ORIGINAL PAPER Comparison of digestion methods

Chemical Papers 68 (8) 1015–1021 (2014) DOI: 10.2478/s11696-014-0555-5

ORIGINAL PAPER

Comparison of digestion methods for determination of total phosphorus in river sediments Jitka Malá*, Marcela Lagová Department of Chemistry, Faculty of Civil Engineering, Brno University of Technology, Veveri 331/95, 602 00 Brno, Czech Republic Received 4 October 2013; Revised 7 January 2014; Accepted 18 January 2014

In this study, four digestion methods used to determine total phosphorus in river sediments, including Na2 CO3 fusion, the H2 SO4 and H2 SO4 + H2 O2 methods and the SMT protocol were investigated. Interference effects of iron, calcium and organic matter in river sediments, and the substances contained in the digestion agents on the photometric determination of the phosphates were analysed. The digestion methods were tested on ten river sediment samples. Statistical analysis of the results showed significant differences between sample treatments relating to the mean total phosphorus concentration. c 2014 Institute of Chemistry, Slovak Academy of Sciences Keywords: sediments, total phosphorus, digestion methods, photometric determination, interferences

Introduction Phosphorus is recognised as one of the key factors responsible for the eutrophication of fresh water and a key element in biogeochemical cycles in river ecosystems (House, 2003). Dissolved phosphorus compounds usually comprise only a small fraction of the total phosphorus (TP) in bodies of water, hence they occur in river waters at low concentrations. Phosphorus accumulates in sediments in both inorganic and organic forms: it is physically adsorbed onto sediment surfaces, chemically bonded in minerals and is biologically assimilated in cells and in the detritus originating from biota (House et al., 2002). Assessment of the TP content in sediments entails decomposition of the sediment matrix followed by determination of the released phosphates. The method most commonly used for the determination of phosphates is a spectrophotometric method with ammonium molybdate. The phosphates react with ammonium molybdate in the acidic medium to form a heterocomplex of phosphomolybdic acid. This heterocomplex is further reduced in the presence of

Sb3+ ions. The resulting molybdenum blue is suitable for spectrophotometric evaluation. Sodium sulphite, tin(II) chloride, 1-amino-2-naphtol-4-sulphonic acid, hydrazine sulphate, ferrous salt and ascorbic acid can be used as reducing agents. Interferences in the analysis of phosphorus may be expected from compounds in the sediment that also react with molybdate (e.g. silicates, arsenates, etc.) or affect the reduction of phosphomolybdic acid (e.g. Fe2+ /Fe3+ ) or from extraction reagents that alter the pH of the acid medium (e.g. HCl, H2 SO4 , NaOH, NH4 OH) (Maly, 1985). Decomposition of the sediment matrix can be achieved in various ways, usually based on methods using strong acids or bases along with thermal degradation (Pardo et al., 1998). The oldest methods of determining TP in soils were developed by soil scientists and were only later adopted by environmental researchers. Among these methods, sodium carbonate fusion and perchloric acid digestion, both introduced by Jackson (1958), are of particular importance. Carbonate fusion is regarded as one of the most effective digestion methods because it also extracts the phosphorus which forms part of in

*Corresponding author, e-mail: [email protected]

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J. Malá, M. Lagová/Chemical Papers 68 (8) 1015–1021 (2014)

the matrix of silicate minerals (Kovar et al., 2009). Combinations of the acids H2 SO4 + HF or HClO4 + HF have similar effects. Another method for determining phosphorus concentrations has been proposed by the Commission of the European Communities within the framework of the Standards, Measurements and Testing Programme (SMT protocol). This protocol was designed to determine five phosphorus fractions (inorganic P, organic P, apatite P, non apatite inorganic P and TP) in sediments, and is based on the Williams method (Williams et al., 1980, Ruban et al., 1999). Other scientists, such as Bíliková (1992), used sulphuric acid as the digestion agent. Decomposition by sulphuric acid can be accelerated by adding potassium or ammonium persulphate (Hupfer, 1995) or hydrogen peroxide (Zwirnmann, 1999). Ostrofsky (2012) compared the efficiency of four selected digestion methods: the combustion method (similar to but not identical with the SMT protocol), the persulphate (K2 S2 O8 ) method, the HClO4 + HNO3 method and the H2 SO4 + H2 O2 method, on a collection of sediments from six lakes. He found no significant differences between the methods with regard to the mean TP concentrations. However, the persulphate method exhibited greater variability than the other three methods. He explained that this resulted from the lower efficiency of the persulphate method in oxidising organic-bound phosphorus to reactive phosphates in samples with a higher organic content (Ostrofsky, 2012). It is obvious that the accuracy of the TP determination depends not only on the efficiency of decomposition of the sediment matrix but also on the potential interfering effects of the reagents used in the decomposition reaction, as well as the substances released in the course of the decomposition of the sediment. The current study sought to compare four different commonly used digestion methods using spectrophotometric determination of the phosphates released.

Experimental The following analytical reagent-grade chemicals: sulphuric acid (Penta, CO); hydrochloric acid, hydrogen peroxide, sodium and ammonium hydroxides, sodium hydrogen carbonate, ammonium molybdate, ascorbic acid (Penta, Czech Republic), sodium carbonate (Mach, Czech Republic), silica (Sigma– Aldrich, Czech Republic), potassium antimonyl tartrate, ferric ammonium sulphate (Merck, Czech Republic) were used. 2,4-dinitrophenol (Aldrich, Czech Republic, 97 mass % purity) was also used All glassware was cleaned five times with tap water prior to use. Impurities were removed with a clean brush used only for this purpose. Next, the glassware was washed once using deionised water before being

immersed into an ultrasonic bath filled with deionised water for 10 minutes. The effect on the photometric determination of phosphates of the reagents used in the digestion and certain substances which can be released from the sediment was examined, using a concentration of P, −1 c(PO3− . Each determination was car4 ) = 0.5 mg L ried out in 10 replicates. The differences between a set of control samples and a set of samples with the tested substance were investigated using the Student t-test at a 5 % significance level. The statistical analyses were performed using the R software (R Development Core Team, 2009). The digestion methods were tested on ten sediment samples collected from three brooks in the Morava river basin, Czech Republic: Troubsky (TRB) – 3 sampling sites, Leskava (LES) – 4 sampling sites and an un-named stream near the village of Hartmanice (HAR) – 3 sampling sites. Wet sediments were passed through a nylon sieve to separate the fractions smaller than 100 m, then these fractions were dried at 105 ◦C to constant mass and manually crushed into powder. The sediments were characterised by the organic matter contents, expressed as the loss on ignition (Czech Office for Standards, 2007), and calcium and iron contents determined using a Prodigy ICP optical emission spectrometer (Teledyne Leeman Labs, USA) in accordance with respective methods (Czech Office for Standards, Metrology and Testing, 2009). In all the digestion methods tested the phosphorus contained in the samples was converted to soluble reactive phosphorus, which was then measured using the molybdenum blue method proposed by Murphy and Riley (1962), using ascorbic acid as reduction agent. The optical density was measured at a wavelength of 710 nm using a Cecil CE 2021 spectrophotometer. The detection limit of this method is c(PO3− 4 ) = 0.005 mg L−1 and the calibration curve is linear up to c(PO3− 4 ) = 0.8 mg L−1 . The first digestion method tested was the sulphuric acid method described by Bíliková (1992). Unfortunately, the author did not provide a detailed account of the parameters used. Accordingly, the following conditions were used: a 100 mg sediment sample was digested with 4 mL of concentrated sulphuric acid at 440 ◦C for 70 min in a 100 mL Kjeldahl flask in a Digesdahl digestion apparatus (Hach–Lange). This was sufficient time for the sample to become almost clear. After cooling, the sample was transferred into a 200 mL volumetric flask, diluted with deionised water, then 2,4-dinitrophenol was added and the sample neutralised with concentrated ammonium hydroxide to attain pH 5. The flask was then made up to 200 mL with deionised water and, once the sediment settled, an aliquot of the clear supernatant was analysed. Several modifications of the SMT protocol have been reported; the method described by Pardo et al. (2003) was used in this study. A 200 mg sediment sam-


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ple was combusted in a porcelain crucible at 450 ◦C for 3 h. After cooling, 20 mL of 3.5 mol L−1 HCl was added and the suspension shaken for 16 h. Finally, the suspension was centrifuged at 5000 min−1 for 10 min and an aliquot of the supernatant was analysed. The third procedure tested was the sodium carbonate fusion method, performed as described by Kovar et al. (2009). A 1000 mg sample of sediment was melted with 4 g of Na2 CO3 in a platinum crucible. After melting, the mixture was heated using the full power of the burner for 20 min in an oxidising atmosphere, then the contents of the crucible were dissolved in 30 mL of 4.5 mol L−1 H2 SO4 . The crucible and the lid were then placed in 25 mL of 1 mol L−1 H2 SO4 and brought to the boil. Both solutions were transferred into a 250 mL volumetric flask and the flask filled with deionised water. After allowing the sediment to settle, a 2 mL aliquot of the clear supernatant was analysed. The pH of the aliquot was adjusted to 5 with 5 mol L−1 NaOH, using 2,4-dinitrophenol indicator. The H2 SO4 + H2 O2 method has been used in our research centre for several years for TP analyses of samples of both water and sediments. The method has been applied under various reaction conditions. For example, Hupfer digested 5–10 mg of sediment with a mixture of 2 mL of 5 mol L−1 H2 SO4 , 2 mL of 30 mass % H2 O2 and 20 mL of distilled water at 150 ◦C for 16 h (Hupfer et al., 2004). The same author later used the same parameters but reduced the digestion time to 8 h (Hupfer et al., 2009). Kleeberg used a reaction time of 3 h at two temperatures (120 ◦C and 160 ◦C) (Kleeberg et al., 2010). Ostrofsky digested a 5–10 mg sediment sample with 2 mL of 5 mol L−1 H2 SO4 and 2 mL of 30 % H2 O2 , with no added water at 120 ◦C for 8 h (Ostrofsky, 2012). In the present study, the conditions were changed so that the reaction time could be substantially reduced. A 100 mg sediment sample was digested with 3 mL of concentrated sulphuric acid in a 100 mL Kjeldahl flask at 440 ◦C in a Digesdahl digestion apparatus (Hach Lange), until white fumes appeared. After heating for a further 5 min, 17 mL of 30 % H2 O2 was added drop-wise through a capillary funnel and the mixture was heated for a further 5 min. After cooling, the volumetric flask was filled to the 100 mL with deionised water and, once the sediment settled, an aliquot of the clear supernatant was analysed. All TP determinations were performed using three replicates. The mean TP concentrations and coefficients of variation were calculated for each method and sediment sample. Both data sets were analysed and no obvious violation of normality or homogeneity and no obvious relationship between group mean and variance were found. In order to compare the mean TP concentrations, the analysis of variance was conducted separately for each stream, with the sampling site as a blocking factor. This procedure is recommended by

Table 1. Reagents used during decomposition methods tested c/(mol L−1 ) Na2 CO3 fusion SMTP H2 SO4 H2 SO4 + H2 O2 Na2 CO3 NaOH HCl H2 SO4 NH4 OH H2 O2

0.15 a

– 0.64 – –

– – 3.5 – – –

– – – 0.37 a

–

– – – 0.55 – 1.65

a) These reagents are added in small amounts to adjust pH of the final solutions.

Crawley (2007); otherwise analysis of pooled data with an auto-correlated structure (streams) results in biased p-values. If a significant difference was detected among the treatments, the Tukey test for multiple comparisons was performed. Analyses were performed using the R software (R Development Core Team, 2009). All the procedures were verified by analysis of a certified reference material BCR -684 – river sediment (European Commission, Joint Research Centre, Institute for Reference Materials and Measurements). In BCR -684, the concentrated HCl-extractable P is certified. The expanded uncertainties of the difference between the results obtained and the certified value (U∆ ) were calculated and compared with the absolute differences between the mean measured values and the certified value (∆m ) (Linsinger, 2010). Standard deviations of the measurements were used as rough estimates of measurement uncertainties.

Results and discussion The reagents used in the various decomposition methods are listed in Table 1. Table 2 shows the concentrations of the reagents that were verified by the procedure described above as not interfering with the photometric determination of phosphates (error of less than 1 %). In addition, Table 2 also lists SiO2 and Fe3+ since they are common components of the sediments. When assessing the possible impact of these substances on photometric determination, both the dilution of the extract prior to the photometric determination, as well as the chemical transformations of reagents during decomposition, must be considered. The H2 SO4 method only uses sulphuric acid, at a concentration in the leachate of 0.37 mol L−1 . Prior to photometric analysis, the sample is diluted and neutralised with NH4 OH to pH 5, hence the H2 SO4 does not interfere with the determination of phosphates. The SMT protocol uses combustion followed by extraction using hydrochloric acid. The HCl concentration in the leachate is 3.5 mol L−1 . So that the acidity does not interfere, the maximum permitted concentration of HCl is 200 mmol L−1 .Hence, the leachate sample to be photometrically assessed must be diluted at least 17.5 times or neutralised.


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Table 2. Concentrations of selected compounds that do not interfere with photometric determination of phosphates Compound NaHCO3 NaOH HCl H2 SO4 NH4 OH H2 O2 Fe3+ SiO2

c/(mmol L−1 )

Error/%

P-value of the Student t-test

10 40 200 120 30 0.88 1.5 0.13

0.7 0.3 0.7 0.8 0.6 0.4 0.1 0.7

0.084 0.531 0.201 0.269 0.203 0.509 0.917 0.085

Fig. 1. Mean TP concentrations (± SD) in sediments obtained by the four analytical methods of decomposition tested.

The Na2 CO3 fusion includes a 25-fold dilution of the leachate sample prior to photometric determination and neutralisation of the acidic sample by the addition of NaOH. There is uncertainty as to whether the acidification of the sample in the process of dissolving the solidified melt removes all of the carbonates in the form of CO2 . If not, residual hydrogen carbonates could be present in the solution and interfere via their buffering capacity. However, at the recommended dilution, the concentration of hydrogen carbonates cannot exceed the critical value of 10 mmol L−1 . The advantage of this method is that, unlike the other methods tested, it extracts phosphorus from apatite inclusions or those that are part of the matrix of silicate minerals (Syers et al., 1967). The silicates released have negligible interfering effect on the photometric determination at the recommended dilution. In the leachate obtained by the H2 SO4 + H2 O2 method, the concentration of sulphuric acid is 0.55 mol L−1 . This means that, in order to fulfil the acidity conditions, the leachate must be either diluted 4.6 times or neutralised prior to photometric determination. Hydrogen peroxide interferes even at very low concentrations. Given the reaction time required, how-

ever, the H2 O2 is fully decomposed during digestion of the sediment. Interference by Fe3+ was observed at concentrations higher than 1.5 mmol L−1 . Since sample portions of 100–200 mg were used and large dilutions of the leachates obtained, the concentrations of iron were substantially lower than this value, even in case of sediments rich in iron. Having demonstrated that neither sediment constituents nor reagents interfered with the analysis of phosphorus, it was possible to compare the efficiencies of the 4 extraction techniques. The mean TP concentrations found in the sediments using the four digestion methods are shown in Fig. 1. The results ranged from 0.52 mg g−1 to 2.80 mg g−1 . Fig. 1 shows that the results obtained by the various digestion methods differ from each other, but the order of the results is not the same for all the sediment samples. The variance calculated for each stream separately, with the sampling site as a blocking factor, showed a significant difference among the treatments of the LES and TRB samples (LES: F = 2.23, P = 0.002; TRB: F = 10.70, P = 0.008). In the case of samples from HAR, the difference between the meth-



Table 3. P-values of simultaneous tests for linear hypotheses concerning mean TP concentrations Linear hypotheses

LES

TRB

HAR

H2 SO4 + H2 O2 – H2 SO4 = 0 0.361 0.028 SMTP – H2 SO4 = 0 0.069 Na2 CO3 fusion – H2 SO4 = 0 0.002 SMTP – H2 SO4 + H2 O2 = 0 Na2 CO3 fusion – H2 SO4 + H2 O2 = 0 0.006 0.926 Na2 CO3 fusion – SMTP = 0

0.237 0.034 0.007 0.423 0.064 0.444

0.927 0.267 0.517 0.517 0.268 0.048

Table 4. Main characteristics of sediments analysed Sediment Loss on ignition/% Fe/(mg g−1 ) Ca/(mg g−1 ) LES8 LES9 LES10 LES12 TRB1 TRB4 TRB7 HAR2 HAR3 HAR4

10.5 7.1 8.1 6.1 3.9 3.9 3.3 9.6 6.5 7.1

± ± ± ± ± ± ± ± ± ±

1.1 0.7 0.8 0.6 0.4 0.4 0.3 1.0 0.7 0.7

13.8 10.4 14.3 10.1 24.4 23.6 24.4 31.1 30.4 25.1

± ± ± ± ± ± ± ± ± ±

1.8 1.4 1.9 1.3 3.2 3.1 3.2 4.0 4.0 3.3

2.9 2.5 2.4 4.2 8.7 2.3 17.8 11.4 11.2 9.0

± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.1 0.3 0.5 0.1 1.1 0.7 0.7 0.5

ods was just above the level of significance (F = 4.22, P = 0.063). Accordingly, the Tukey test for multiple comparisons was performed on the data from all three brooks. The results are shown in Table 3. Differences in the results can be explained by the relatively large diversity of the sediments, which differed in their composition. The details are shown in Table 4. The values measured in the three brooks are typical for river/brook sediments. Similar data were reported by House et al. (2002). Statistical analysis suggested that, for the LES samples, the SMT protocol detected significantly lower levels of TP than the H2 SO4 + H2 O2 and H2 SO4 methods. In addition, the Na2 CO3 method detected significantly lower levels of TP than the H2 SO4 + H2 O2 method. The LES samples were rich in organic matter and had low iron and calcium contents. The H2 SO4 method, which is thought to be ineffective in consistently oxidising all organic-bound phosphorus to reactive phosphates, especially in samples that are rich in organic matter (Ostrofsky, 2012), was shown to be sufficiently effective up to loss on ignition of 10.5 % (sample LES8). Like the TRB samples, which contained little organic material and medium amount of iron, the SMT protocol and the Na2 CO3 fusion provided lower amounts of phosphorus than the H2 SO4 method. No statistically significant difference was found between the H2 SO4 and H2 SO4 + H2 O2 methods. However, more detailed analysis of the results showed that comparable results were measured only for the TRB4

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sample, which differed from the remaining two samples in its low calcium content. In the case of the TRB1 and TRB7 samples, the results of the H2 SO4 + H2 O2 methods were lower. It can be assumed that, in the case of the samples with a high content of calcium (possibly phosphorus bound in apatite), a larger dose of sulphuric acid and a longer contact time for the H2 SO4 method in comparison with the H2 SO4 + H2 O2 methods would be more effective than the addition of a strong oxidising agent (H2 O2 ). For the HAR samples, a significant difference was found only between the SMT protocol (the highest TP concentrations measured) and the Na2 CO3 fusion (the lowest TP concentrations measured). These sediments were rich in phosphorus, with the HAR2 sample having the highest concentration of all the sediments tested. The HAR samples also had the highest concentrations of iron, calcium and medium-to-high concentrations of organic matter. From these results, it may be concluded that the H2 SO4 and H2 SO4 + H2 O2 digestion methods were efficient for all of the sediments analysed. At higher concentrations of organic substances, the H2 SO4 + H2 O2 method was more appropriate due to the addition of an oxidising agent. The SMT protocol was most efficient for the HAR samples, where it gave results comparable with the H2 SO4 and H2 SO4 + H2 O2 methods. The HAR sediments differed from the other samples in their high calcium and iron contents. During its retention in bed sediments, P is initially adsorbed, particularly on the surfaces of Fe oxides, edges of calcite and calcium carbonate surfaces of calcareous sediments, and can be released to the water phase again (Jalali et al., 2013). Hence, it can also be easily desorbed by the extraction procedure. Although the alkaline melting is undoubtedly a very effective digestion method, especially because of the decomposition of the silicate matrix, for this set of sediments analysed, the Na2 CO3 fusion gave low results. This was probably the result of the experimental design in which the melting apparently decomposed the organic matter. Phosphorus compounds were most probably converted to phosphates, which, at the high temperatures, may have reacted with other components, such as iron and calcium, to form poorly soluble phosphorus compounds which were not dissolved. Another possible explanation is that the phosphates released were adsorbed on the silicic acid precipitate. The mean coefficients of variation of the methods tested were 7.70 % (Na2 CO3 fusion), 6.27 % (H2 SO4 ), 11.64 % (SMT protocol) and 6.15 % (H2 SO4 + H2 O2 ). According to one-way ANOVA, there was no significant statistical difference between the methods with regard to the coefficients of variation (F = 1.69, P = 0.182). This suggests that there was no significant difference in relative variability of the results obtained by the methods tested.


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Table 5. TP in BCR -684. Comparison of measurement results with a certified value (concentrated HCl – extractable P) Measurement Certified value H2 SO4 H2 SO4 + H2 O2 Na2 CO3 SMTP

TP/( g g−1 ) ∆m /( g g−1 ) U∆ /( g g−1 ) 1373 1316 1342 1329 1467

± ± ± ± ±

35 18 33 19 38

– 31 44 57 94

– 96 80 78 200

Table 5 shows the results of verification of all the procedures by analysis of a certified reference material BCR -684 (three parallel determinations). As ∆m ≤ U∆ for all the procedures tested, no significant differences were found between the measurement results obtained and the value certified for BCR -684. From a comparison of the digestion methods, the H2 SO4 + H2 O2 method is recommended for analysing TP in sediments. This method, using the present experimental design (i.e. concentrated sulphuric acid and a high digestion temperature), is simple, safe and time-efficient (total sample decomposition takes approximately 15 minutes). The basic laboratory safety principles need to be observed. Sulphuric acid must be present in the digestion flask when the hydrogen peroxide is added, and the flask must be placed behind a safety shield during digestion (the shield is part of the Digesdahl–Hach–Lange apparatus). Attention must be paid to the quality of the hydrogen peroxide. Some producers use phosphoric acid to stabilise this highly unstable compound. However, this chemical must not be used. Furthermore, a portion of at least 100 mg of the sediment sample is recommended for use in all the methods tested. Even if the sediment is homogenised, the use of an extremely small sample portion extends the variability of the results.

Conclusions Statistically significant differences were found between the digestion methods tested (the Na2 CO3 fusion, the H2 SO4 method, the H2 SO4 + H2 O2 method and the SMT protocol) used for TP analyses in river sediments. This implies that the digestion methods are not universal, and the composition of the sediment should be taken into account when choosing an appropriate method. The H2 SO4 + H2 O2 method, which was effective for most of the analysed sediments, is recommended for use. Acknowledgements. This research was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic, grant no. FAST-S-11-14/1161.

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