can affect solubility of P significantly. This affects in turn steps of digestion and selection of P determination method.
The matrix is also important for aqueous samples. For example arsenate, silicate and nitrate interfere with the molybdenum blue reaction (Grasshoff et al. 1999, chapter 5.2.3). Nitrate causes problems in millimolar concentrations, also in solutions with nitric acid or Aqua regia. Furthermore, a salt matrix effect exists; e.g. salt concentrations deepen colour intensity of molybdenum blue. The pH value is important mostly for acidic extracts. A strong self-coloration (e.g. by plant pigments) also interferes with photometric P determination.
Soils, sediments and muds are mostly classified by their concentration of organic matter (table 2.2-1). Most mineral soils in Central Europe/Germany have soil organic matter (SOM) concentrations of less than 2 %. Exceptions are Tschernosems/Chernozems (rare in Central Europe) with around 6 % of organic matter (Wikipedia: Chernozem, WRB classification). All soils with concentrations of SOM of less than 15 % are classified as minerals soils. According to Blume et al. (2010) and Ad-hoc-AG Boden (2005), soils with SOM concentrations between 15 and 30 % are defined as (in German) "Anmoor" and with SOM concentration > 30 % they are defined as peat.
Mineral soils normally can be digested with aqua regia (or hydrofluoric acid and/or perchloric acid) in a microwave without previous ashing. Previous ashing of soil should be preferred at 10 % or more of SOM. "Anmoor" soils and peat are not digested such as mineral soils but as organic material. That means that they are ashed before digestion with aqua regia or HNO3 + H2O2.
Before the digestion of mineral soils or mineral sediments (especially calcareous mud) by aqua regia carbonate concentrations have to be tested. Carbonate concentration up to 2 % should not be problematic. If carbonate concentrations are higher than 2 %, adding of concentrated HCl (for aqua regia) would decompose carbonate to CO2, the sample could bubble out the vessel, and part of the HCl would have been consumed. In such calcareous soils and sediments carbonate should be eliminated previously to digestion. This can be done by HCl or by heating up to 900 °C. If carbonate concentrations are higher than 30 %, it is not possible and appropriate to digest samples with aqua regia.
Analogous to mineral soils, muds are classified by their soil texture according to their grain size distribution in sand-, silt- and clay-muds. Sand is defined as separate particle < 2 mm to > 63 µm, silt as < 63 µm to > 2 µm and clay as < 2 µm (Ad-hoc-AG Boden 2005). The percentage of grain size classes determines the soil texture (Ad-hoc-AG Boden 2005, p. 142).
| Material | SOM (%) |
| mineral soil | < 15 |
| "Anmoor" | 15-30 |
| moor/peat | > 30 |
| mineral sediments | < 5 |
| organic sediments | > 5 |
In soil science the term "mud" for sediments with more than 5 % of SOM is classified in far greater detail (tables 2.2-2 and 2.2-3). However, such sediments have not been widespread in lakes. Therefore, less data about phosphorus concentrations are available; more data exist for ponds and moors (cf. chapter 1.6).
| Type of mud |
Texture of mud |
Material composition | ||
| SOM (%) | Carbonate (CaCO3) % |
Silicate (%) |
||
| organo- mineral mud (Fm) |
Sand mud (Fms) | 5…< 30 | no information | mainly |
| Silt mud (Fmu) | ||||
| Clay mud (Fmt) | ||||
| Diatom mud (Fmi) | ||||
| Calcareous mud (Fmk) |
||||
| organic mud (Fh) |
Algae mud (German: Lebermudde) (Fhl) |
≥ 30 | no information | |
| Peat mud (Fhh) | ||||
| Detritus mud (Fhg) | ||||
| Texture of mud |
Description |
| Sand mud | with clearly visible parts of organic substances, thin horizon |
| Silt mud | with clearly visible parts of organic substances, especially in old moraine areas and "Thüringer Becken" |
| Clay mud | with clearly visible parts of organic substances, plastic, soapy or greasy consistence, mostly thin horizon |
| Diatom mud | from Diatom residues, can be distinguished from calcareous mud by HCl addition and from clay mud only by microscope |
| Calcareous mud |
in fresh condition plastic or elastic, disaggregates not completely by HCl addition, a plenty of undissolved material (> 20 mass-%), from sedimented calcareous particles or calcareous material formed from dead organisms (e.g. stoneworts, snails) |
| Algae mud | homogeneous from elastic (liver-like), gelatinous consistency, conchoidal cracking, formed by dead, decomposed algae residues (phytoplankton) and characterizes deep, calm areas rich of algae, but poor in higher aquatic plants lakes |
| Peat mud | with clearly visible peat residues, brown-black |
| Detritus mud | most common lake sediment, often with seeds and visible residues of aquatic plants, homogeneous dense, plastic to a bit elastic material, from very fine decom- posed organic substances |
The typical reference value for P concentrations is dry matter mass of the material (chapter 3.2). For samples with similar water content and similar density this value is well-suited to compare different samples. When only fertilizer effects have to be evaluated the dry matter mass works well. However, soil volume, wet and dry matter and ash mass have to be determined, if available P in area or volume (of soil) have to be evaluated.
If muds or sediments have to be digested, the dry matter density (of the undisturbed sample) and the water content have to be determined, because different pore volumes and water content can limit comparison of different samples. Samples with high organic matter (OM) concentrations often have high water content. For digestions of ashes the loss of ignition has to be determined. In calcareous samples carbonate should be destroyed by heating to 900 °C.
The water content can affect dry matter density significantly (figure 2.2-1). Therefore, P concentrations should refer to volume or in the case of sediments to the ecosystem effecting area (sediment – water – interface). Interpretations of P concentration in relation to dry matter mass can result in misunderstanding/miscalculation of P availability or interrelation between P und OM concentrations (cf. chapter 1.7). Furthermore, water content and OM concentrations are correlated. That means that high P concentrations in muddy sediments cannot be interpreted as "organically bound" (figure 2.2-2). This is especially the case, if the dry matter mass is the reference value.
Generally, it is possible to digest samples with high OM concentrations (or even biomass) such as minerals samples. However, ashing before digestion is recommended (table 2.2-4).
| Preparation | |
| Material | Working steps |
| Soils | air-drying or drying at 40 °C/60 °C in drying oven |
| < 2 mm sieving | |
| if necessary, homogenisation and/or ashing | |
| if necessary, destroying of carbonate | |
| Sediments | drying at 60 °C in drying oven |
| if necessary, homogenisation and/or ashing | |
| if necessary, destroying of carbonate | |
In soil science only the fine soil (< 2 mm) is analysed. All material > 2 mm are called soil skeleton. It is analysed separately only in exceptional cases!
If samples with high OM concentrations are digested directly, density and especially water content have to be determined (exception charcoal). Bone chars, bone chips and other bio chars are water poor (perhaps hydrochar is not) and do not need to be dried. Bone char can be digested finely crushed or as discrete particles of 1 to 4 mm. If single particles of char shall be digested, long digestion times concerning the microwave (45 min) are necessary to ensure a high energy input. Bone char is, in contrast to other biochar, P-rich. That means that such bone char extracts must be diluted very strongly.
Organically bound phosphates are hard to extract (e.g. Svendsen et al. 1993). Therefore, extraction methods are very strict and include high energy input (pressure and temperature), strong acids and if necessary high concentrations of an oxidizing agent. If ashes are extracted, the organic matrix is destroyed, and method of P quantification can be selected more freely.
| Preparation | |
| Material | Working steps |
| peat and muds | perhaps sieving <2 mm after drying |
| perhaps ashing | |
| destroying carbonate if necessary | |
| plants and litter | ground to dust fineness after drying (woody plants have to be ground in each case |
| and/or ashing | |
| potato tuber, liquid manure and the like |
better freezing and Lyophilisation |
| ashing if necessary | |
| bones | ground finely after drying |
| bone char and the like |
perhaps crush finely, no other preparation necessary |
| Preparation of extracts | |
| some extracts | dilution |
Animal biomass is mostly analysed as dry matter or ash. Most materials are P-rich, because most economically interesting organisms have P-rich endoskeleton or exoskeleton (vertebrates with bones, mussels with shells). For evaluation of results it is very important to know the scientific question: if total P amount removed from ecosystem (and therefore with bones) or if only the P amount in the used biomass/product is interesting. This also has to be considered for literature studies.
During preparation of fatty tissues, a strong odour is released. That means that such tissues should be at least lyophilized before ashing. A fat solvent extraction can be tried with hexane or a similar solvent to extract fat from bones (e.g. Lamoureux et al. 2011, Murden et al. 2017). High concentrations of OM are correlated with strong soot formation. For this reason, biomass could be incinerated outdoors. However, some losses of small ash particles are possible, which is why this method is not suited for the determination of loss of ignition. Either, very large masses are ashed and the loss is low or very small samples are ashed to decrease the formation of soot. Alternatively, a new method for ashing in a microwave system is available (Phönix, Fa. CEM).
For the analysis of animal tissues, the dilution of extracts is essentially before measurement, since most analytical methods work in the µmol range. Therefore, secondary errors for multiple dilutions have to be calculated (table 2.2-6).
| Preparation | |
| Material | Working steps |
| all materials | drying and ashing |
| soft tissues | perhaps lyophilisation and fragmentation |
| only measure without ashing, if special digestion protocols exist | |
| Preparation of extracts | |
| all extract | dilution |
Such matrices are water samples (seston), rainfall with or without dust and aerosols. Because of the mixed liquid and solid phases aliquoting for measurement replication is problematic. Either aliquots are stored separately (frozen) and are digested completely or suspension is accurately mixed after thawing and before digestion (Table 2.2-7).
Digestates are water- and organic-rich materials, such as liquid manure, silage and sewage sludge. According to their amount such samples should be prepared as samples in table 2.2-2 or 2.2-3.
| Preparation | |
| Material | Working steps |
| Seston | Freezing water samples at -20 °C according to the number of replicates and digestion method 50-100 ml |
| Thawing quickly before measurement (in warm water) | |
| shake intensively before aliquoting | |
References
Ad-hoc-AG Boden, Bodenkundliche Kartieranleitung, 5th ed., 438 pages, Hannover 2005, Ed. Federal Institute for Geosciences and Natural Resources in cooperation with the State Geological Surveys of Germany, ISBN: 9783510959204
bgr.bund.de, KA 5 Geländeformblatt
Blume H-P, Brümmer GW, Horn R, Kandeler E, Kögel-Knabner I, Krezschmar R, Stahr K, Wilke B-M, Thiele-Bruhn S, Welp G (2010) Scheffer/Schachtschabel. Lehrbuch der Bodenkunde. Spektrum Akademischer Verlag, 16th ed., ISBN: 9783827414441
Meier-Uhlherr R, Schulz C, Luthardt V (2015) Steckbriefe Moorsubstrate. 2nd unchanged ed. HNE Eberswalde (ed.), Berlin
Phönix, CEM, https://cem.de/applikation/veraschung-/-muffelofen, last accessed 16.09.2024
Schlungbaum G, Nausch G, Stolle S (1979) Sedimentchemische Untersuchungen in Küstengewässern der DDR. VIII. Spezielle Untersuchungen zur Verteilung von Phosphaten und Eisenverbindungen in der Sedimentoberflächenschicht des Barther Boddens. Meer Beitr Sekt Biol Univ Rostock 7: 499-505
Svendsen LM, A Rebsdorf P Nornberg (1993) Comparison of methods for analysis of organic and inorganic phosphorus in river sediment. Wat Res 27: 77-83, DOI: 10.1016/0043-1354(93)90197-P
Wikipedia https://de.wikipedia.org/wiki/Schwarzerde
For citation: Zimmer D, Schumann R (year of download) Chapter 2.2 Influence of the Matrix (Version 1.1) in Zimmer D, Baumann K, Berthold M, Schumann R: Handbook on the Selection of Methods for Digestion and Determination of Total Phosphorus in Environmental Samples. DOI: 10.12754/misc-2020-0001
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Dana Zimmer, Karen Baumann
After sampling, environmental samples such as sediments and soils have to be prepared (and stored) before using them for analysis. The way of sample preparation affects the binding forms and extractability of P in the samples. Generally, effects of preparation (e.g. drying) and storage are more pronounced for wet samples with suboxic redox potential and/or higher percentages of organic matter (e.g. digestates, sewage sludge, peat soils, water-rich and/or suboxic, organic-rich sediments, see chapter 2.3) than for relatively dry samples such as from terrestrial mineral soils (e.g. Ajiboye et al. 2004, Bayens et al. 2003, Qi et al. 2014, Rapin et al. 1986, Styles and Coxon 2006).
Potential changes of P forms in solid samples
Generally, the following opportunities are possible for preparation (points (1) to (4)) and storage (points (5) to (6)) of relatively solid samples such as soils:
Traditionally, soil samples are dried in the air or in a drying oven (mostly 60 °C) and sieved < 2 mm (fine soil). If the equipment is available, the samples can also be lyophilised. In different experiments, it was verified that drying, irrelevant if in a drying oven or by lyophilisation, and re-wetting for extraction can shift the P concentrations between P fractions and also change the totally extractable P in comparison to naturally wet samples (Ajiboye et al. 2004, Condron and Newman 2011, Dail et al. 2007, Schlichting and Leinweber 2002, Styles and Coxon 2006, Xu et al. 2011).
It is supposed that the changed extractability of dried samples is caused by a changed particle size by sieving and grinding of the dried samples (e.g. breaking down of aggregates, increase of surface area), and by microbial processes/transformations during drying (Condron and Newman 2011, Jager and Bruins 1975, Schlichting and Leinweber 2002). Additionally, drying can break down organic substances and cause a lysis of microbial cells releasing P and subsequently changing the available and extractable percentage of P (Khan et al. 2019, Sparling et al. 1985, Srivastava 1998, Turner and Haygarth 2001). Such drying effects are more pronounced in soil samples with a higher percentage of organic matter (such as peat soils) than in mineral soil samples (Styles and Coxon 2006).
By drying at 60 °C, aging of poor crystalline Fe (hydr)oxides is strengthened, that means that their crystallinity increases (Landa and Gast 1973). Already at 50 °C ferrihydrite is transformed into better crystalline goethite and hematite (Das et al. 2011). This might be the explanation why the percentage of P in the NaOH fraction decreases in sequential extractions of sewage sludge, liquid manure and suchlike, since NaOH mainly extracts P from Fe oxides and organic substances (Ajiboye et al. 2004, Dail et al. 2007). Additionally, the oxidation of anaerobic sediments increases the crystallinity of Fe and Mn oxides. For this reason, the percentage of poorer crystallin oxides (stronger binding partners for P and other elements) is shifted to more crystalline oxides, which are weaker binding partners for P (Bordas and Bourg 1998, Rapin et al. 1986). If S is a binding partner in anaerobe samples, the oxidation of the samples and thereby the oxidation of sulfides to sulfate can change the bioavailability of S-bound elements as well (Rapin et al. 1986 and chapter 1.1 and 2.3). High S and N concentrations have to be considered, mainly in swine manure, because both can outgas as H2S respectively NH4-N by oxidation (and drying) and therefore affect P binding forms.
The usage of naturally wet samples can be problematic as well, since without sieving the soil samples, roots and soil animals are in the samples. Their removal is especially complicated for organic rich soil samples because they cannot easily be distinguished from soil matrix (Condron and Newman 2011). For this reason, Condron and Newman (2011) suggest sieving of naturally wet soil or sediment samples through 6 to 10 mm sieves for the removal of coarse material such as stones, roots and mussel shells. Such sample preparation and usage of samples in the naturally wet state can be especially advantageous for the analysis of potential hotspots such as the rhizosphere (chapter 1.2 and Feng et al. 2005). If samples were taken in a reductive state and redox-sensitive P forms (e.g. Fe-P) have to be analysed (Condron and Newman 2011), the redox potential has to be maintained during preparation. This can be realised by a N2 protective atmosphere in a glove box.
If samples (irrelevant if in reductive or oxidative state) are stored in a wet state until analysis, this should be realised only for some days (e.g. at 4 °C in a refrigerator). For a longer time, samples should be frozen (if necessary, by maintenance of redox potential) to reduce microbial transformations (Rapin et al. 1986). In anaerobe samples small changes of binding forms are caused by the freezing of naturally wet samples (Rapin et al. 1986). Oven drying of sewage sludge and liquid manure can cause changes in binding forms and extractability in comparison to frozen and wet samples (Ajiboye et al. 2004, Dail et al. 2007). Besides changes of binding forms, especially for peat samples it has to be considered that drying changes the wettability of the samples. That means that a re-wetting of peat samples for extraction can be very problematic.
Recommendations: In order to minimise the effects of drying on P binding forms, environmental samples should either be analysed in naturally wet state (if necessary sieved, parallel dry matter determination) or drying should be as gently and fast (decrease microbial transformations) as possible. Lyophilisation or drying at room temperature (preferably small sample amounts for fast drying) have to be preferred to drying in the drying oven at > 40 °C. Since the effects of drying are more pronounced for samples with high percentages of organic matter, such as peat samples, drying effects should be considered especially for such samples and subsequently whole sample preparations should be adapted as well (use naturally wet or lyophilised samples). Grinding of samples increases homogeneity of samples (potentially lower standard deviation) but also changes extractable P percentages by an increase of the surface area and a break down of aggregates. Therefore, it has to be decided on the basis of scientific question and potential analyses, if grinded or only sieved samples are used.
There is specific expertise about the various environmental samples in the individual working groups of the P-Campus. The working groups Soil Science and Agronomy (both AUF, University of Rostock) especially have expertise in analytics of samples of soils, plants, biochar and biomass ashes. The expertise in sampling and preparation of water and sediment samples can be found in the working groups of the IOW, the working group Applied Ecology & Phycology (Institute of Biosciences, University of Rostock), especially there also on the Biological Station Zingst, and in the working group Soil Physics (AUF, University of Rostock).
References
Ajiboye B, Akinremi OO, Racz GJ (2004) Laboratory characterization of phsphorus in fresh and oven-dried organic amendments. J Environ Qual 33, 1062-1069, DOI: 10.2134/jeq2004.1062
Baeyens W, Monteny F, Leermakers M, Bouillon S (2003) Evalution of sequential extractions on dry and wet sediments. Anal Bioanal Chem 376, 890–901, DOI: 10.1007/s00216-003-2005-z
Bordas F, Bourg ACM (1998) Critical evaluation of sample pretreatment for storage of contaminated sediments to be investigated for the potential mobility of their heavy metal load. Water Air Soil Poll 103, 137–149, DOI: 10.1023/A:1004952608950
Condron LM, Newman S (2011) Revisiting the fundamentals of phosphorus fractionation of sediments and soils. J Soils Sediments 11, 830–840, DOI: 10.1007/s11368-011-0363-2
Dail HW, He Z, Erich MS, Honeycutt CW (2007) Effect of drying on phosphorus distribution in poultry manure. Comm Soil Sci Plant Anal 38, 1879-1895, DOI: 10.1080/00103620701435639
Das S, Hendry MJ, Essilfie-Dughan J (2011) Transformation of Two-Line Ferrihydrite to goethite and hematite as a function of pH and temperature. Environ Sci Technol 45, 268-275, DOI: 10.1021/es101903y
Feng M-H, Shan X-Q, Zhang S, Wen B (2005) A comparison of the rhizosphere-based method with DTPA, EDTA, CaCl2, and NaNO3 extraction methods for prediction of bioavailability of metals in soil to barley. Environmental Pollution 137, 231-240, DOI: 10.1016/j.envpol.2005.02.003
Jager G, Bruins EH (1975) Effect of repeated drying at different temperatures on soil organic matter decomposition and characteristics, and on the soil microflora. Soil Biol Biochem 7, 153 to 159, DOI: 10.1016/0038-0717(75)90013-9
Khan SU, Hooda PS, Blackwell MSA and Busquets R (2019) Microbial biomass responses to soil drying-rewetting and phosphorus leaching. Front. Environ. Sci. 7: 133, DOI: 10.3389/fenvs.2019.00133
Landa ER, Gast RG (1973) Evaluation of crystallinity in hydrated ferric oxides. Clays and Clay Minerals 2 I, 121-130
Qi Y, Huang B, Darilek JL (2014) Effect of Drying on Heavy Metal Fraction Distribution in Rice Paddy Soil. PLoS ONE 9(5): e97327, DOI: 10.1371/journal
Rapin F, Tessier A, Campbell PGC, Carignan R (1986) Potential artifacts in the determination of metal partitioning in sediments by a sequential extraction procedure. Environ Sci Technol 20, 836-840, DOI: 10.1021/es00150a014
Schlichting A, Leinweber P (2002) Effects of pretreatment on sequentially extracted phosphorus fractions from peat soils. Commun. Soil Sei. Plant Anal. 33: 1617-1627, DOI: 10.1081/CSS-120004303
Sparling GP, Whale KN, Ramsay AJ (1985) Quantifying the contribution from the soil microbial biomass to the extractable P levels of fresh and air-dried soils. Austral J Soil Res 23, 613–621, DOI: 10.1071/SR9850613
Srivastava SC (1998) Microbial contribution to extractable N and P after air-drying of dry tropical soils. Biol Fertil Soils 26, 31–34, DOI: 10.1007/s003740050339
Styles D, Coxon C (2006) Laboratory drying of organic-matter rich soils: phosphorus solubility effects, influence of soil characteristics, and consequences for environmental interpretation. Geoderma, 136, 120–135, DOI: 10.1016/j.geoderma.2006.03.017
Turner BL, Haygarth PM (2003) Changes in bicarbonate-extractable inorganic and organic phosphorus by drying pasture soils. Soil Science Society of America Journal, 67, 344–350, DOI: 10.2136/sssaj2003.3440
For citation: Zimmer D, Baumann K (year of download) Chapter 2.4 Effects of Drying and Storage on P Binding Forms in Environmental Samples (Version 1.0) in Zimmer D, Baumann K, Berthold M, Schumann R: Handbook on the Selection of Methods for Digestion and Determination of Total Phosphorus in Environmental Samples. DOI: 10.12754/misc-2020-0001
Last updated: 2025-04-09