Preparation of Samples
Dana Zimmer, Rhena Schumann
Representative sampling is necessary for especially heterogeneous samples such as soils and sediments. This is feasible by several punctures for soil samples or by collecting several sediment cores and the subsequent mixing of several samples. Samples can be filled in plastic bags or transported directly in the sediment tube. If further analyses are necessary, e.g. concentrations of plant protection products, transport bags/boxes have to be checked for possibility of (de)sorption of analytes and replaced if necessary. A very inert material is Teflon (polytetra fluor ethylen). If heavy metal concentrations have to be determined parallelly each contact with metal-containing material have to be avoided (e.g. sampler, sieves, spatula, …).
After sampling, material has to be chopped coarsely and dried in flat bowls. For very loamy or clayey soils as well as for peat chopping is essentially because such material will otherwise cake to hard lumps during drying, making chopping nearly impossible.
If other analyses are necessary, e.g. molecular biological analyses, samples have to be prepared according to the most sensitive analysis method. This may be the immediate preparation/analysis of fresh tissues but also freezing. Soil samples are sieved < 2 mm after drying and if necessary, sub samples are grinded. After drying, sediments are grinded as well and optionally ashed depending on digestion method (e.g. persulfate digestion). In this case the loss of ignition has to be determined.
Phosphorus compounds in liquid samples can react from bond phosphorus to phosphate (desorption, phosphatases) or free phosphate can be bonded (absorbed in cells, adsorption). To avoid such processes or at least reduce them samples have to be transported refrigerated and be stored in a refrigerator at 4 °C (maximum a few days). The best storage is freezing (-20 °C). In samples with high water content the percentage of dry matter or water content have to be determined. After lyophilisation or drying samples are milled. Elemental concentrations are determined after ashing at 550 °C.
The loss of ignition has to be determined for ashed samples to calculate the P concentrations in dry matter or sample volume from those in ash. Water samples are not dried but prepared wet chemically. Particulate organic matter (POM) can be concentrated on glass fibre filter. However, it has to be considered that the (low) P concentrations in these filters increase the blanks significantly.
In comparison to higher plant algae have less supporting tissues. Therefore, it is easy to chop and homogenise algae biomass. Due to the lack of differentiation of thalli it is not necessary and not possible to analyse different parts of the organism.
The different composition of plant tissues (supporting tissue or storage tissue) result in different analyses of the different plant parts according to the scientific question. Very coarse or wet plant material such as potato tubers have to be chopped (the peel optional separately) before drying (by air or in drying oven) or lyophilisation. After drying plant material has to be grinded stepwise (first coarse, then finer mills, especially for woody material) and/or ashed.
Animals such as mussels or fishes have to be transported refrigerated and must be frozen quickly. Under certain circumstances, separation in meat and bones for fishes or meat and shells for mussels can be necessary. Bones and shells can be dried in a drying oven and be milled subsequently. Meat of fishes and mussels produces a lot of odour during ignition in moist status and a lot of soot by fat burning. It can be tried to decrease the problems by previous freezing and lyophilisation. Alternatively, a new method for ashing in a microwave system is available (Phönix, Fa. CEM). The exceptionally high concentrations of P in bones means that small amount of ash have to be weigh in (poor reproducibility) or measurement solutions have to be strongly diluted (poor reproducibility and dilution secondary errors). For this reason, it is interesting to work on digestions of dry matter and optimize the digestion method as well as quantifying yield.
Reference
DIN 19747 (2009) Investigation of solids - Pre-treatment, preparation and processing of samples for chemical, biological and physical investigations. DOI: 10.31030/1527573
For citation: Zimmer D, Schumann R (year of download) Chapter 3.1 Sampling, Preparation and Storage (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
For soils, percentage of dry matter is only determined, if for specific analyses, e.g. enzyme activity, weigh-in of wet matter is necessary. Otherwise, soil samples are air-dried or dried in an oven at 40 or 60 °C to accelerate drying.
For sediments, the elemental concentrations per dry matter are often only determined. However, it is recommended to note water and dry matter content as well as dry matter density for each sample and add them to elemental analyses. Thereby, different reference values can be selected.
A complete air-drying has to be done in a desiccator, as otherwise residual water would remain in the material depending on humidity. All dried or ashed samples have to cool down in the desiccator before weigh-out (Tab. 3.2-1).
| Dry matter | ||
| Temperature °C | Degree of Dryness | Material |
| Lyophilisation | if sensitive organic compounds or P fractions have to be measured |
|
| ca. 20 | soil | |
| 40 (60) | ||
| 60 | seston | |
| 90 | plant biomass | |
| 105 | without crystal water | sediment |
Protocol
► Empty mass1 of crucible made of porcelain or aluminium trays, if they are further annealed
► Permanent labelling
► Apply liquid cobalt salt solution (e.g. Co-nitrate) or Fe(III)-chloride with sharpened thin wood sticks (toothpick, shashlik skewer) on the crucible base and let dry.
► Annealing at 550 °C; permanent black or rusty brown labels develop (Fig. 3.2-1 to 3.2-3).
► Weigh in some g of sediment in 1-4 crucibles or trays each.
► Weigh in dabbed biomass (estimation of amount in tab. 3.2-2).
► Following masses are noted: crucible empty, crucible with wet mass and crucible with dry mass
► Place crucibles for 10 - 16 hours (at listed temperature, tab. 3.2-1) in the drying oven
► Cool down in desiccator and weigh
► Before calculation of water content, calculate all masses without crucible mass (equation A and B)!
► In case of standard error > 5 % for water content: repeat determination.
► Minimum masses of ashes should not be lower than 1 g for sediments and 100 mg for tissue, to make sure that possible contamination or losses only have minimal effects on results (light ash flakes).
1 Mass or weight: All data in gram refer to the mass. This mass is also measured as weight force. For this reason, outside physics the mass is also called weight. Weight force can be calculated as a product from mass with gravity acceleration. However, the mass is an absolute physical measurand. Therefore, mass is the exact term.
| Drying | Digestion | |||
| Wet matter weigh-in (ca. g) |
Dry matter percentage (%) |
Weigh-in per replicate (mg) |
Volume of extract of digestion (ml) |
|
| mineral soils | 10 | > 90 | < 500 DM | 50-1003 |
| organic rich dry matter |
100-200 DM | 50-1003 | ||
| sediment | 3 | 20-90 | 50-100 Ash | 10-152 |
| algae | 1-2 | 10-30 | 3 Ash | 10-152 |
| plants | < 10 DM | 50-1003 | ||
| animal tissues | < 10 DM | 50-1003 | ||
| bone char | < 50 | 50-1003 | ||
2 Subboiling digestion
3 Microwave Mars Xpress
Synonyms for dry matter percentages are percentages of dry substances or suspended (filterable) material.
In contrast to density the dry matter density is calculated by dry matter and not by wet matter and volume (equation C). Very different water contents of sediments have a large influence on dry matter density (Fig. 2.2-1 and 3.2-8).
Protocol
► Take sediment sample with sampling tube (or box corer) as big as possible.
► Keep surface as undisturbed as possible.
► Push the sediment upward with a plunger until the overlaying water is drained.
► Prepare the syringe (Fig. 3.2-7)
► Cut front side by scalpel
► Mark 1 cm around
► The volume of a 20 ml syringe (inner diameter 1,9 cm) is than 2,8 cm3
► draw up syringe plunger to 1,5 - 2 cm
► prick syringe around 1 cm into sediment and pull out. In doing so, build up negative pressure with plunger.
► Press out sediment sample up to 1 cm, push away supernatant (horizon < 1 cm) with spatula (Fig. 3.2-4).
► Transfer punched sample completely into crucible, weigh wet matter, dry (see above) and determine dry matter.
Reference
Schlungbaum G (1979) Untersuchungen über die Sedimentqualität in den Gewässern der Darß- Zingster Boddenküste unter besonderer Berücksichtigung der Stoffaustauschprozesse zwischen Wasser und Sediment. Postdoctoral Thesis, University of Rostock
For citation: Zimmer D, Schumann R (year of download) Chapter 3.2 Dry matter and dry matter density (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
Dana Zimmer, Rhena Schumann
Since concentrations of soil organic matter (SOM) in soils and some muds are < 15 %, ashing is not necessary and sometimes counterproductive. The conversion of minerals such as goethite to hematite can result in stronger binding of P (covalent, Cornell & Schwertmann 2006). Crystal water from clay minerals is removed and particles can shrink and include elements. Samples with > 15 % SOM (Blume et al. 2010) can be ashed, but samples with > 30 % (peat) should be ashed. If ashing is not possible, such organic rich samples have to be digested very strongly (microwave, H2O2 plus HNO3). Animal material (except bones) should be ashed or if digested by microwave they should be lyophilized and milled very fine.
High Fe concentrations in soils and sediments cause a red sample colour during ashing, since between 500 and 600 °C yellow-brown ferrihydrites or goethite is converted to red hematite (Derie et al. 1976). Such red colouring can complicate neutralisation after persulfate digestion, since the indicator for the pH value is yellow. At high Fe concentrations in the extract, Fe flakes can appear during neutralisation with NaOH or ammonia. These Fe flakes can absorb parts of the P and remove it from photometric detection or increase turbidity during photometric detection (if not completely dissolved before).
During ashing of animal material, such as fish or mussels, huge amounts of soot develop in muffle furnace. This is caused by the high percentages of fat in the biomass. Due to the differences in composition of matrix, mussels have to be separated into mollusc flesh and shells. High carbonate concentrations can disturb the digestion of samples (cf. chapter 2.2). Phosphorus concentrations in vertebrates are often only presented for (human) used tissues. If balances are needed, P concentrations of bones are necessary as well. In the ScienceCampus Phosphorus Research there is only little experience with pre-treatment and extraction of animal products such as fish.
Before ashing, samples have to be dried (or lyophilised) until constant weight. Longer stored material (weeks to months) have to be dried again before weigh-in. For ashing dry matter weigh and ash weigh have to be noticed to determine the loss of ignition. Only by this value elemental concentrations can be calculated for dry matter.
Protocol
► Determine empty mass of crucibles
► Weigh in sediment or dry biomass (estimation of amount in table 3.3-1)
► Dry longer stored dry matter for some hours in desiccator before weigh-in
► Note following masses: crucible empty, crucible with dry matter and crucible with ash
► Put crucible in the muffle furnace for 4 hours at 550 °C,
► Attention: very long time for cooling down in oven > 12 h, transfer to desiccator only with crucible pliers!!!
► Cooling down crucible in desiccator and weigh
► Before calculation of loss of ignition, calculate all masses without crucible mass!
► Minimum mass of ashes is 50 mg, to avoid complications by contaminations
► It is safer to calculate stepwise (see example), because neither crucible masses nor conversion of ash (g absolute), loss of ignition (% dry matter) can easily be excluded or reformulated.
| Drying | ||
| Weigh-in of dry matter (ca. g) |
Loss of ignition (%) | |
| Soils | less common | >90 |
| Sediment | 1 | 20-90 |
| Algae | 0,5 | 10-40 |
| Plants | 0,5-1 | |
| Animal tissue | ||
The loss of ignition is the organic matter of samples which is lost by ignition (ashing) (elements C, H, O and N).
References
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/Schachtschnabel. Lehrbuch der Bodenkunde. Spektrum Akademischer Verlag, 16th ed., ISBN: 9783827414441
Cornell RM, Schwertmann U (2006) The iron oxides: Structure, properties, reactions, occurrences and uses. Wiley VCH Verlag, Weinheim, 2nd completely and revised ed., ISBN: 9783527606443
Derie R, Ghodsi M, Calvo-Roche C (1976) DTA study of the dehydration of synthetic goethite αFeOOH. J thermal Analysis 9: 435-440, DOI: 10.1007/BF01909409
For citation: Zimmer D, Schumann R (year of download) Chapter 3.3 Ashing and Loss of Ignition (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
Rhena Schumann, Dana Zimmer
If carbonate concentrations – especially in soil and sediment samples – exceed 1 to 2 %, this carbonate has to be removed before digestion with aqua regia. Otherwise, carbonate reacts to CO2 after addition of HCl und the sample bubbles outside the vessel and some of the HCl is consumed as well. Carbonate-containing samples have to be tested before adding concentrated HCl; slight bubbling in vessels could be acceptable.
If several grams of material are necessary, carbonate removal such as for grain size analysis is applicable (DIN ISO 11277). For small amounts of samples (mg) carbo¬nate removal according to Harris et al. (2001) is more suitable. However, this method has not been tested so far in the labs of the working groups Soil Science or Applied Ecology and Phycology. The necessary sample mass has to be estimated from carbonate concentrations in samples, since carbonate is lost as CO2 and sample material has to be sufficient for digestion. For soil samples with more than 50 % carbonate, which were prepared for grain size analysis, the procedure according to DIN ISO 11277 did not work. Even after several days with repeated HCl addition, large quantities of CO2 bubbles build up. Therefore, the test for carbonate removal was aborted. For sediment samples with high concentrations of organic matter (OM) and carbonate, which would be ashed anyway, it could be considered to decompose carbonate by temperatures between 900 and 1000 °C. The three described methods below for carbonate removal have not been tested before in the labs. Therefore, it might be necessary to adjust the methods.
Decomposition of carbonate by acid washing according to DIN ISO 11277 for larger amounts of samples (up to ~50 g)
► Procedure:
► Weigh in airdried and ground soil (< 2 mm) in a beaker, note weigh-in mass
► put on protective clothing (lab coat, gloves, glasses)
► put the beaker with the soil sample under the fume cupboard and add ~10 % HCl (around 3 parts of water + 1 part conc. HCl, around 3 M HCl) in excess, to create a suspension
► mix with a glass rod from time to time until no CO2 bubbles develop (normally ca. 30 to 60 minutes)
► add again some ml of HCl, mix carefully and observe
► repeat procedure as long until no visible bubbles build up
► weigh filter paper, if filter cake shall be dried in it (see below)
► build up a filtration aid under the fume cupboard: place funnels + filter over a big beaker
► transfer soil sample quantitatively into the filter, wash the beaker with ultrapure water into the filter as long until no soil is in the beaker
► take care, that the underlying beaker is not overflowed when catching the filtrate
► add some ml of HCl to the sample
► after filtration of part of the sample suspension, catch some drops of the filtrate with a test tube. Attention, acid!
► Add some drops of saturated NH4-oxalate into the test tube
► Observe if Ca-oxalate precipitates as white turbidity (white streaks), if yes,
► Ad some new HCl to the filter cake after complete filtration and repeat test with NH4-oxalat until no turbidity can be observed
► Wash filter cake at least 2 times with ultrapure water, until the filtrate is clear
► Either dry filter cake in the filter paper (60 °C in drying oven) and weigh out, notice the mass of filter paper,
or
► Wash filter cake from the filter with water into a weight beaker, dry the sample, (60 °C in drying oven) and weigh out
► Difference between mass before carbonate decomposition and after carbonate decomposition is the mass of carbonate removed as CO2
► This mass difference has to be considered for calculation of elemental concentrations after sample digestion
It is unknown, which amounts of P are washed out with HCl. Therefore, HCl volume should be measured and P should be determined in the filtrate. Alternatively, HCl could be evaporated to a smaller defined volume.
According to DIN ISO 11277, four ml of 1 M HCl (around 3 % HCl) are necessary for each % of carbonate, plus an acid excess of 25 %. Around 250 ml ultrapure water are added to the soil; after that the HCl is added. The suspension is mixed up to 15 min or until the end of reaction (no bubble development) at 80 °C on a sand bath or heating plate and mixed from time to time.
Alternatively, the method of Harris et al. (2001) can be used for small samples amounts. In this method carbonate is decomposed by HCl vapours.
Carbonate decomposition according to Harris et al. (2001) by HCl vaporisation for small sample amounts (~30 mg)
► Procedure:
► Weigh in 30 mg of oven-dried and grinded soil sample in Ag-foil-capsules (8-5 mm; sample scoop), take no Sn-capsules, since they will be damaged by HCl vapour
► Put the open capsules into microtiter plate and add around 50 µl ultrapure water, to moisture the soil to field capacity
► Place the microtiter plate(s) into a 5-liter-vacuum-desiccator
► Place the desiccator under the fume cupboard
► put on protective clothing (lab coat, gloves, glasses)
► place a 150 ml beaker with 100 ml conc. HCl (12 M) in the desiccator
► close the desiccator and fumigate the soil sample around 6 hours with HCl
► open the desiccator under the fume cupboard. Attention, acid vapour!
► Remove sample scoops with soil samples and dry them at 60 °C in a drying oven for around 4 hours
► Dhillon et al. (2015) recommended drying of soils samples at 105 °C for 16 hours, in order to remove all HCl residues and avoid corrosion on C-analyser. If samples are digested by aqua regia this is not necessary
► Weigh out samples and calculate mass difference for determination of element concentrations
Carbonate decomposition by heating to around 1000 °C
According to safety data sheets, the decomposition temperature of CaCO3 is 825 °C. Peters and Wiedemann (1959) and Narsimhan (1961) report, that a stronger decomposition starts at around > 890 °C. At this temperature CaCO3 reacts to CaO and CO2. During lime burning, temperatures of around 1200 °C are reached in order to produce CaO from CaCO3. The decomposition temperature for MgCO3 is around 550 °C (Liu et al. 2011, Sawada et al. 1979); in safety data sheets it is > 350 °C.
CaCO3 and MgCO3 are the most common carbonates in soils. Heating of soil samples to > 900-1000 °C causes therefore also decomposition of carbo¬nates, so that such samples can be analysed as carbonate-free samples.
In the labs of the Biological Station Zingst there is a muffle furnace for ashing of soil samples at 550 °C. This oven can be heated to temperatures of 1000 °C. Especially for samples with high amounts of organic substances and carbonate the usage of parallel decomposition of organic substances and carbonate at high temperatures can be considered. According to Wang et al. (2011), sediment samples could be heated stepwise (with exception of marine sediments): first to 500 °C (with weigh out) for 12 hours and after weighing a second heating to 800 °C for 12 h (with further weigh out), not only to ash the samples and remove the carbonate before digestion but also to determine percentage of organic matter and carbonate. For marine sediments 550 °C for 12 hours or 500 °C for 15 hours for determination of organic substances are recommended by Wang et al. (2011). According to Burlakovs et al. (2015), Heiri et al. (2001) and Santisteban et al. (2004), the following temperatures are recommended: 105 °C (12 to 24 hours) for gypsum concentration, 550 °C (for 4 hours) for organic substances and 900 °C (for 2 hours) for carbonates.
Sampling ash can be used to digest samples with acid persulfate or aqua regia and to determine P concentrations. If these methods are planned to be used for parallel determination of organic and carbonate C (not only for ashing and carbonate decomposition), the following points have to be considered:
► Crystal water from clay minerals is removed at around 500 °C (e.g. Dean 1974, Grim 1953, Santisteban et al. 2004)
► Gypsum, sulphide minerals and metal-oxyhydroxides can be oxidised and/or dehydrated (e.g. Ralska-Jasiewiczowa et al. 2003)
► at temperatures between 425 °C and 520 °C minerals such as siderite (FeCO3), magnesite (MgCO3), rhodochrosite (MnCO3) and dolomite (CaMg(CO3)2) are decomposed (e.g. Duval 1963, Brauer and Negendank 1993, Ralska-Jasiewiczowa et al. 2003, Weliky et al. 1983)
► Goethite (FeOOH) is dehydrated at temperatures between 280 °C and 400 °C and converted to hematite (Derie et al. 1976, Prasad et al. 2006, Schwertmann 1959)
► Gibbsite (Al(OH)3) loses water at temperatures of around 300 °C (Davies 1974)
► Generally, the mineralogy of the sample is changed significantly.
Depending on the mineralogy of the sample (e.g. clay-%, other carbonates, Fe-(hydr)oxides) the parallel determination of concentrations of organic substances and carbonate during ashing or carbonate decomposition can be affected by errors. For solely ashing and carbonate decomposition temperatures between 900 to 1000 °C for 4 hours are recommended.
Samples are weighted in in porcelain crucibles (determine empty mass of crucible) and ashed in muffle furnace for 4 hours. Subsequently, ash-containing crucibles are weighted, and the mass of the empty crucible is subtracted. The difference between sample weigh-in and out is the mass loss, which must be considered for digestion and subsequent determination of P concentrations in the samples.
References
Brauer, A, Negendank, JFW (1993) Paleoenvironmental reconstruction of the Late- and Postglacial sedimentary record of Lake Weinfelder Maar. Lect Notes Earth Sci 49, 223-235, DOI: 10.1007/BFb0117599
Burlakovs, J, Ozola, R, Kostjukovs, J, Kļaviņš, I, Purmalis, O, Kļaviņš, M (2015) Properties of the jurassic clayey deposits of southwestern Latvia and northern Lithuania. Mater Sci Appl Chem. DOI: 10.1515/msac-2015-0001
Davies, B. E. (1974). Loss on ignition as an estimate of soil organic matter. Soil Sci Soc Am Pro 38, 150–151, DOI: 10.2136/sssaj1974.03615995003800010046x
Dean Jr, WE (1974) Determination of carbonate and organic matter in calcareous sediments and sedimentary rocks by loss on ignition: comparison with other methods. J Sediment Petrol 44, 242-248, DOI: 10.1306/74D729D2-2B21-11D7-8648000102C1865D
Derie R, Ghodsi M, Calvo-Roche C (1976) DTA study of the dehydration of synthetic goethite αFeOOH. J Thermal Anal 9: 435-440, DOI: 10.1007/BF01909409
Dhillon, GS, Amichev, BY, de Freitas, R, Van Rees, K (2015) Accurate and precise measurement of organic carbon content in carbonate-rich soils. Commun Soil Sci Plant Anal 46, 2707-2720, DOI: 10.1080/00103624.2015.1089271
DIN ISO 11277 Soil quality - Determination of particle size distribution in mineral soil material - Method by sieving and sedimentation, DOI: 10.31030/9283499
Duval, C (1963) Inorganic thermogravimetric analyses. Elsevier Publishing, Amsterdam‐London‐New York, 2nd ed., DOI: 10.1126/science.141.3586.1169-b
Grim, RE (1953) Chapter 9 Dehydration, Rehydration and the changing taking place on heating. In Clay Mineralogy, pp. 190-249
Harris, D, Horwáth, WR, van Kessel, C (2001) Acid fumigation of soils to remove carbonates prior to total organic carbon or carbon-13 isotopic analysis. Soil Sci Soc Am J 65, 1853–1856, DOI: 10.2136/sssaj2001.1853
Liu, XW, Feng, YL, Li, HR (2011) Preparation of basic magnesium carbonate and its thermal decomposition kinetics in air. J Cent South Univ Technol 18, 1865−1870, DOI: 10.1007/s11771-011-0915-z
Narsimhan, G (1961) Thermal decomposition of calcium carbonate. Chem Eng Sci 16, 7-20, DOI: 10.1016/0009-2509(61)87002-4
Peters, H und Wiedemann, HG (1959) Untersuchung des thermischen Zerfalls von Calciumoxalat und Calciumcarbonat auf einer Thermowaage hoher Genauigkeit. Z anorg. allg. Chem. 298, 142-151, DOI: 10.1002/zaac.19593000305
Prasad, PSR, Shiva Prasadad, K, Krishna Chaitanya, V, Babua EVSSK, Sreedhar, B, Ramana Murthy, S (2006) In situ FTIR study on the dehydration of natural goethite. J Asian Earth Sci 27, 503-511, DOI: 10.1016/j.jseaes.2005.05.005
Ralska-Jasiewiczowa, M, Goslar, T, Różński, K, Wacnik, A, Czernik, J, Chróst, L (2003) Very fast environmental changes at the Pleistocene/Holocene boundary, recorded in laminated sediments of Lake Gościaż, Poland. Palaeogeogr Palaeoclim Palaeoecol 193, 225–247, DOI: 10.1016/S0031-0182(03)00227-X
Santisteban, JI, Mediavilla, R, López Pamo, Dabrio, CJ, Ruiz Zapata, MB, Gil García, MJ, Castaño, S, Martínez-Alfaro, PE (2004) Loss on ignition: a qualitative or quantitative method for organic matter and carbonate mineral content in sediments? J Paleolimnol, 32, 287-299, DOI: 10.1023/B:JOPL.0000042999.30131.5b
Sawada, Y, Yamaguchi, J, Sakurai, O, Uematsu, K, Mizutani, N, Kato, M (1979) Thermal decomposition of basic magnesium carbonates under high-pressure gas atmosphere. Thermochim Acta 32, 277-291, DOI: 10.1016/0040-6031(79)85115-1
Schwertmann, U (1959) Die fraktionierte Extraktion der freien Eisenoxyde in Boden, ihre mineralogischen Formen und ihre Entstehungsweisen. J Plant Nutr Soil Sci 84, 194-204, DOI: 10.1002/jpln.19590840131
Wang, Q, Li, Y, Wang, Y (2011) Optimizing the weight loss-on-ignition methodology to quantify organic and carbonate carbon of sediments from diverse sources. Environ Monit Assess 174, 241–257, DOI: 10.1007/s10661-010-1454-z
Weliky, K, Suess, E, Ungerer, CA, Muller, PJ, Fischer, K (1983) Problems with accurate carbon measurement in marine sediments and particulate matter in seawater: A new approach. Limnol Oceanogr 28, 1252–1259, DOI: 10.4319/lo.1983.28.6.1252
For citation: Schumann R, Zimmer D (year of download) Chapter 3.4 Removal of Carbonate (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, Sebastian Marcus Strauch, Theresa Zicker, Maximilian Berthold, Michael Oster
Environmental samples are often very heterogeneous. Especially small weigh-ins would be affected by high standard deviation for repetitions of P determinations without sample homogenisation. This is especially true for suspensions, solid plant or mineral samples and animal tissues. In a lot of cases a multi-step homogenisation is necessary.
Because suspensions are not real solutions, solid particles sediment in dependency on time, which means a certain separation between solid and liquid phase. Examples for suspensions are liquid manure, digestates, slurry and sewage sludge. If digestion and P determination is planned from wet matter (cannot be recommended), the suspension has to be mixed by vigorous stirring. This should be done before every subsample. Parallelly, beside samples for P determination, subsamples have to be taken for determination of dry matter percentage (DM-%). Extremely heterogenous samples (e.g. undecomposed, bulky plant parts) cannot be digested as wet samples. Such samples have to be dried and further pre-treated. There are different possibilities for preparation/homogenisation. The selection of possibilities depend on scientific questioning and the character of the suspension (e.g. DM-%). After the drying of suspension, the sample can be treated such as a solid sample (see 3.5.2 homogenisation of solid samples). For samples with high concentrations of organic matter (OM) a homogenisation by ashing is possible (chapter 3.3). Take care that masses of the complete suspension and of the wet and dry solid and liquid phases are determined.
(a) Separation between solid and liquid phase of suspensions
Especially for suspensions such as liquid manure and digestates, which are used for fertilisation, questions arise from distribution of nutrients between solid and liquid phase. Under these circumstances a separation between solid and liquid phase is urgently necessary before drying.
Separation is possible by (a.1) gravity-related sedimentation of the solid phase. However, a long waiting time is necessary and the separation is incomplete. Additionally, microbial conversions are possible due to long waiting time. After separation solid and liquid phase have to be treated separately. This method cannot be recommended due to the incomplete separation and long waiting time.
The second possibility of separation is for example (a.2) screw-press (Mönicke et al. 1978). This type of separation is especially used in the professional agricultural sector for large sample volumes. The solid and liquid phase can subsequently be used separately in farming. After separation by a screw press the solid phase has a DM concentration between 18 and 25 % and the liquid phase of 3 to 7% (Burgstaler et al. 2017). In digestates P is more accumulated in the solid phase compared to the liquid phase (Bachmann et al. 2015). The consistency of the solid phase is comparable to that of bought potting soil or peat. This solid phase only needed to be dried and eventually ground and ashed. The liquid phase is a suspension, in which particles can sediment. This suspension has to be stirred vigorously if digested in a wet state (parallel determination of dry matter) or has to be dried before further analysis (points b and c).
Separation of solid and liquid phase can also be done by (a.3) centrifugation. For representative sampling larger sample amounts should be centrifuged. This is much faster than gravity-related sedimentation and separation is more completely than with a screw-press. In the labs of the working group soil science (in AUF) up to 750 ml per vessel can be centrifuged by the centrifuge (Heraeus Multifuge 3 SR+ Centrifuge, Thermo Scientific). In one pass 4 vessels of 750 ml can be processed in this centrifuge. Alternatively, adapters can be used (4 per rotor) for filling with 12 x 15 ml centrifuge tubes (48 per rotor), 5 x 50 ml centrifuge tubes (20 per rotor) or 2 x 100 ml centrifuge tubes (8 per rotor). Other adapters, for example for microtiter plates, are not available. Since the centrifuge has a swing-out rotor the maximum centrifugal acceleration is 4500 x g. Additionally, a cooling (≤ -9 °C) is possible in this centrifuge. In case of uneven loading the centrifuge stops automatically due to their imbalance detection. Acceleration and deceleration can be set separately. After centrifugation the supernatant has to be decanted carefully or has to be removed by a syringe. When solid and liquid phase are separated after centrifugation the solid phase can be dried completely (see points b and c). Decision has to be made if liquid phase has to be dried or can be digested in the wet state.
A separation of phases can also be done by (a.4) filtration. The most complete separation between solid and liquid phase is possible by appropriate selection of filters. Direct filtration of suspension can only be recommended for suspensions with very high percentages of the liquid phase. Otherwise, the filters will be clogged by the developing filter cake. A previous centrifugation is therefore recommended. After filtration the filter cake has to be separated from the filter. Therefore, selection of the filter is also affected by this possibility. Before filtration the mass of the filter has to be determined. After filtration the filter can be dried and weigh with the filter cake. The dried filter cake can be brushed off carefully from the filter.
(b) Drying of sample in drying oven
If the suspension is not separated in solid and liquid phase, it is recommended to dry the suspension completely or a large subsample of it (vigorously stirring as well as possible). If the solid phase was separated previously, this solid phase has to be dried completely. It has to be decided individually if the liquid phase has to be dried as well or can be digested in the wet state. Take care that for all samples the weigh-in and out before and after drying is noted (note empty mass of the vessels), to determine DM-%. Flat bowls should be preferred for drying in the drying oven (if possible, with circulating air). Drying with circulating air is much faster than without. The selected temperature depends on scientific questioning and subsequent analyses. If only total element concentrations have to be determined, a temperature of 60 °C can be recommended. To prevent samples from “sticking/caking”, the samples have to be stirred from time to time during drying or should be crumbled by hand. If liquid manure, digestates or something similar is dried, an odour nuisance is possible. For such samples lyophilization (c) is recommended or the drying oven has to be in a separate room. After drying the sample has to be grounded (see chapter 3.5.2 homogenisation of solid samples). In the working groups soil science, soil physics and agronomy several drying ovens are available. One of the drying ovens (with circulating air) is separated in the basement.
(c) Lyophilisation (freeze drying)
If a freeze dryer is available, it should be selected instead of oven-drying for odorous samples or for subsequent temperature-sensitive analyses (e.g. microbial decomposition of samples). Depending on sample amount and type of freeze-dryer the samples can be filled in centrifuge tubes (e.g. 50, 100 or 500 ml). For all samples weigh-in (or samples volume) and weight out after drying have to be noted (note also empty mass of tubes), to determine DM-%. Tubes are closed with a lint-free paper (laboratory cloth) which is fixed by a rubber band. Tubes are set in a holder and frozen (at -20 °C) in a freezer. Note that frozen water has a larger volume than liquid water. Therefore, sufficient volume in the tube is necessary. If samples are completely frozen, the paper can be removed and the tubes can be set in the freeze dryer. It is not allowed to use the freeze dryer alone. Before usage a detailed briefing by a professional laboratory assistant is mandatory or the equipment is operated by the laboratory assistant. Depending on the sample volume, complete drying can take a week or longer. After drying the sample is mostly a powdery substance. The solid can be digested directly or has to be homogenised such as peat (chapter 3.5.2). However, sieving to < 2 mm is not necessary. In the working group soil science (AUF, University of Rostock) is a freeze dryer (Alpha 2-4 by Christ Gefriertrocknungsanlagen; Martin Christ Gefriertrocknungsanlagen GmbH; https://www.martinchrist.de/en/). The ice condenser has a maximum capacity of 4 kg and a temperature of -85 °C. That means that it is possible to dry solvent-based or other low-eutectic samples as well.
Solid samples such as plants, peat, < 2 mm sieved mineral soils, sediments and bio chars can be very heterogeneous as well. In plant and peat heterogeneity is caused by bulkiness and/or different tissues. In soils and sediment heterogeneity is mainly caused aggregates and/or different sized individual particles (mainly sands).
Plant material and peat
After drying, plant material has to be milled (if necessary stepwise: first coarse, then fine). Herbaceous plants (surface biomass, roots), seaweed and similar can be crumbled by hand and subsequently milled fine (e.g. Ultra-Centrifugal mill ZM1000 by Retsch, Pulverisette 23 and 6 by Fritsch) or ashed directly (see chapter 3.3). In the working group soil science for fine milling the “Ultra-centrifugal mill ZM1000” by Retsch can be used. The mill ZM1000 has a speed of 15000 rotations per minute and a sieve < 0.25 mm. Material for this mill has to be < 5/10 mm. The real size depends on the structure and strengths of the material. In the working group Agronomy (AUF, University of Rostock) the ball mill Pulverisette 23 (by Fritsch) can be used for volumes of up to 5 ml (grain size < 6 mm) to mill to final grain size of 5 µm. Generally, this mill can be used for cryo-milling, but this has not been tested before. For larger sample volumes the planetary ball mill Pulverisette 6 (by Fritsch) can be used. This mill holds a sample volume between 10 and 225 ml; the maximum grain size is 10 mm. The grinding fineness is < 1 µm. Fine grinding is not necessary if larger sample amounts will be ashed (see chapter 3.3).
Cereal whole plants and suchlike are separated before drying in straw and corn by threshing. The straw is cut by scissors in short pieces before grinding. Alternatively, the straw can be grinded by a coarse shredder. In the working group Soils Science (AUF, University of Rostock) the mill “Schneidmühle SM200” by Retsch can be used. The final fineness is between 0.25 and 0.20 mm, depending on hole wide of the sieves. It was possible to grind corncobs with this mile. The coarse crushed straw and suchlike can be further milled with fine mills (see above). For a representative straw sample several subsamples should be milled and pooled together again. Cereal corns can be grinded with different mills such as knife mills for animal tissue (see below) or grinder for nuts and alike or can be ashed without further grinding. Grinding of oil-containing seeds can be problematic (e.g. rape), if samples warm up during grinding. This has to considered for time of grinding and other settings for the mills. In the working group Agronomy (AUF, University of Rostock) the ball mill Pulverisette 23 is used for grinding of rape seed. For these seeds 35 rotations min-1 and a time of 20 sec is used. The milling chamber is filled to a maximum of ¾, so that mill balls are well visible. Additionally, in the experimental station Satower Strasse a cutting mill combination Pulverisette 25/19 by Fritsch is available. In this mill corn cobs can be grinded as well. It is a combination mile for pre-crushing and fine grinding in one step. Pieces of a maximum of 120 mm x 85 mm can be processed. After completely automatic two-step crushing the final fineness is 0.2 to 0.6 mm. The sample is cooled during grinding. The crushing is as follows: in the mill Pulverisette 25 the whole sample is pre-crushed, subsequently the sample fell via a hopper on a sample divider, where the sample normally is subdivided in the relation 1:13. The split ratio is variable. This amount-reduced subsample is automatically milled in the Pulverisette 19 to a final fineness of 0.2 mm and automatically aspirated by a Fritsch Zyklon into the sample vial. It has to be decided specifically if fineness is sufficient or if further grinding with a fine mill (see above) is necessary. Request for theses mills is possible at the working group Agronomy or the leader of the experimental station (chapter 10).
Woody plant material with leaves (e.g. branches/twigs from willows) has to be separated by hand before drying in woody parts and leaves. Subsequently, it has to be milled separately. The wood is grinded in a coarse mill and subsequently in a fine mill (see above). According to the producer the mill SM200 by Retsch is suited. The combination mill Pulverisette 25/19 might be suited as well. This has to be requested in the working group Agronomy. For grinding the wood in the fine mill, the material must be of adequate fineness (< 5/10 mm). Only small samples amounts should be milled, because these fine mills warm up very fast. Leaves of the twigs are processed such as herbaceous plant material. That means that normally no coarse pre-grinding is necessary; fine grinding or direct ashing is possible.
Tubers such as from potatoes are often peeled and separated in peel and crushed potato pulp (depending on scientific question). Peel and potato pulp are crushed and subsequently dried separately. Grinding of potato material in common mills for plant material, such as the ultra-centrifugal mill, is not possible. In the working group Agronomy, the cutting mill combination Pulverisette 25/19 by the firm Fritsch is used (see above). It can be tried to crush potatoes in a knife mill (see chapter 3.5.3) as well. If this is not possible the material has to be ashed (see chapter 3.3).
Peat material has to be processed differentially after drying due to fibrousness. Ideally, peat material is sieved to < 2 mm after drying such as mineral soil. Subsequently, it is milled in a mortal mill (see below). If this is not possible due to long/coarse fibres it can be tried to process peat in knife mills. If this is not possible, the peat samples have to be ashed (chapter 3.3).
Mineral soil, sediments, biochar (also bone char)
Minerals soil has to be sieved < 2 mm after drying. If very large and solid aggregates are in soil samples (e.g. in clay soils) it may be necessary to crush the aggregates with mortal and pestle by hand. Crushing of large aggregates may never be done on the 2 mm sieve because the sieve would be destroyed! Soils and sediments cannot be milled in mills for plant material. After sieving < 2 mm subsamples of the soil are milled in a mortar mills (e.g. in the working group Soil Science 2 mortar mills: RM100 by Retsch and Pulverisette 2 by Fritsch), to destroy small aggregates and homogenise the sample. The final fineness of the Pulverisette 2 is 10 to 20 µm. Sample volumes of maximum 190 ml with a particle size of maximum 6 to 8 mm can be milled. For RM100 no more details were found, because only successor model RM200 is on the market. According to own experience the sample volume, particle size and final fineness is similar to the Pulverisette 2. In the working group Soil Science in both mills soil volumes of around 1 to 2 tablespoons are milled.
Mineral sediments can be processed like mineral soil. Sediments with too high OM percentages and from which subsamples cannot be milled have to be ashed. Bone chars and probably other biochar cannot be milled in mortar mills for soils according to own tests. Such samples have to be crushed by mortal and pestle by hand dust-finely, if possible. This crushing is not necessary if different sieve fractions of chars are processed and the digestion of single particles is planned.
Animal tissue can be differentiated in water-rich soft tissue (e.g. muscle meat), (fish) skin and solid tissues such as bones, cartilage, horns and mussel shells. Only a few experiences are available for these materials. It has to be kept in mind that for all pieces the wet mass, the dry mass and the ash mass have to be determined.
If not separated previously, birds have to be plucked and other animals have to be skinned. Muscle meat has to be separated from bones and offal. First, the animal is opened, and the offal are removed separately. Offal (especially stomach and intestines) have to be washed separately to remove for example digestive residues. Subsequently, the muscle meat has to be removed from bones by a sharp blade. Bones have to be processed separately. Fishes are processed as whole fish or separated in pieces according to the scientific question. If the fish has to be separated, the head has to be removed first, followed by the offal. Afterwards, the fish is fileted to separate fish meat (filet) and bones (carcass). The fish skin either remains with the filet or has to be removed before fileting.
Soft tissue such as fish meat and fish skin
Water-rich soft tissue has to be crushed separately as well as possible (muscle meat, skin, offal) before drying. A first pre-crushing can be done by hand with scissors or a knife. Generally, fish muscle meat can be digested in a fresh state in a microwave with HNO3. This should only be done if the material is relatively homogeneous. Parallelly, the DM-% has to be determined. In the working group Aquaculture & Sea-Ranching (AUF, University of Rostock) fish were frozen as whole fish but also crushed. Whole fish were frozen at -20 °C, subsequently sawn coarsely in small pieces and finally minced 3 times. For fish pieces the frozen filet (due to the leathery skin) as well as the carcass were minced separately. According to the experience of the working group Aquaculture, fish meat has to be crushed in a frozen state, because in a fresh state too much water would be lost! Crushing in knife mills was not possible, because fish pieces rotated in the mill but were not crushed. After crushing, the material was frozen again at -20 °C and subsequently lyophilised for around 3 days. The lyophilised material was milled first by an Agate ball mill (at 200 rotations per minute for 20 minutes) and subsequently milled finely in a ceramic mill (IKEA 365+ IHÄRDIG). Barrento et al. (2009) crushed fresh fish meat with a knife mill (Grindomix GM200 by Retsch; 5000 rotations per minute) until complete visible destruction of material. Polypropylene cups and stainless steel knifes were used. Subsequently, the material was frozen at -20 °C. A subsample was lyophilised for 48 hours at -50 °C and low pressure (10-1 atm). Finally, samples were ashed and digested by HNO3. Ersoy and Çelik (2009) also crushed fresh fish samples in a mill by stainless steel knifes but digested the homogenised material directly without drying.
Generally, fishes can be dried in a drying oven or lyophilised (see chapter 3.5.1 points b and c). In both cases the weigh-in and out must be noted to determine DM-%. Drying in a drying oven causes a lot of odour. Therefore, drying by lyophilisation is recommended (chapter 3.5.1 point c).
According to Schoo (2010), larvae of lobster can be completely frozen directly after harvest, lyophilised subsequently pulverized and digested. No further information is presented about pulverisation by Schoo (2010).
Generally, fish meat can also be ashed in a muffle furnace in a wet state at 450 to 550 °C for homogenisation (e.g. Engmann and Jorhem 1998, Jorhem et al. 1996). According to experiments at the Biological station Zingst this causes a lot of odour and a strong sooting of the oven (see chapter 3.3).
Hard tissue, e.g. bones, cartilage and horns
Bones, cartilage and horns cannot be milled in common mills or crushed by hand with mortar and pestle for homogenisation. Special bone mills are necessary. According to the experience of the working group Aquaculture, carcasses of fishes can be crushed by a mincer. Possibly, the jaw crusher (https://www.retsch.com/products/milling/jaw-crusher/) of the firm Retsch is suited to crush bones, since with this crusher ores, ceramic and suchlike is crushed. The mill BB50 by Retsch processes material < 40 mm and mill to particles of < 0.5 mm, whereas the BB200 can mill particle sizes of < 90 mm to < 2 mm. Bone material < 10 mm can partly be crushed in ball mills. If bones, cartilage and horn are chips < 5 mm they can be digested directly with HNO3 + H2O2 in a microwave. If this material is visible heterogeneous it has to be crushed like coarse material or should be ashed in a muffle furnace for homogenisation. Ashing of bones has not been tested before in the labs. Since during pyrolysis (free of O2) at 500 °C bone chips form a relatively crumbly material, it is supposed that during ashing in a muffle furnace a material is formed, which can be crushed by mortar and pestle by hand.
Carbonate-containing biogenic material, e.g. mussel shells, chitinous carapace of arthropods
Mussel shells mainly consist of calcium carbonate (in the form of aragonite), which is stabilised by the organic substance conchiolin (Wikipedia conchiolin). The first reference for conchiolin is Frémy (1855). It has to be considered to clean mussel shells before crushing from adhesive dirt and other organic substances, according to the scientific question. Generally, mussel shells can be crushed by hand with mortar and pestle (Pilkey & Goodell 1963, Ragland et al. 1979). Alternatively, they could be ashed in a muffle furnace at 550 °C, since organic substances would be ashed leaving only a fragile calcium carbonate, which could be crushed easily (400 °C, in Elsaesser 2014). Analyses of mussel shells have not been done before in the labs. According to Kost (1853), a strong formation of ammonia could be possible during ashing.
Chitin-containing exoskeleton (e.g. crustacean) can be coarsely crushed by hand, and, according to Cárdenas et al (2004), dried in porcelain cups at 105 °C (to weight constancy) and subsequently ashed at 900 °C for homogenisation. Boßelmann et al. (2007) milled exoskeletons of lobsters in a ball mill with 500 rotations per minute for 3 h at room temperature and with liquid nitrogen in a mortar. They found no chemical differences due to different forms of homogenisation.
A further possibility to mill samples being difficult to mill and heat-sensitive is milling in cryogenic mills. During milling, samples are cooled by immersion of the milling cup in liquid nitrogen. Additionally, the sample is embrittled by the nitrogen cooling and volatile substance remain in the sample. For these mills special safety regulations have to be considered during processing and the staff has to be trained in handling liquid nitrogen. Such mills are for example the cryogenic vibrating tube mill CryoMill by Retsch or the cryogenic mill C3 (Prozess- und Analysentechnik GmbH). The cryogenic mill from Retsch has a closed LN2-system (autofill), which avoids direct contact of the user with liquid nitrogen and can therefore increase safety. Until now, there is no experience in the labs with such cryogenic mills.
Comparison of P concentrations between differentially pre-treated soil samples: < 2 mm and not mortared vs. additionally mortared (homogenised) soil samples
In this experiment 4 soils (BS1 to BS4) each < 2 mm sieved, either not mortared or additionally mortared for homogenisation (n = 5). Soil samples of 0.50 g were digested with aqua regia in a microwave. P concentrations were measured at ICP-OES at a wave length of 214.914 nm.
For all 4 soils no significant differences in P concentrations between soil < 2 mm and additionally mortared soil were verified (Tab. 3.5-1). However, relative standard deviation was lower in additionally mortared samples compared to the of only sieved samples. This is also visible in the smaller range between minimum and maximum for mortared samples. The smallest range between minimum and maximum for < 2 mm sieved samples was 32 mg P kg-1 (BS1) and the largest was 1214 mg P kg-1 (BS2), whereas for mortared samples the smallest range was 16 mg P kg-1 (BS2) and the largest 43 mg P kg-1 (BS3). Additionally, no outliers were found for mortared samples. Mortaring of soils samples increased homogeneity of samples, since differentially big soil aggregates were destroyed. This effect was also evident for other elements such as Al, Ca, Fe, K, Mg, Mn, Zn (not shown).
| soil | pre-treatment | minimum | maximum | mean | S % |
| BS1 | <2 mm | 598 | 630 | 615 | 2.1 |
| mortared | 602 | 620 | 612 | 1.2 | |
| BS2 | <2 mm | 792 | 2006 | 1051 | 51 |
| mortared | 797 | 813 | 803 | 0.8 | |
| BS3 | <2 mm | 938 | 1004 | 975 | 2.7 |
| mortared | 936 | 979 | 962 | 2.2 | |
| BS4 | <2 mm | 655 | 719 | 681 | 4.3 |
| mortared | 672 | 691 | 678 | 1.1 |
References
Bachmann, S, Uptmoor, R, Eichler-Löbermann, B (2016) Phosphorus distribution and availability in untreated and mechanically separated biogas digestates. Sci. Agric. 73 (1), 9-17, DOI: 10.1590/0103-9016-2015-0069
Barrento, S, Marques, A, Teixeira, B, Carvalho, ML, Vaz-Pires, P, Nunes, ML (2009) Accumulation of elements (S, As, Br, Sr, Cd, Hg, Pb) in two populations of Cancer pagurus: Ecological implications to human consumption. Food and Chemical Toxicology 47, 150–156, DOI: 10.1016/j.fct.2008.10.021
Boßelmann, F, Romano, P, Fabritius, H, Raabe, D, Epple, M (2007) The composition of the exoskeleton of two crustacea: The American lobster Homarus americanus and the edible crab Cancer pagurus. Thermochimica Acta 463, 65–68, DOI: 10.1016/j.tca.2007.07.018
Burgstaler, J, Wiedow, D, Kahnswohl, N (2017) Presentation „Gärresteaufbereitung – Restgaspotentiale und Erfahrungswerte aus der Praxis“, last accessed 09.05.2018
Cárdenas, G, Cabrera, G, Taboada, E, Miranda, SP (2004) Chitin Characterization by SEM, FTIR, XRD, and 13C Cross Polarization/Mass Angle Spinning NMR. Journal of Applied Polymer Science 93, 1876–1885, DOI: 10.1002/app.20647
Edmond Frémy: Recherches chimiques sur les os. In: Annales de chimie et de physique. Series 3, Vol. 43, 1855, pp. 47–107 (Gallica), p. 96
Elsaesser, WN (2014) Influence of diet on element incorporation in the shells of two bivalve molluscs: Argopecten irradians concentricus and Mercenaria mercenaria. Dissertation at the University of South Florida
Engman, J & Jorhem, L (1998) Toxic and essential elements in fish from Nordic waters, with the results seen from the perspective of analytical quality assurance. Food Additives & Contaminants 15, 884-892, DOI: 10.1080/02652039809374725
Ersoy, B & Çelik, M (2009) Essential elements and contaminants in tissues of commercial pelagic fish from the Eastern Mediterranean Sea. J Sci Food Agric 89, 1615–1621, DOI: 10.1002/jsfa.3646
Jorhem, L , Sundström, B, Engman, J, Åstrand‐Yates, C, Olsson, I (1996) Levels of certain trace elements in beef and pork imported to Sweden. Food Additives & Contaminants 13, 737-745, DOI: 10.1080/02652039609374462
Kost, H (1853) Über die Struktur und chemische Zusammensetzung einiger Muschelschalen. Dissertation at the University of Würzburg
Mönicke, R Köditz, K, Döhler, K (1978) Fest-Flüssig-Trennung von Gülle mit einer Schneckenpresse. Agrartechnik 28, 69-70
Pilkey, OH & Goodell, HG (1963) Trace elements in recent mollusk shells. Limnology and Oceanography 8, 137-148, DOI: 10.4319/lo.1963.8.2.0137
Ragland, PC, Pilkey, OH, Blackwelder, BW (1979)Diagenetic changes in the elemental composition of unrecrystallized mollusk shells. Chemical Geology 25, 123-134, DOI: 10.1016/0009-2541(79)90088-3
Schoo, KL (2010) Stoichiometric constraints in primary producers affect secondary consumers. Dissertation at Kiel University
For citation: Zimmer D, Schumann R, Strauch SM, Zicker T, Berthold M, Oster M (year of download) Chapter 3.5 Homogenisation (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