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Article

Comparative Assessment of Three Methods for Soil Organic Matter Determination in Calcareous Soils, Eastern Algeria

by
Hadjer Laoufi
1,*,
Hakim Bachir
2,
Samir Hadj-Miloud
3 and
Kerry Clark
4,*
1
Laboratory of Characterization and Valorisation of Natural Resources, University of Mohamed El Bachir El Ibrahimi, El-Anasser, Bordj Bou Arreridj 34030, Algeria
2
Research Division of Bioclimatology and Agricultural Hydraulics, National Institute of Agronomic Research (INRAA), Algiers 16200, Algeria
3
Laboratory of Water Management in Agriculture (LMEA), Soil Science Department, National Higher Agronomic School (ENSA), Avenue Hassan Badi, BP El Harrach, ES1603, Algiers 16200, Algeria
4
School of Natural Resources, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211, USA
*
Authors to whom correspondence should be addressed.
Land 2025, 14(10), 2030; https://doi.org/10.3390/land14102030
Submission received: 28 August 2025 / Revised: 30 September 2025 / Accepted: 8 October 2025 / Published: 10 October 2025
(This article belongs to the Section Land – Observation and Monitoring)
Browse Figures
Versions Notes

Abstract

Soil organic matter (SOM) plays a fundamental role in soil fertility and ecosystem functioning. In calcareous soils, SOM quantification is often challenging due to carbonate interference. This study aimed to compare three common analytical methods for SOM determination: the Anne method, the modified Walkley–Black method, and the Loss on Ignition (LOI) method, with and without decarbonation. Twenty-five soil samples were collected from a calcareous parcel in the Bordj Bou Arreridj region (Algeria), and SOM content was analysed using all methods. The results revealed substantial variability in SOM content across methods, reflecting differences in sensitivity to carbonates and efficiency of organic carbon oxidation. The Anne method, considered the reference technique, yielded the highest mean SOM content (3.61%), followed by LOI without decarbonation (3.41%), the modified Walkley–Black method (2.96%), and LOI with decarbonation (2.55%). Strong correlations were observed between methods, particularly between the Anne method and LOI with decarbonation (R2 = 0.91), confirming the latter as a reliable alternative. Decarbonation significantly reduced SOM overestimation, as demonstrated by paired statistical tests and a large effect size (Cohen’s d = 2.95). Linear regression models were established to estimate SOM from LOI results, providing a cost-effective approach for rapid assessment. These findings highlight the importance of method selection according to soil type, the need for standardised protocols, and the value of LOI with decarbonation as a robust, practical, and economical method for SOM determination in calcareous soils.

1. Introduction

Soil organic matter (SOM) is a fundamental pillar of soil quality, exerting a decisive influence on the soil’s fertility, structure, water retention, and biological activity [1]. Its role in the biogeochemical carbon cycle makes it a key component in atmospheric CO2 sequestration, offering promising perspectives for climate change mitigation [2,3]. SOM is characterised by marked heterogeneity, comprising both well-defined organic molecules (such as amino acids, polysaccharides, and lipids) and more complex humic substances, whose formation and accumulation mechanisms remain subjects of scientific debate [4]. Recent technological advances in NMR spectroscopy and pyrolytic chromatography have significantly improved the structural characterisation of humus [5]. Nevertheless, quantitative assessment of SOM remains an essential preliminary step, though still hindered by persistent methodological challenges.
Soil represents the third largest carbon reservoir on Earth, after the oceans and terrestrial biomass [6]. This distribution varies geographically: humid temperate regions favour organic carbon storage, whereas arid and semi-arid zones tend to accumulate carbon in inorganic forms (SIC), mainly as carbonates [7]. This dichotomy necessitates differentiated analytical approaches for accurate quantification of carbon stocks under varied pedoclimatic conditions.
The measurement of SOC is a standard procedure in soil science. Several methods are commonly used to estimate SOM or its primary component, SOC, each presenting specific advantages and limitations [8,9]. Traditional SOM estimation is based on an indirect approach: measuring Corg and applying a historical conversion factor (1.724), originally proposed by van Bemmelen and rooted in the 19th-century work of E. Wolf. This factor assumes that SOM contains a constant 58% carbon [10]. However, this assumption is challenged by the diversity of soil types, with actual Corg/SOM ratios varying significantly from 1/1.4 to 1/2.8), depending on soil characteristics and environmental conditions [11,12,13].
The methodological landscape for SOM quantification remains fragmented [14,15], although consensus generally converges on the oxidation of organic carbon. Among recent innovations, automated elemental analysers have become reference tools in modern pedology. These instruments allow complete mineralisation of samples at very high temperatures (>1000 °C), with accurate detection of the CO2 produced [16]. Nevertheless, their adoption is limited by financial and technical constraints [17,18].
In agricultural contexts, monitoring SOM stocks has emerged as a key sustainability indicator [19]. The complexity of the organic matrix, which integrates both rapidly cycling labile fractions and stable humic compounds, makes its evaluation particularly challenging [20]. This difficulty is exacerbated in calcareous soils, where abundant carbonates introduce major methodological artefacts. The loss-on-ignition (LOI) method, for example, can lead to significant overestimation of SOM in the absence of prior carbonate removal [21,22]. Traditional methods [23,24] are based on chemical oxidation using potassium dichromate. While the Walkley–Black method remains widely used, it presents notable drawbacks: hazardous reagents, time-consuming procedures, and the need to apply a correction factor (1.32), based on the assumption that 76% of SOM is organic carbon [12]. The systematic use of the 1.724 conversion factor for Corg to SOM is also strongly criticised, given the variability in the true carbon content of SOM depending on humification processes and environmental conditions [25,26].
The LOI method, despite its operational simplicity and low cost, has important limitations in calcareous environments. Consistent findings demonstrate that it can significantly overestimate SOM without prior decarbonation [27]. However, acid pretreatment can substantially improve its reliability, making it a promising option for laboratories with limited resources [28,29,30]. Despite their widespread use, these methods have rarely been systematically compared in calcareous soils [31], resulting in problematic heterogeneity in the available data. This compromises both result comparability and the reliability of carbon balance assessments across scales.
This study aims to address this methodological issue through a comparative evaluation of four analytical approaches—Anne’s method, LOI with and without decarbonation, and the modified Walkley–Black method-applied to calcareous soils. The specific objectives are (1). To compare rigorously SOM levels obtained by each method and analyse their interrelationships; (2). To assess the robustness of each protocol in the specific context of calcareous soils; (3). To quantify the impact of decarbonation on thermal methods; (4). To formulate practical recommendations for more reliable SOM estimation in carbonate-rich soils.

2. Materials and Methods

2.1. Study Area

The study was conducted in the agricultural commune of El Hammadia, located in Bordj Bou Arreridj, in northeastern Algeria. The experimental site is located at 35°58′47″ North latitude and 4°44′51″ East longitude, at an altitude of 680 m above sea level (Figure 1). This region experiences a semi-arid climate, characterised by hot, dry summers and cool winters. The northeast of Algeria is subjected to very irregular spatio-temporal variation, rainfall varies faster toward the north/south direction [32]. Annual rainfall averages around 365 mm, with marked seasonal variations [33]. The soils in this area are predominantly calcareous, with a high carbonate content resulting from the weathering of limestone parent material.
The selected study site was a cultivated plot within the Laabachi farm. The studied plot has an area of 2490 m2. The land had previously been amended with organic materials such as compost and crop residues, which influence the SOM content. The field was lying fallow at the time of sampling, allowing for undisturbed sampling conditions. The study plot exhibits a crop rotation pattern typical of semi-arid agricultural systems. In 2021, it was cultivated with tomatoes, followed by rapeseed (Brassica napus) in 2022. The fallow period implemented in 2023 represents an agronomic practice aimed at restoring soil fertility following two years of intensive production.

2.2. Soil Sampling and Preparation

Soil sampling was carried out during the winter of 2022 following a systematic grid design (Figure 2). Twenty-five samples were collected from the surface horizon (0–20 cm) using a hand auger. The samples were placed in airtight sampling bags and transported to the laboratory for processing. Upon arrival, the samples were air-dried for 15 days in a shaded environment to limit microbial activity and prevent alteration of organic properties. After drying, the samples were manually ground and sieved.
Subsamples intended for analysis using the Walkley–Black and Anne methods were sieved through a 0.2 mm mesh. For the LOI method, samples were sieved to 2 mm. In the case of LOI with decarbonation, a 1:3 diluted hydrochloric acid (HCl) solution was gradually added to each soil sample until effervescence ceased, indicating the complete removal of calcium carbonate (CaCO3). The decarbonated samples were then air-dried again prior to analysis.

2.3. Analytical Methods

2.3.1. Determination of Soil Organic Carbon by Anne Method

Soil organic matter (SOM) was estimated based on the measurement of Corg, assuming it represents approximately 58% of SOM. The method relies on the chemical oxidation of Corg using an excess of potassium dichromate in a concentrated sulphuric acid medium, followed by back-titration of the remaining oxidant with a ferrous ammonium sulphate (Mohr’s salt) solution. SOM was obtained by converting the measured organic carbon content using the Van Bemmelen factor (1.724) [23].
A 0.5 g sample of air-dried, finely sieved soil was weighed into a 250 mL flask. Then, 10 mL of potassium dichromate solution (K2Cr2O7) and 15 mL of concentrated sulphuric acid (H2SO4) were added. The flask was covered with a watch glass and heated for 5 min, timed from the first appearance of condensation. After cooling, the contents were transferred to a 100 mL volumetric flask and made up to volume with distilled water.
An aliquot of 20 mL was pipetted into a 400 mL beaker, followed by the addition of 200 mL of distilled water, 1.5 g of sodium fluoride (NaF), and a few drops of diphenylamine indicator. The excess dichromate was then titrated with a standard solution of Mohr’s salt [Fe(NH4)2(SO4)2·6H2O] until a blue–green end point was observed. A blank titration (without soil) was also carried out to correct the results.

2.3.2. Determination of Soil Organic Carbon by the Modified Walkley–Black Method

This method involves cold oxidation of organic carbon with potassium dichromate in sulfuric acid. The Cr3+ ions produced give a green colour whose intensity is proportional to organic carbon content. Absorbance was measured at 590–600 nm using a SHIMADZU UVmini-1240 spectrophotometer (Kyoto, Japan). SOM was calculated using a correction factor (1.32) applied to the measured carbon content [24].
A 0.2 g sample of finely ground soil (<0.2 mm) was placed in a 100 mL beaker and treated with 5 mL of potassium dichromate solution and 10 mL of concentrated sulphuric acid. The mixture was stirred for 3 min and then left to react for 30 min. Afterwards, 25 mL of distilled water was added, the solution was agitated again and left to settle overnight. Absorbance was measured using a spectrophotometer, alongside a standard calibration curve prepared with glucose. The organic carbon thus obtained was then converted into soil organic matter (SOM) using the Van Bemmelen factor (1.724).
The calibration curve was prepared from a stock glucose solution at 1.25 g/L, with increasing volumes from 1 to 15 mL distributed over five beakers. The standard solutions were dried in a MEMMERT-type oven (108 L, model UN110) (Bavaria, Germany) at 80 °C for 24 h, then subjected to the same chemical treatment as the soil samples before spectrophotometric analysis.

2.3.3. Loss on Ignition (LOI)

LOI method is commonly used to assess SOM. However, experimental protocols are not standardised, particularly regarding combustion duration and temperature. According to [34], LOI and its correlation with TOC are primarily governed by temperature. The selection of this parameter must therefore be precise: sufficiently high to ensure the complete elimination of SOM, yet not excessive so as to prevent the dehydroxylation of clay minerals and the alteration of carbonates. Studies indeed report varying conditions, with temperatures ranging between 150 °C and 900 °C and heating times spanning from 2 to 17 h [22]. In this study, a calcination temperature of 450 °C for four hours was chosen [35], as this temperature is sufficient to destroy organic matter without affecting calcium carbonate or structural water [9,36]. For improved comparison, a decarbonation step was performed on a separate set of samples (complete removal of calcium carbonates using hydrochloric acid diluted 1:3). These samples were then air-dried and subsequently combusted at 450 °C for four hours in a muffle furnace (Nabertherm L 40/11/B180 type) (Lilienthal, Germany). (Table 1).
These findings support the use of LOI (with or without decarbonation) as a practical method, especially in resource-constrained contexts.

2.4. pH and Carbonate Determination

Soil pH was measured using a digital pH metre (Phs-3CB Bench type) (Jiangsu, China) in a 1:2.5 soil–water suspension. Calcium carbonate was quantified using the Bernard calcimeter, based on the volume of CO2 released upon acid addition to the soil [35].

2.5. Statistical and Spatial Analysis

Statistical analysis and graphs were calculated using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA, USA). GIS analysis using Arc GIS 10.8 (ESRI, Redlands, CA, USA) allowed for spatial visualisation of SOM distribution using IDW interpolation.
The results of the comparison revealed that the correlation coefficient can be applied to determine the relationship between the methods studied. This coefficient also indicates the strength or weakness of the relationship and its direction. In this section, we aimed to develop an empirical model for estimating SOM content based on the results obtained from the fastest and least costly method, namely the LOI method.
The resulting empirical model allows for the correction of data obtained through this method, without the need to resort to the Anne method or the modified Walkley–Black method, which, although considered reference methods, are significantly more time-consuming and expensive.

3. Results

3.1. Characterisation of the Soil Samples

The general physico-chemical characterisation of the soil samples (Table 2) revealed notable variability in total limestone content, with an average of 39.04% and a coefficient of variation (CV) of 24.16%, indicating significant heterogeneity among samples. According to [35], samples 1, 2, 3, 9, 16, and 17 are classified as calcareous, whereas samples 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 18, 19, 20, and 25 are considered highly calcareous. In contrast, pH values were relatively stable across the dataset, ranging narrowly from 7.17 to 7.90 with a low standard deviation (SD = 0.23) and a very low CV (3.11%), reflecting a slightly alkaline [35], but homogeneous pH environment.
The soil organic matter (SOM) content obtained from the 25 samples varies depending on the analytical method used, but all indicate notable variability between samples. The Anne method yielded an average organic matter content of 3.61%, with a standard deviation of 0.73% and a coefficient of variation of 20.31%, reflecting moderate variability among samples. The modified Walkley–Black method produced an average of 2.96%, with a standard deviation of 0.72% and a coefficient of variation of 24.33%, indicating greater heterogeneity in measured values. Using the LOI method with decarbonation, the average organic matter content was 2.55%, with a standard deviation of 0.59% and a coefficient of variation of 22.81%, suggesting relatively high dispersion around the mean despite generally lower values. Finally, the LOI method without decarbonation gave an average organic matter content of 3.41%, with a standard deviation of 0.73% and a coefficient of variation of 21.67%, showing variability comparable to the other methods. Overall, coefficients of variation exceeding 20% for all methods suggest strong variability in organic matter content between samples, likely due to natural soil heterogeneity as well as differences in the sensitivity and precision of each method.
The Anne method yielded the highest average SOM content (3.61%), followed by the Walkley–Black method (2.96%), LOI without decarbonation (3.41%), and LOI with decarbonation (2.55%) (Figure 3). According to [38], the modified Walkley–Black method achieves a SOC recovery rate close to 100%, eliminating the need to multiply TOC by the correction factor of 1.32. Thus, many modified dichromate oxidation techniques involving external heating, such as the Anne method, do not require a correction factor.
The study by [22] demonstrates that dichromate oxidation with external heating allows near-total SOC recovery in the tested calcareous soils. However, this method may overestimate SOC in soils with very low organic carbon content [39], likely due to reactions between potassium dichromate (K2Cr2O7) and certain inorganic soil constituents [40].

3.2. Normality Test

Based on Kolmogorov–Smirnov and Shapiro–Wilk normality tests (Table 3), the data follow a normal distribution.
The results (Figure 4) indicate that the median values obtained using the various methods for estimating SOM content are not identical but remain within a comparable range, suggesting reasonable analytical consistency across approaches. The Anne method shows a noticeably higher median than the other techniques, while the Modified Walkley–Black method exhibits the lowest values overall, though maintaining acceptable inter-sample reproducibility. A particularly strong similarity is observed between the Anne method and the LOI method with decarbonation, confirming the compatibility of these two techniques in calcareous contexts. Moreover, the LOI method with decarbonation displays a narrow interquartile range, indicating low data dispersion and enhanced reproducibility. In addition, the close agreement between the mean and median values suggests a symmetrical distribution of results, with few significant outliers. The LOI method without decarbonation tends to overestimate SOM content compared to the LOI method with decarbonation, likely due to the inclusion of carbonates, and it also exhibits slightly greater dispersion, which may reflect reduced analytical precision.
Finally, the consistency between the Anne method and the LOI method with decarbonation supports the validity of the latter as a robust alternative, particularly suitable for laboratories with limited resources.

3.3. Statistical Analysis of the Effect of Decarbonation on Soil Organic Matter Content

To assess whether decarbonation significantly influenced the measurement of soil organic matter by the LOI method, a statistical analysis was conducted comparing the results obtained with and without carbonate removal.
A moderate decrease in average soil organic matter content was observed following decarbonation (Δ = −0.82%), highlighting the importance of carbonate removal to avoid overestimation when using the loss on ignition method (Table 4).

3.4. Inferential Tests

The results (Table 5) show a statistically significant difference between the SOM values measured with and without decarbonation. The paired Student’s t-test (t(24) = 14.75, p < 0.001) clearly indicates that decarbonation has a highly significant effect on the values obtained using the LOI method.
This finding is strongly supported by the non-parametric Wilcoxon test (W = 0.0, p < 0.001), which reinforces the robustness of the conclusion, even under non-normal data distributions.
Consistently, both tests confirm that SOM values significantly decrease after carbonate removal. The one-tailed test result (p = 1.000) is not meaningful in this context and was therefore not considered for interpretation.
Finally, the effect size analysis (Cohen’s d = 2.95) reveals a very large magnitude of the observed difference. This indicates that, beyond statistical significance, decarbonation has a substantial practical impact on SOM estimates and should be considered a critical step in the LOI method, particularly in calcareous soils.

3.5. Overview of Spatial Distribution of Organic Matter

A spatial distribution map of SOM was developed to provide an overview of its distribution according to the different analytical methods used. The results obtained through spatial interpolation (Figure 5) show that the distribution of SOM varies depending on the method applied.
According to the Anne method, the majority of the plot exhibits a high SOM content, while the remaining area shows a moderate content. The modified Walkley–Black method indicates that more than half of the land is moderately rich in SOM, with the rest being very rich.
The distribution maps based on the loss on ignition methods, whether with or without decarbonation, display relatively similar patterns, with a predominance of moderately SOM-rich areas.

3.6. Comparative Analysis of Empirical Relationships Between Methods for Estimating Soil Organic Matter

Statistical analysis was conducted to establish empirical models linking different analytical methods. These models allow estimation of organic matter content using faster and less expensive methods (e.g., LOI) rather than relying on the classical Anne reference method.
Three linear correlation models were established to evaluate the agreement between different methods of estimating organic matter in calcareous soils. The coefficient of determination (R2) was used as the main criterion to assess the quality of the fit. The results are summarised below.

3.6.1. Correlation Between the OM Contents Measured with the Anne Method and Walkley–Black Method

The relationship between soil organic matter (SOM) contents determined by the Anne method and those obtained using the Walkley–Black method (Figure 6), together with the corresponding regression analysis (Table 6) y = 0.8385x − 0.0539; R2 = 0.7232; p < 0.0001, revealed a moderate linear correlation compared to the other methods.
This relationship shows that, although both approaches generally follow the same trend in estimating SOM, the regression slope below 1 (0.8385) with a standard error of ±0.1082 indicates that the Walkley–Black method systematically underestimates values relative to the Anne method. The slightly negative intercept (−0.0539) suggests a consistent underestimation across the entire SOM range.
The relatively low coefficient of determination (R2 = 0.7232) highlights a weaker agreement between the two methods. Nevertheless, the average difference (Δ = +0.65%) remains small, suggesting that, although variability is higher, the systematic bias is less pronounced.
Overall, these results confirm the limitations of the Walkley–Black method, particularly its tendency to underestimate SOM in calcareous soils due to incomplete oxidation of soil organic carbon.

3.6.2. Correlation Between the OM Contents Measured with the Anne Method and LOI Method Without Decarbonation

The relationship between SOM contents determined using the Anne method and those obtained by the LOI method without decarbonation (Figure 7) reveals a strong linear correlation, expressed by the regression equation: y = 0.9487x − 0.0138, with a coefficient of determination R2 = 0.8848.
This relationship indicates that both methods provide generally consistent results for estimating SOM, although the LOI method without decarbonation tends to give slightly lower values than those obtained by the Anne method, as reflected by the slope slightly below 1 (0.9487). The negative intercept (−0.0138) suggests a moderate underestimation by the LOI method without decarbonation, particularly at low SOM levels.
The relatively high coefficient of determination (R2 = 0.8848) confirms strong agreement between the two datasets, despite the differing analytical principles. The average difference between the two methods (Δ = +0.31%) further supports this underestimation by the LOI method without decarbonation.

3.6.3. Correlation Between the OM Contents Measured with the Anne Method LOI Method with Decarbonation

A very strong linear correlation was observed between the SOC contents estimated by the Anne method and those obtained with the LOI method after decarbonation (Figure 8). The regression equation (Table 6) obtained is as follows:
y = 0.7691x − 0.1808. The coefficient of determination R2 = 0.9089 (p < 0.0001) indicates a strong relationship between the two methods, confirming that the SOC contents obtained by the Anne method are closely related to those measured with the LOI method applied to decarbonated samples.
However, the slope clearly lower than 1 (0.7691) and the low standard error (±0.0508) show that the LOI method with decarbonation systematically underestimates SOC contents compared to the Anne method, particularly for high SOC levels. The negative intercept (−0.1808) reinforces this trend, indicating a general underestimation across the entire range of SOC contents.
The mean difference between the two methods (Δ = +0.67%) also highlights this systematic bias. This underestimation can be explained by the partial loss of non-organic fractions during combustion, even after decarbonation, which may reduce the apparent SOC content.

4. Discussion

The results of this study highlight the significant variability of SOM content depending on the analytical method used, particularly in calcareous soils rich in carbonates. This variability confirms previous findings underscoring the lack of standardised protocols for SOM determination [14,41].
In this study, the Anne method was adopted as the reference method, as it is recognised for its accuracy and reliability in calcareous soils. The other methods were therefore evaluated in comparison with it.
Among the methods tested, the Anne method yielded the highest mean SOM content (3.61%), followed by LOI without decarbonation (3.41%), the modified Walkley–Black method (2.96%), and finally LOI with decarbonation (2.55%). These differences reflect the specific principles of each method.
The difference between SOM contents determined by the Anne method and those obtained by LOI without decarbonation can be explained by the presence of carbonates in calcareous soils, which decompose partially during ignition [42]. This decomposition contributes to the weight loss that is not exclusively due to organic matter, thereby distorting the results [20,21].
Compared to the Anne method, the lower SOM values obtained with the modified Walkley–Black method can be attributed to its partial oxidation of organic carbon when applied without external heating, which limits its effectiveness in certain soil types [38].
In contrast, LOI without decarbonation yielded slightly higher SOM contents than LOI with decarbonation, mainly due to the thermal decomposition of carbonates, which artificially inflates weight loss [21,25].
LOI with decarbonation produced the lowest SOM values, as removing carbonates before ignition reduces this overestimation but may also result in the loss of part of the more labile organic matter. The observed difference between the two LOI protocols (Δ = +0.82%) clearly reflects the effect of carbonate decomposition on SOM estimates.
These systematic differences highlight the respective strengths and limitations of each method relative to the Anne method, which generally provided the highest SOM values but may itself overestimate SOM in soils with low carbon content due to interactions with inorganic soil constituents [39,40]. In addition, it should be noted that sample preparation differs between the methods: the Anne and LOI methods are performed on soil sieved to <2 mm, whereas the modified Walkley–Black method is applied to soil sieved to 0.2 mm, which may also contribute to variability in the results.
Calcareous soils present a specific challenge for SOM measurement, as carbonates interfere with both combustion and wet oxidation methods. Our results showed that LOI without decarbonation systematically overestimates SOM, in line with the findings of [9,36]. In contrast, prior decarbonation significantly improved measurement accuracy. The statistically significant difference (p < 0.001) between the two LOI approaches, with a Cohen’s d of 2.95 (indicating a large effect size), clearly demonstrates the need to remove carbonates prior to combustion to avoid biased results.
Correlations between the methods provide insight into their relative performance. A strong correlation was observed between the Anne method and LOI with decarbonation (R2 = 0.91), suggesting that LOI, when applied carefully, can serve as a viable alternative to wet oxidation methods, particularly in carbonate-rich soils. Conversely, the modified Walkley–Black method showed lower correlations with LOI (R2 = 0.72), likely due to partial oxidation of organic carbon [28] and chemical interferences with inorganic soil components [39]. Despite its limitations, the LOI method stands out for its simplicity, reproducibility, and accessibility, especially in low-resource laboratories.
However, controlling analytical conditions such as temperature, duration, and decarbonation is crucial. The optimal conditions (450 °C for 4 h) confirmed in this study, minimise clay dehydroxylation while ensuring effective combustion of SOM [25].
The reliability of LOI can be improved through appropriate pretreatments, particularly decarbonation to avoid carbonate interference, fine sieving to ensure sample homogeneity, and strict control of ignition conditions. The precise control of calcination temperature and duration determines which fractions of the solid material are volatilized. Excessive temperature or prolonged ignition time may cause mineral mass losses (mineral carbon decomposition or clay dehydroxylation), leading to an overestimation of the results. These steps contribute to more accurate and reproducible SOM estimates in calcareous soils.
The absence of a universal analytical protocol for SOM estimation remains a barrier to the comparability of results between studies [43]. While elemental analysers offer high precision [16], their high-cost limits widespread use. Traditional methods such as dichromate oxidation and LOI therefore remain essential, provided they are calibrated and adjusted to the specific properties of the soil studied.
Several recommendations emerge to improve data comparability. First, the selection of method must be well considered: for calcareous soils, LOI with decarbonation or the Anne method are preferable. Second, the conventional conversion factor of 1.724 used to estimate SOM from organic carbon may not be universally valid. Soil-specific conversion factors should be developed [12,13]. Finally, researchers must explicitly report the method used, including sample preparation and analytical conditions.
Results suggest that the Anne method provides the most accurate estimation of SOM in calcareous soils, owing to its capacity for more complete oxidation of organic carbon. Nevertheless, its use may be constrained by cost, reagent handling, and the potential risk of overestimation in soils with low organic content. In contrast, LOI with decarbonation, despite a systematic underestimation, shows strong concordance with the reference method and appears to be a reliable, robust, and cost-effective alternative when analytical conditions are carefully controlled. This highlights that the choice of method should be guided not only by accuracy but also by practical considerations such as resources, laboratory infrastructure, and soil characteristics.
These findings confirm the need to consider the mineralogical composition of soils when selecting analytical methods, especially in carbonate-rich environments, in order to avoid biased estimations and misinterpretation of organic matter dynamics.
In summary, this study confirms that the choice of method significantly affects SOM values, particularly in calcareous soils. When rigorously applied, LOI with decarbonation appears to be a reliable, robust, and cost-effective alternative to more advanced instrumental techniques. The development of harmonised analytical protocols tailored to local pedological conditions should be a priority for future research on soil organic carbon dynamics.

5. Conclusions

This study compared the effectiveness of four commonly used methods for estimating SOM in calcareous soils: the Anne method, the modified Walkley–Black method, and LOI with and without decarbonation. The findings revealed significant variability in SOM values depending on the analytical method, largely due to the sensitivity of the protocols to the presence of carbonates and the efficiency of organic carbon oxidation.
The Anne method, considered as the reference in this work, proved the most effective in the studied conditions, although it may overestimate SOM under certain circumstances. The modified Walkley–Black method showed lower recovery of organic carbon. LOI, on the other hand, showed high potential when pre-decarbonation was applied. The strong correlation observed between LOI with decarbonation and the Anne method suggests that this approach is a reliable and accessible alternative, especially in resource-limited laboratories.
These findings emphasise the importance of selecting appropriate methods according to soil type and support the need for standardised protocols, particularly for calcareous soils. Developing soil-specific conversion factors and ensuring greater transparency in methodological reporting are also critical to improving data comparability across studies. As accurate SOM measurement becomes increasingly central to sustainable soil management and climate change mitigation strategies, harmonising analytical practices remains a key research priority.

Author Contributions

Conceptualization, H.L.; methodology, H.L.; validation, H.L., H.B. and S.H.-M.; formal analysis, H.L.; investigation, H.L.; resources, H.L., H.B. and S.H.-M.; data curation, H.L.; writing—original draft preparation, H.L.; writing—review and editing, H.L., H.B., S.H.-M. and K.C.; visualisation H.B. and S.H.-M.; supervision, H.B., S.H.-M. and K.C.; project administration, H.B., S.H.-M. and K.C.; funding acquisition, H.B., S.H.-M. and K.C. All authors have read and agreed to the published version of the manuscript.

Funding

The APC was funded by the United States Department of Agriculture Foreign Agricultural Service Grant FX24BF-10777R003.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We would like to thank B. Laabachi, owner of the farm that served as our study site, for his availability and support during the conduct of this research.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
SOMSoil Organic Matter
SOCSoil Organic carbon
SICSoil Inorganic Carbon
TOCTotal Organic Carbon
LOIloss on ignition
CorgCarbon Organic
IDWInverse Distance Weighting

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Figure 1. Geographical location of the study area.
Figure 1. Geographical location of the study area.
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Figure 2. Overview of the sampling plan.
Figure 2. Overview of the sampling plan.
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Figure 3. Representation of soil organic matter content according to the different methods analysed.
Figure 3. Representation of soil organic matter content according to the different methods analysed.
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Figure 4. Statistical comparison of SOM content using four analytical methods.
Figure 4. Statistical comparison of SOM content using four analytical methods.
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Figure 5. Map of the spatial distribution of organic matter.
Figure 5. Map of the spatial distribution of organic matter.
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Figure 6. Correlation between the OM contents measured with the Anne method and OM contents measured by the LOI method without decarbonation.
Figure 6. Correlation between the OM contents measured with the Anne method and OM contents measured by the LOI method without decarbonation.
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Figure 7. Correlation between the OM contents measured with the Anne method and OM contents measured by the LOI method without decarbonation.
Figure 7. Correlation between the OM contents measured with the Anne method and OM contents measured by the LOI method without decarbonation.
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Figure 8. Correlation between the OM contents measured with the Anne method and the OM measured by the LOI method with decarbonation.
Figure 8. Correlation between the OM contents measured with the Anne method and the OM measured by the LOI method with decarbonation.
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Table 1. Comparative analysis of SOM methods.
Table 1. Comparative analysis of SOM methods.
MethodAdvantagesDisadvantages
Loss on ignition (LOI) method- Previously widely used
- Easy to apply
- Low cost [37] 		- Unreliable due to reactions unrelated to organic matter (e.g., interference from carbonates or bound water)
- Overestimates OM content (may include oxides and carbonates decomposed at high temperatures) Corg is derived from OM using a fixed conversion factor (0.58), which proves inaccurate for organic horizons [37]
Anne method - Oxidation is performed at boiling temperature, ensuring more complete oxidation if maintained for 5 min
- More thorough attack of organic matter
- Residual dichromate solution is clearer 		- Requires heating
- More costly
- Use of chemical reagents
Modified Walkley–Black method- Previously widely used
- Inexpensive
- Allows for rapid approximate evaluation Targets specific OM pools
- Slight interference from [37]		- Destructive
- Incomplete oxidation: requires correction factor
- Tends to underestimate Corg
- Interference from chlorides, Mn2+ and Fe2+ oxides
- Uses hazardous chemical compounds [37]
Table 2. Descriptive statistics of the studied parameters.
Table 2. Descriptive statistics of the studied parameters.
ParameterMinMaxMeanSDCV (%)
Total Limestone (%)27.1660.3239.049.4324.16
pH7.177.97.510.233.11
OM (Anne Method) (%)2.435.133.610.7320.31
OM (Walkley–Black Method) (%)1.664.232.960.7224.33
OM (LOI with decarbonation) (%)1.543.972.550.5922.81
OM (LOI without decarbonation) (%)2.265.013.410.7321.67
Table 3. Normality test.
Table 3. Normality test.
Kolmogorov–SmirnovShapiro–Wilk
StatisticsdfSig.StatisticsdfSig.
OM (Anne Method) (%)0.097250.200 0.958250.384
OM (Walkley–Black Method) (%)0.120250.200 0.964250.511
OM (LOI with decarbonation) (%)0.092250.200 0.952250.277
OM (LOI without decarbonation) (%)0.125250.200 0.949250.234
Table 4. Descriptive statistics for SOM content with decarbonation and without decarbonation.
Table 4. Descriptive statistics for SOM content with decarbonation and without decarbonation.
GroupMean ± SDMedianRange (Min–Max)
With decarbonation2.59 ± 0.592.541.54–3.97
Without decarbonation3.41 ± 0.743.342.26–5.01
Table 5. Statistical test results comparing groups with and without decarbonation.
Table 5. Statistical test results comparing groups with and without decarbonation.
Statistical TestStatisticp-Value
Paired Student’s t-testt(24) = 14.75p = 0.000
Paired Wilcoxon testW = 0.0p = 0.000
One-tailed test (hypothesis of decrease)—p = 1.000
Effect size (Cohen’s d)d = 2.95—
Table 6. Regression parameters between SOM measurement methods and Anne method.
Table 6. Regression parameters between SOM measurement methods and Anne method.
ComparisonSlopeInterceptR2p-ValueStandard ErrorRegression Quation
Walkley–Black vs. Anne0.8385−0.05390.7232<0.0001±0.1082y = 0.8385x − 0.0539
LOI (with decarb.) vs. Anne0.7691−0.18080.9089<0.0001±0.0508y = 0.7691x − 0.1808
LOI (without decarb.) vs. Anne0.9487−0.01380.8848<0.0001±0.0714y = 0.9487x − 0.0138
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Laoufi, H.; Bachir, H.; Hadj-Miloud, S.; Clark, K. Comparative Assessment of Three Methods for Soil Organic Matter Determination in Calcareous Soils, Eastern Algeria. Land 2025, 14, 2030. https://doi.org/10.3390/land14102030

AMA Style

Laoufi H, Bachir H, Hadj-Miloud S, Clark K. Comparative Assessment of Three Methods for Soil Organic Matter Determination in Calcareous Soils, Eastern Algeria. Land. 2025; 14(10):2030. https://doi.org/10.3390/land14102030

Chicago/Turabian Style

Laoufi, Hadjer, Hakim Bachir, Samir Hadj-Miloud, and Kerry Clark. 2025. "Comparative Assessment of Three Methods for Soil Organic Matter Determination in Calcareous Soils, Eastern Algeria" Land 14, no. 10: 2030. https://doi.org/10.3390/land14102030

APA Style

Laoufi, H., Bachir, H., Hadj-Miloud, S., & Clark, K. (2025). Comparative Assessment of Three Methods for Soil Organic Matter Determination in Calcareous Soils, Eastern Algeria. Land, 14(10), 2030. https://doi.org/10.3390/land14102030

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