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Validation of an analytical methodology to determine humic substances using low-volume toxic reagents

  • Isabel Brás EMAIL logo , Ana Tavares , José Pereira and Elisabete Silva
Published/Copyright: September 12, 2022
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Open Agriculture
From the journal Open Agriculture Volume 7 Issue 1

Abstract

The 12 principles of green chemistry were stated to pursue good practices and techniques that minimize the use of hazardous substances in the production and application of chemicals. To minimize the risks and the pollution generated using chemicals, new methodologies must be developed that avoid the application of dangerous products or its amount. Considering the principle of pollution prevention, this work proposes the development and application of a different methodology for the quantification of humic substances (HS), applying a spectrophotometric methodology that reduces the quantity of hazardous reagents (sodium dichromate and sulfuric acid) and minimizes the waste production. To deeply acknowledge the method, a validation of the methodology was done to allow the evaluation of its effectiveness to report reliable results. It was done by the evaluation of the parameters often used for this purpose (analytical limits, precision, accuracy, selectivity, robustness, and method working range). The detection limit attained was 0.03 g glucose/L and the quantification limit was 0.09 g glucose/L. The precision and accuracy were also evaluated using the repeatability limit and through performance testing, respectively, with a repeatability limit of 0.045 g/L and a Z-score less than 2. For selectivity and robustness, differences in variances were not significant, and the working range is well adjusted, with no differences regarding the precision of the lower and higher standard. The results obtained showed that the method under study, adapted to the green chemistry principles, can be applied to quantify HS in impure and complex samples.

Keywords: green chemistry; humic substances; spectroscopy; validation of analytical methods

Abbreviations

a

linear regression intercept

b

linear regression slope

C a

concentration attained in the assay, g/L

C s

spiked concentration, g/L

C Glucose

glucose concentration, g/L

COD

chemical oxygen demand, mg O2/L

F

F-test ratio

FA

fulvic acids

HA

humic acids

HS

humic substances

IUPAC

International Union for Applied Chemistry

LOD

limit of detection, g/L

LOQ

limit of quantification, g/L

m compost

mass of compost under evaluation, g

R

product–moment correlation coefficient

r

repeatability limit, g/L

RSD

relative standard deviation, %

S

standard deviation of the n measurements done

S 1 2

variance of the n measurements done for a calibration curve standard solution (1)

S 2 2

standard deviation of the n measurements done for a calibration curve standard solution (2)

S a

standard deviation of the intercept

S b

standard deviation of the slope

S y/x

estimation of the linear regression random errors in the y-direction

S ri 2

variance of the n measurements done for a standard in repeatability conditions

x lab

results obtained for the analyzed sample, g/L

x m

average concentration of the n measurements done, g/L

x t

theoretical concentration of the analyzed standard, g/L

y ˆ i

values calculated by regression line corresponding to the individual x-values

Z

score

1 Introduction

In recent years, issues of environmental preservation, emissions reduction, and sustainable development have been widely discussed because of the need to preserve the environment and ensure a healthy quality of life for present and future generations. In this context, chemical activities, both laboratorial and industrial, require a constant search for cleaner technologies and processes that seek solutions to reduce waste or, when this is not possible, minimize the production of hazardous substances.

In the end of the twentieth century, the International Union for Applied Chemistry started to engage efforts to develop the concept of green chemistry. Green chemistry was defined as the “development of chemical products and processes that seek to reduce or eliminate the use and production of hazardous substances” [1]. In fact, the release of chemical products is one of the main cause of environmental degradation and health problems in living beings because of each of their characteristics: corrosivity, inflammability, explosivity, reactivity, toxicity with high risk to behave as carcinogens, neurotoxins, immune agents, dermatologic agents, and reproductive toxins. These principles of green chemistry are being applied to several fields. Hashem et al. proposed a greener technique to preserve goatskin instead of chlorine salts, decreasing its content from discharges in tannery wastewater [2].

To follow the principles of green chemistry, several changes must be done in the process or procedures, namely the use of alternative reagents, that are sustainable and produce less toxic waste, the improvement of natural synthesis processes and the development of new substances that do not pollute the environment. Other example very important is the refitment of chemical s reactions in order to achieve higher yields, produce less waste, minimizing energy consumption and dangerousness.

In this context, this article validates a methodology based on the principles of green chemistry, a method for quantification of humic substances (HS), which reduces the use of hazardous substances, namely sulfuric acid and sodium dichromate, reduces the production of toxic waste, such as chromium VI, and reduces the time and electrical energy spent.

HS are macromolecules composed of carbon, oxygen, small percentages of nitrogen, and other elements such as phosphorus or sulfur and are defined by a series of brown or black amorphous polymers of moderately high molecular weight, formed by secondary synthesis reactions by biotic and abiotic factors [3,4]. HS have in their composition amino acids, lipids, mono- and polyaromatic compounds, polyphenols, fatty acids, aromatics rings, and several functional groups that give them various properties [5]. These compounds are present in the environment, namely in soils and correspond to its organic matter and are the result of microbial action and the chemical and biological degradation of animal and plant remains.

According to MacCarthy [6], it is possible to extract HS from soils, sediments, and water through laboratory procedures. Such extraction results in three main fractions: fulvic acids, humic acids, and humin. As mentioned by Tombácz and Meleg [7], these fractions are divided according to their solubility in an aqueous medium as a function of the pH of the extracting solution. Current methods for its quantification exploit these properties, such as the method described by Jodice et al. [8] in which the quantification of organic matter is done by oxidation with dichromate (Na2Cr2O7) in an acidic medium followed by spectrophotometric reading at 590 nm against a calibration with glucose standards.

Following the principles of green chemistry, the work carried out proposes the HS quantification through an analytical protocol that reduces the use of quantities of toxic reagents, namely sodium dichromate, and less production of chromium VI wastes, while reducing the use of energy and maintaining its analytical quality. For this, in addition to the study of the method and its adaptations, its validation was performed to ensure the operational quality and analytical performance of the method to produce valid and reliable results [9,10].

In the laboratory, an experimental protocol accounts for several procedures that play an important role in the result accuracy and precision. The deviation of the result from the real value, indicated by the accuracy, and the dispersion of consecutive values of a sample result, demonstrated by the precision, arise from systematic errors, originated in equipment, technicians, environmental or other conditions, or even random errors subsequent to distractions and external causes. The knowledge of the method response, given by the method validation is a key factor to the laboratory quality [11]. Relevant overall performance indicators in method validation are the limit of detection (LOD), the limit of quantification (LOQ), accuracy, precision, linearity, selectivity, specificity, ruggedness, and robustness [12]. The validation process ends with a conclusion and statement of whether the analytical requirement is met [13].

In this context, and with a focus on the pollution prevention, this work proposes a HS quantification method, applying a methodology that reduces the quantity of hazardous reagents (sodium dichromate and sulfuric acid) and minimizes waste production. Both methods were tested to evaluate their agreement and after the new method was validated with the evaluation of the aforementioned parameters, it is important to have confidence in the analytical methodologies correct results when reporting data. The convenience of the method was tested in the determination of HS in composts.

2 Materials and methods

This study involves experimental work to validate a more sustainable analytic method to HS quantification and it was followed by its application in compost samples. The modification from the classical method [14] was done to decrease the volume of toxic reagents and toxic laboratory wastes produced. Figure 1 shows all analytical procedures for the classic method (Method 1) and the method proposed here (Method 2).

Figure 1 Analytical procedure of Method 1 (left) and Method 2 (right).
Figure 1

Analytical procedure of Method 1 (left) and Method 2 (right).

2.1 Analytical methods

The method under evaluation is intended to become a greener routine methodology, adapted from the procedure for fertilizing organic products [14]. Alkaline extraction of the sample HS is used for obtaining the total humic extract [8,14]. Carbon quantification is performed by oxidation with dichromate (Na2Cr2O7), in an acidic medium, followed by reading in a Lambda 25 spectrophotometer (PerkinElmer) at 590 nm. For this, a glucose calibration line was developed, with standards from 0.01 to 1.00 g glucose/L. In this study, the calibration was achieved for glucose as it has a known carbon content. The adaptation proposed here proposes to reduce the amount of sulfuric acid and dichromate used, as well as the amount of chromium wastes generated and the reduction of time and electrical energy spent for this analysis.

Therefore, 1.5 mL of Na2Cr2O7 instead of 5.0 and 3.5 mL of H2SO4 instead of 15 mL were used for the oxidation of organic matter, and the oxidation process was done in a chemical oxygen demand digester for 90 min at 90°C instead of an oven (Figure 2).

Figure 2 (a) Digestion of total humic extract in oven digestion; (b) digestion of the total humic extract in a CQO digester.
Figure 2

(a) Digestion of total humic extract in oven digestion; (b) digestion of the total humic extract in a CQO digester.

2.2 Samples for HS extraction

The quantification of HS was performed in two composts obtained through composting of mixtures of straw and grass and two distinct wastes: elderberry and grape stalk, as shown in Figure 3.

Figure 3 Composts (a) grape stalk + grass + straw; (b) elderberry stalk + grass + straw.
Figure 3

Composts (a) grape stalk + grass + straw; (b) elderberry stalk + grass + straw.

2.3 Analytical method validation

For the validation process, the minimum validation requirements were evaluated: analytical limits, precision, accuracy, selectivity, robustness, and working range of the method [15]. Glucose was used to carry out the validation process, namely the development of the calibration curve, because this substance is constituted by carbon (C), representing 40% of the C in the soil. To evaluate the concentration of HS, in terms of carbon concentration, equation (1) was used.

(1) HS ( Carbon content , % ) = C Glucose m compost ⁎ 20 .

2.3.1 Quantification

Calibration indicates a process by which the response of a measurement system is related to a concentration or a known amount of substance. In spectroscopy, this is a usual procedure, where the measurement is the radiation absorbance according to the Lambert-Beer law, for standards in the same conditions of the samples to be analyzed. A mathematical regression is applied and is represented by equation (2).

(2) y = a + b x ,

where y represents the measurement and x is the standard concentration. There are several conditions to accept the calibration curve, namely values of the correlation coefficient greater than 0.995. After evaluating the random errors in the values for the slope and intercept, equations (4) and (5) [9], the acceptance of the linear regression attains the percentual value of the standard deviation of the slope by the slope (S b/b * 100) that should be less than 5%, and that the intercept should be inside the interval between its value and standard deviation of the intercept (a ± S a) [15].

(3) S y / x = ∑ i ( y i − y ˆ i ) 2 n − 2 ,

(4) s b =   S y / x ∑ i n ( x i − x ¯ ) 2 ,

(5) s a = S y / x ∑ i n x i 2 n ∑ i n ( x i − x ¯ ) 2 .

2.3.2 Analytical limits

The determination of analytical limits, the LOD and the LOQ, were performed by measuring the glucose concentration of 10 blanks (distilled water), obtained from the calibration line constructed using glucose standards of concentrations from 0.01 to 1 g glucose/L. The LOD indicates an analyte level that is significantly different from zero, whereas the LOQ is the lowest level at which the performance is acceptable for a typical application and it is possible to state the concentration of the analyte in the sample. When the method involves the use of a linear calibration, equations (6) and (7) are used.

(6) LOD =   3.3 ⁎ S y / x b ,

(7) LOQ = 10 ⁎ S y / x b ,

where b is the calibration curve slope and S y/x is the estimation of the linear regression random errors in the y-direction.

2.3.3 Work range

The HS quantification involves the construction of a calibration line with glucose standards, obtained through the spectrophotometric reading of each standard of different concentrations used.

The glucose concentrations of ten standards with concentrations ranging between 0.01 g and 1 g/L were determined. The working range of the method was assessed by the test of variances homogeneity following the two-sided significance test F-Snedecor/Fisher. Using the variances obtained, a test of homogeneity of variances was performed to assess the working range of the method and whether it was well adjusted. The parameter F is calculated according to equation (8), where S 1 and S 2 are the higher and lower variances of repeated analysis of the calibration curve limit standards, in intermediate precision conditions. Subscripts 1 and 2 are allocated in the equation so that F is always superior than one. If the value obtained for this test is lower than the established F value, then the differences in variances are not significant and the working range of the method is well matched. The test assumes that the populations from which the samples are taken are normal [9].

(8) F = S 1 2 S 2 2 .

2.3.4 Sensitivity

Sensitivity is the change in instrument response that corresponds to a change in the measured quantity. Thus, it is defined as being the first-order derivative of the calibration curve, that is, a linear calibration corresponds to the slope of the calibration curve. Although is not usually a mandatory parameter in method validation, it is useful because it assesses the ability of a method to distinguish small differences in concentration from analysis and can be used as a check of the instrument performance, namely in spectrophotometric analysis.

2.3.5 Accuracy

The accuracy of the method was evaluated by the Z-score performance test. Spiked blanks were performed in intermediate precision conditions and the respective concentrations of glucose were determined. To evaluate the accuracy by Z-score, the results obtained (x lab) were compared with a known glucose standard (x t), and the results were obtained using equation (9). For the accuracy of the method to be acceptable, the value obtained by the Z must be lower than 2.

(9) Z = ( x lab − x t ) S .

2.3.6 Precision

Precision indicates the dispersion of results between independent tests repeated over the same sample, under the same conditions. It can be evaluated by the repeatability, with the evaluation of the repeatability limit (r) and the relative standard deviation (RSD). It is performed with at least ten determinations within the range of three different concentrations and three replicates each, or with at least six determinations for a single concentration. To evaluate the precision, ten measurements of 0.01 g glucose/L standard were made under repeatability conditions, and the variance of the results was evaluated to calculate the aforementioned parameters. The repeatability limit (r) between two consecutive assays was calculated by equation (10). The results of two determinations made under repeatability conditions are accepted if |xi − x(i − 1)| ≤ r. Also, RSD was evaluated by equation (11), where S is the standard deviation of the 10 measurements done and x m is their average glucose concentration.

(10) r = 2.8 × S ri 2 ,

(11) RSD ( % ) = S x m .

2.3.7 Selectivity

One analytical methodology is specific or selective when it allows the analyte to be quantified against other substances. The interference of other substances that may be present in the sample must be evaluated using complex samples for the purpose [16].

To evaluate the method selectivity, samples spiked with 0.01 and 1 g glucose/L were prepared with distilled water and water collected from a natural lake. The selectivity can be assessed by the test of variances in homogeneity following the F-Snedecor/Fisher significance test (equation (8)), where the variances are calculated from the assays from both spiked samples. If the test result is less or equal to the established F value, there are no differences regarding their accuracy, the method is selective and can be used in impure/complex samples.

Another important parameter to evaluate the selectivity is the recovery, evaluated according to equation (12), where C a is the attained concentration and C s is the spiked concentration.

(12) Recovery ( % ) = C a C s ⁎ 100 .

2.3.8 Robustness

The robustness of an analytical method indicates its ability to tolerate small variations in analytical parameters. The robustness of a method is a measure of its ability to remain unchanged under small but studied variations in method parameters and indicates its independence during normal use [16].

To perform this parameter, five replicates of samples were analyzed and the analysis was performed according to a five-factor control plan that could influence the process.

According to the RELACRE 13 guide, to determine the robustness of a method, the Youden test can be performed, which allows not only to assess the robustness of the method, but also to classify the variations in the results and indicate the type of influence in each variation – whether by excess or by default [9].

3 Results and discussion

3.1 Availability assessment of the classical and new method

Green chemistry concerns the use of a set of principles that reduce or eliminate the use of hazardous substances in the production and application of chemicals. Within the 12 principles of green chemistry, the first is the principle of prevention, where it is stated that it is better to prevent the production of waste than to treat them after their production.

In fact, green chemistry is closely connected with environment preservation once there is intent to reduce or eliminate the use or generation of hazardous substances in the design, production, and application of chemicals [17]. Considering the classical method (Method 1) for HS determination, 50.00 mL of sample are used with the addition of 5.00 mL of Na2Cr2O7, 15.00 mL of H2SO4 and distilled water until 100.00 mL. Although 100.00 mL of solution are produced, only 5 mL are used for the spectrophotometric analysis. In the proposed low volume methodology (Method 2), the oxidation phase is performed in a digester with reduction of sample and reagent volumes as well as wastes generated, i.e., 2.50 mL of sample, 1.50 mL of Na2Cr2O7, and 3.50 mL of H2SO4, decreasing 92.5% of the wastes produced and 80% of the hazardous reagents needed.

To evaluate the availability for the low-volume method (Method 2) to be applied to the same extent as the traditional, two calibration curves were performed (Figure 4). It is possible to see that the sensibility of both methods is similar once they have similar slopes of the calibration curve. The higher value for the intercept and higher absorbance for the standards with Method 1 are related to a higher number of molecules that absorb energy at 590 nm. So, it is possible to say that both methods have similar results and is more adequate to use the method that requires less hazardous reagents and produces less hazardous wastes, that is, Method 2. So, it can be proposed as a main finding that the new method can be used instead of the classical with much lower environmental and economic impact in the laboratory activity.

Figure 4 Calibration curves done with both methods under study.
Figure 4

Calibration curves done with both methods under study.

3.2 Validation process of method 2

Several interfering factors may arise in the quantification of HS in environmental samples like the matrix complexity, solutions pH, extraction time, stability of the analytical solutions and the occurrence of incomplete reactions. Thus, the proposed modifications, namely the amount of oxidants used and oxidation time, deserve special attention, so that the final results obtained by the method preserve its accuracy, precision, repeatability, reproducibility, in addition to guaranteeing that the method is selective and robust. The method validation process as well as the requirements studied follow the RELACRE 13 guide – Validation of results in chemical laboratories.

First, the relationship between concentration and the instrumental response was established from the absorbance readings of five glucose standards of concentrations ranging from 0 to 1 g glucose/L. The data obtained from Figure 5 show a correlation coefficient of 0.99, which indicates a strong correlation between the two measurements (0.91 < R < 0.99) and the RSD of the slope is less than 5%.

Figure 5 Glucose calibration at 590 nm.
Figure 5

Glucose calibration at 590 nm.

Table 1 shows the results of the analytical method validation requirements applied to Method 2.

Table 1

Results obtained for the minimum analytical validation requirements

Parameter Results Observations
Calibration y = 0.3135x + 0.0076
Work range (g/L) 0.01–1.00 F test OK (F = 2.507 F, critic = 3.179)1
R 2 0.9996 OK
Acceptance of slope 0.01 OK
Acceptance of intercept 0.00446–0.0106 It does not include zero
LOD (g/L) 0.03
LOQ (g/L) 0.09
Repeatability limit (absorbance) 0.014
Repeatability limit (g/L) 0.045
RSD (%) 19 High
Accuracy (Z test) Z = 1.07 <2, OK
Selectivity (recovery test, %) 140 Sample with high amount of dissolved carbon
Robustness OK F test OK (F = 2.656 F, critic = 6.388)2

  1. 1 F Snedecor for 9 degrees of freedom (95% confidence); 2 F Snedecor for 4 degrees of freedom (95% confidence).

The calibration curve for the quantification of glucose was accepted according to the criterium previously described, except for the intercept that does not include zero. This situation is very common and should not jeopardize the method of application. The values obtained for the method detection and quantification limits were 0.03 and 0.09 g glucose/L, respectively, that is only above 0.03 g/L is possible to consider the presence of glucose in the sample and only above 0.09 g/L is possible to present the concentration in a sample.

The parameters precision and accuracy were evaluated by the repeatability limit and through performance test, respectively, having obtained the repeatability value of 0.065 and Z-score less than 2. For accuracy, the condition of the test score less than 2 was verified, and for precision, the consecutive values obtained not exceeding the repeatability limit were found. Compliance with these conditions demonstrates the method’s accuracy and precision.

As for selectivity and robustness, evaluated by the concentrations of glucose found in standards of 0.01 and 1 g/L prepared using water collected from a lake, the values of the F-test were lower than the established Fisher–Snedecor F values (9% freedom at 95% confidence). This indicates that the differences in variances were not significant, which proves the suitability of these criteria when using complex matrices and facing small variations in the parameters of analysis.

The variances of the glucose standards of lower and higher concentrations are reduced, respecting the condition for the F-test, and therefore, the working range is well adjusted, showing no differences regarding their accuracy.

Overall, the study of alternative quantification methods that use less reagents, mainly hazardous reagents and consequently fewer wastes, is crucial for environmental sustainability. The results obtained showed that the method under study (Method 2), adapted to the principles of green chemistry, has accuracy and precision, is selective, presents sensitivity, selectivity, and robustness, and can thus be applied to impure and complex samples. This Method 2 complies with one of the principles proposed by green chemistry, which aims to minimize the impacts on the environment and human health, the reduction of hazardous reagents, and the reduction of waste generated by them in laboratory activities, at the end of each process, thus developing cleaner methodologies [1]. Moreover, it is possible to reach the economic and environmental savings obtained by the replacement of the equipment used in the digestion process. Other authors referred that there is no accurate and precise method for humic acid determination [18]. The method now presented shows the applicability of the method for HS determination.

3.3 Application of method 2

HS are important to agriculture and the environment, and nowadays, even to medicine and technology [19]. These substances are essential to maintain healthy ecosystems and they contribute to minimize major environmental problems, namely through carbon sequestration and therefore global warming. The HS represent a huge amount of the organic matter of the soil and is a product of chemical and biological transformation of animal and plant residues [3]. HS are nowadays not only natural, but extracted and engineered, and in the past 20 years, such products have been widely used in soil improvement and environment governance [19]. In the case of HS quantification, standard protocols and quality criteria are lacking to scale-up their production and use. The present protocol may have an important role in this field, engaging the readiness with the environmental sustainability.

Olk et al. [20] formulated four main reasons that thwart the widespread use of humic materials in agriculture, namely an insufficient number of field studies addressing the effects on humic product efficacy depending on environmental and management factors, a need for a mechanistic explanation of HS activity, a lack of quality control of humic products, and an insufficient number of long-term field trials. This is corroborated by Yang et al. [19] who reported the low interest in this class of sustainable polymers, although their unparalleled redox capacities and unexpected biological interactions could be a base for a new agricultural chemistry. Thus, it is important to improve and spread this knowledge.

In this study, to verify the method application, two composts were sampled and analyzed for HS quantification using the modified dichromate digestion method (Method 2). As can be seen in Table 2, the composts have HS content like expected for this type of soil amendments.

Table 2

Results obtained for HS quantification

Sample HS (g/L) HS (%)
Compost 1 0.202 ± 0.045 10.09 ± 1.80
Compost 2 0.144 ± 0.028 7.16 ± 1.12

The HS content was higher for compost 1 compared with compost 2. The only difference between them was the raw materials used for their production, elderberry stalk and grape stalk for compost 1 and 2, respectively. According to Silva et al. [21], it is common to obtain HS in composts between 2.7 and 6.6%. With these results, it is possible to realize the effect of the raw materials used in the compost production and its influence on the HS final content, probably related to their chemical composition, namely with the presence of refractory compounds. The processes of humification of organic matter is poorly understood, and for this reason, the application of HS in soils becomes complex, also considering its fractionation in humic and FA [21]. In the soil, the HS can be widely applied for improving availability of soil nutrients (ensuring food security), softening and opening soil texture (increasing soil quality), regulating carbon sequestration (controlling global warming), and promoting plant and microbial growth (enhancing soil micro-environment) [21].

4 Conclusion and recommendation

Applying this new methodology, it is possible to conclude that the reduction of the amount of sample, of hazardous reagents used and the consequent production of extremely toxic waste such as acidic chromium VI, leads to results with good precision (repeatability limit of 0.045 g/L), good accuracy (Z-score below 2), and robustness (F test acceptable). It was also verified that a satisfactory selectivity with a recovery of 140%. The perfect knowledge of the random or systematic error effect in the data achieved with laboratorial experiments, and their quantification, is a key factor for achieving reliable results. The implementation of this low-volume method follows the principles of green chemistry, namely the first that states for prevention – it is better to prevent the production of waste than to treat them after their production. With the proposed changes, the analysis costs can be reduced, the degradation of volumetric flasks exposed to high temperatures can be mitigated, about 92.5% of wastes produced and 80% of hazardous reagents can be reduced to achieve a good environmental performance.

The quantification of HS is important to select soil amendments, with high interest to increase the soil fertility. In fact, there has been an increasing effort in the production of these materials from waste, by composting, with the need to evaluate their effective characteristics, namely by the evaluation of the HS amount.

In laboratories, further work must be done to follow greener principles and corrective actions/modifications to achieve more ecological and sustainable activities.

Acknowledgments

The authors would like to thank the Research Centre in Digital Services (CISeD) and the Polytechnic of Viseu for their support.

  1. Funding information: This work is funded by National Funds through the FCT – Foundation for Science and Technology, I.P., within the scope of the project Refª UIDB/05583/2020.

  2. Conflict of interest: The authors state no conflict of interest.

  3. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

[1] Anastas PT, Warner JC. Green chemistry: theory and practice. Oxford University Press; 1998. ISBN: 0198502346, 9780198502340.Search in Google Scholar

[2] Hashem MA, Payel S, Hasan M, Momen MA, Sahariar MS. Green preservation of goatskin to deplete chloride from tannery wastewater. HighTech Innov J. 2021;2(2):99–107. 10.28991/HIJ-2021-02-02-0.Search in Google Scholar

[3] Stevenson FJ. Humus Chemistry: genesis, composition, reactions. New York: John Wiley & Sons; 1994. ISBN: 978-0-471-59474-1.Search in Google Scholar

[4] Janos P. Separation methods in the chemistry of humic substances. J Chromatogr A. 2003;983:1–18. 10.1016/s0021-9673(02)01687-4.Search in Google Scholar

[5] Malcolm RL. The uniqueness of humic substances in each of soil, stream and marine environments. Anal Chim Acta. 1990;232:19–30. 10.1016/S0003-2670(00)81222-2.Search in Google Scholar

[6] Maccarthy P. The Principles of Humic Substances. Soil Sci. 2001;166:738–51. 10.1097/00010694-200111000-00003.Search in Google Scholar

[7] Tombácz E, Meleg E. A theoretical explanation of the aggregation of humic substances as a function of pH and electrolyte concentration. Org Geochem, Kidlington. 1990;15:375–81. 10.1016/0146-6380(90)90164-U.Search in Google Scholar

[8] Jodice R, Barberis R, Conciglis B, Ferrara R, Nappi P, Vicentino E. Método analitici: Fertilizanti organici, compost, fhangi di depurazione, riffute organici, sustrati in fermantazione metanica & biogás. Instituto per el Piante da legno e l’ Ambiente, Torino; 1984.Search in Google Scholar

[9] Miller JN, Miller JC. Statistics and Chemometrics for Analytical Chemistry. England: Pearson Education Limited; 2010. ISBN: 978-0-273-73042-2.Search in Google Scholar

[10] Leite F. Validação em análise química. Editora Átomo; 1996. ISBN: 978-85-7670-077-7.Search in Google Scholar

[11] Welac & Eurachem. Accreditation for Chemical Laboratories: Guidance on the interpretation of the EN 45000 series of standards and ISO Guide 25. WELAC Guidance Document n. 2 & EURACHEM Guidance Document n. 1, Teddington; 1993.Search in Google Scholar

[12] Rao TN. Validation of analytical methods. Intech Open Book Ser; 2017. 10.5772/intechopen.72087.Search in Google Scholar

[13] Eurachem. The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics. Eurachem; 2014. https://www.eurachem.org/index.php/publications/guides/mv.Search in Google Scholar

[14] Real Decreto 1110/1991. de 12 de jutia, por e/quese \ aprueban los métodos oficiala. de· análisis de productos orgánicos fertilizantes. Bolétin Oficiale del Estado. 170, de 17 de julio de; 1991. p. 23725–30.Search in Google Scholar

[15] Associação de Laboratórios Acreditados de Portugal. Guia RELACRE 13 – Validação de Métodos Internos de Ensaio em Análise Química; 2000. ISBN: 972-8574-02-9. http://www.relacre.pt/assets/relacreassets/files/commissionsandpublications/Guia%20RELACRE%2013.pdf.Search in Google Scholar

[16] Portuguese Accreditation Institute. Guia para a Aplicação da NP EN ISO/IEC 17025; 2018. http://www.ipac.pt/docs/publicdocs/requisitos/OGC001_GuiaAplicacao17025_v20181231.pdf.Search in Google Scholar

[17] EPA. Green Chemistry. US EPA; 2013. https://www.epa.gov/greenchemistry.Search in Google Scholar

[18] Shamia IS, Halabi MN, El-Ashgar NM. Humic acid determination in some compost and fertilizer samples. IUG J Nat Stud. 2017;252:42–50. ISSN 2409-4587.Search in Google Scholar

[19] Yang F, Tang C, Antonietti M. Natural and artificial humic substances to manage minerals, ions, water, and soil microorganisms. Chem Soc Rev. 2021;50:6221–39. 10.1039/D0CS01363C.Search in Google Scholar

[20] Olk DC, Dinnes DL, Scoresby JR, Callaway CR, Darlington JW. Humic products in agriculture: Potential benefits and research challenges–A review. J Soil Sediment. 2018;18:2881–91. 10.1007/s11368-018-1916-4.Search in Google Scholar

[21] Silva MEF, Lemos LT, Bastos MMSM, Nunes OC. Recovery of humic-like substances from low quality composts. Bioresour Technol. 2013;128:624–32. 10.1016/j.biortech.2012.11.013.Search in Google Scholar PubMed

Received: 2022-03-15
Revised: 2022-06-12
Accepted: 2022-06-19
Published Online: 2022-09-12

© 2022 Isabel Brás et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

  1. Regular Articles
  2. Foliar application of boron positively affects the growth, yield, and oil content of sesame (Sesamum indicum L.)
  3. Impacts of adopting specialized agricultural programs relying on “good practice” – Empirical evidence from fruit growers in Vietnam
  4. Evaluation of 11 potential trap crops for root-knot nematode (RKN) control under glasshouse conditions
  5. Technical efficiency of resource-poor maize farmers in northern Ghana
  6. Bulk density: An index for measuring critical soil compaction levels for groundnut cultivation
  7. Efficiency of the European Union farm types: Scenarios with and without the 2013 CAP measures
  8. Participatory validation and optimization of the Triple S method for sweetpotato planting material conservation in southern Ethiopia
  9. Selection of high-yield maize hybrid under different cropping systems based on stability and adaptability parameters
  10. Soil test-based phosphorus fertilizer recommendation for malting barley production on Nitisols
  11. Effects of domestication and temperature on the growth and survival of the giant freshwater prawn (Macrobrachium rosenbergii) postlarvae
  12. Influence of irrigation regime on gas exchange, growth, and oil quality of field grown, Texas (USA) olive trees
  13. Present status and prospects of value addition industry for agricultural produce – A review
  14. Competitiveness and impact of government policy on chili in Indonesia
  15. Growth of Rucola on Mars soil simulant under the influence of pig slurry and earthworms
  16. Effect of potassium fertilizer application in teff yield and nutrient uptake on Vertisols in the central highlands of Ethiopia
  17. Dissection of social interaction and community engagement of smallholder oil palm in reducing conflict using soft system methodology
  18. Farmers’ perception, awareness, and constraints of organic rice farming in Indonesia
  19. Improving the capacity of local food network through local food hubs’ development
  20. Quality evaluation of gluten-free biscuits prepared with algarrobo flour as a partial sugar replacer
  21. Effect of pre-slaughter weight on morphological composition of pig carcasses
  22. Study of the impact of increasing the highest retail price of subsidized fertilizer on rice production in Indonesia
  23. Agrobiodiversity and perceived climatic change effect on family farming systems in semiarid tropics of Kenya
  24. Influences of inter- and intra-row spacing on the growth and head yield of cabbage (Brassica oleracea var. capitata) in western Amhara, Ethiopia
  25. The supply chain and its development concept of fresh mulberry fruit in Thailand: Observations in Nan Province, the largest production area
  26. Toward achieving sustainable development agenda: Nexus between agriculture, trade openness, and oil rents in Nigeria
  27. Phenotyping cowpea accessions at the seedling stage for drought tolerance in controlled environments
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  29. Influence of season and rangeland-type on serum biochemistry of indigenous Zulu sheep
  30. Meta-analysis of responses of broiler chickens to Bacillus supplementation: Intestinal histomorphometry and blood immunoglobulin
  31. Weed composition and maize yield in a former tin-mining area: A case study in Malim Nawar, Malaysia
  32. Strategies for overcoming farmers’ lives in volcano-prone areas: A case study in Mount Semeru, Indonesia
  33. Principal component and cluster analyses based characterization of maize fields in southern central Rift Valley of Ethiopia
  34. Profitability and financial performance of European Union farms: An analysis at both regional and national levels
  35. Analysis of trends and variability of climatic parameters in Teff growing belts of Ethiopia
  36. Farmers’ food security in the volcanic area: A case in Mount Merapi, Indonesia
  37. Strategy to improve the sustainability of “porang” (Amorphophallus muelleri Blume) farming in support of the triple export movement policy in Indonesia
  38. Agrarian contracts, relations between agents, and perception on energy crops in the sugarcane supply chain: The Peruvian case
  39. Factors influencing the adoption of conservation agriculture by smallholder farmers in KwaZulu-Natal, South Africa
  40. Meta-analysis of zinc feed additive on enhancement of semen quality, fertility and hatchability performance in breeder chickens
  41. Meta-analysis of the potential of dietary Bacillus spp. in improving growth performance traits in broiler chickens
  42. Biocomposites from agricultural wastes and mycelia of a local mushroom, Lentinus squarrosulus (Mont.) Singer
  43. Cross transferability of barley nuclear SSRs to pearl millet genome provides new molecular tools for genetic analyses and marker assisted selection
  44. Detection of encapsulant addition in butterfly-pea (Clitoria ternatea L.) extract powder using visible–near-infrared spectroscopy and chemometrics analysis
  45. The willingness of farmers to preserve sustainable food agricultural land in Yogyakarta, Indonesia
  46. Transparent conductive far-infrared radiative film based on polyvinyl alcohol with carbon fiber apply in agriculture greenhouse
  47. Grain yield stability of black soybean lines across three agroecosystems in West Java, Indonesia
  48. Forms of land access in the sugarcane agroindustry: A comparison of Brazilian and Peruvian cases
  49. Assessment of the factors contributing to the lack of agricultural mechanization in Jiroft, Iran
  50. Do poor farmers have entrepreneurship skill, intention, and competence? Lessons from transmigration program in rural Gorontalo Province, Indonesia
  51. Communication networks used by smallholder livestock farmers during disease outbreaks: Case study in the Free State, South Africa
  52. Sustainability of Arabica coffee business in West Java, Indonesia: A multidimensional scaling approach
  53. Farmers’ perspectives on the adoption of smart farming technology to support food farming in Aceh Province, Indonesia
  54. Rice yield grown in different fertilizer combination and planting methods: Case study in Buru Island, Indonesia
  55. Paclobutrazol and benzylaminopurine improve potato yield grown under high temperatures in lowland and medium land
  56. Agricultural sciences publication activity in Russia and the impact of the national project “Science.” A bibliometric analysis
  57. Storage conditions and postharvest practices lead to aflatoxin contamination in maize in two counties (Makueni and Baringo) in Kenya
  58. Relationship of potato yield and factors of influence on the background of herbological protection
  59. Biology and life cycle Of Diatraea busckella (Lepidoptera: Crambidae) under simulated altitudinal profile in controlled conditions
  60. Evaluation of combustion characteristics performances and emissions of a diesel engine using diesel and biodiesel fuel blends containing graphene oxide nanoparticles
  61. Effect of various varieties and dosage of potassium fertilizer on growth, yield, and quality of red chili (Capsicum annuum L.)
  62. Review Articles
  63. Germination ecology of three Asteraceae annuals Arctotis hirsuta, Oncosiphon suffruticosum, and Cotula duckittiae in the winter-rainfall region of South Africa: A review
  64. Animal waste antibiotic residues and resistance genes: A review
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  66. The evolving state of food security in Nigeria amidst the COVID-19 pandemic – A review
  67. Short Communication
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  69. Special Issue on the International Conference on Multidisciplinary Research – Agrarian Sciences
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  72. Influence of different parameters on the characteristics of hazelnut (var. Grada de Viseu) grown in Portugal
  73. Organic food consumption and eating habit in Morocco, Algeria, and Tunisia during the COVID-19 pandemic lockdown
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  80. Behavioral response of breeder toward development program of Ongole crossbred cattle in Yogyakarta Special Region, Indonesia
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  82. Perceived attributes driving the adoption of system of rice intensification: The Indonesian farmers’ view
  83. Value-added analysis of Lactobacillus acidophilus cell encapsulation using Eucheuma cottonii by freeze-drying and spray-drying
  84. Investigating the elicited emotion of single-origin chocolate towards sustainable chocolate production in Indonesia
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https://doi.org/10.1515/opag-2022-0120
Keywords for this article
green chemistry; humic substances; spectroscopy; validation of analytical methods
Creative Commons
BY 4.0

Articles in the same Issue

  1. Regular Articles
  2. Foliar application of boron positively affects the growth, yield, and oil content of sesame (Sesamum indicum L.)
  3. Impacts of adopting specialized agricultural programs relying on “good practice” – Empirical evidence from fruit growers in Vietnam
  4. Evaluation of 11 potential trap crops for root-knot nematode (RKN) control under glasshouse conditions
  5. Technical efficiency of resource-poor maize farmers in northern Ghana
  6. Bulk density: An index for measuring critical soil compaction levels for groundnut cultivation
  7. Efficiency of the European Union farm types: Scenarios with and without the 2013 CAP measures
  8. Participatory validation and optimization of the Triple S method for sweetpotato planting material conservation in southern Ethiopia
  9. Selection of high-yield maize hybrid under different cropping systems based on stability and adaptability parameters
  10. Soil test-based phosphorus fertilizer recommendation for malting barley production on Nitisols
  11. Effects of domestication and temperature on the growth and survival of the giant freshwater prawn (Macrobrachium rosenbergii) postlarvae
  12. Influence of irrigation regime on gas exchange, growth, and oil quality of field grown, Texas (USA) olive trees
  13. Present status and prospects of value addition industry for agricultural produce – A review
  14. Competitiveness and impact of government policy on chili in Indonesia
  15. Growth of Rucola on Mars soil simulant under the influence of pig slurry and earthworms
  16. Effect of potassium fertilizer application in teff yield and nutrient uptake on Vertisols in the central highlands of Ethiopia
  17. Dissection of social interaction and community engagement of smallholder oil palm in reducing conflict using soft system methodology
  18. Farmers’ perception, awareness, and constraints of organic rice farming in Indonesia
  19. Improving the capacity of local food network through local food hubs’ development
  20. Quality evaluation of gluten-free biscuits prepared with algarrobo flour as a partial sugar replacer
  21. Effect of pre-slaughter weight on morphological composition of pig carcasses
  22. Study of the impact of increasing the highest retail price of subsidized fertilizer on rice production in Indonesia
  23. Agrobiodiversity and perceived climatic change effect on family farming systems in semiarid tropics of Kenya
  24. Influences of inter- and intra-row spacing on the growth and head yield of cabbage (Brassica oleracea var. capitata) in western Amhara, Ethiopia
  25. The supply chain and its development concept of fresh mulberry fruit in Thailand: Observations in Nan Province, the largest production area
  26. Toward achieving sustainable development agenda: Nexus between agriculture, trade openness, and oil rents in Nigeria
  27. Phenotyping cowpea accessions at the seedling stage for drought tolerance in controlled environments
  28. Apparent nutrient utilization and metabolic growth rate of Nile tilapia, Oreochromis niloticus, cultured in recirculating aquaculture and biofloc systems
  29. Influence of season and rangeland-type on serum biochemistry of indigenous Zulu sheep
  30. Meta-analysis of responses of broiler chickens to Bacillus supplementation: Intestinal histomorphometry and blood immunoglobulin
  31. Weed composition and maize yield in a former tin-mining area: A case study in Malim Nawar, Malaysia
  32. Strategies for overcoming farmers’ lives in volcano-prone areas: A case study in Mount Semeru, Indonesia
  33. Principal component and cluster analyses based characterization of maize fields in southern central Rift Valley of Ethiopia
  34. Profitability and financial performance of European Union farms: An analysis at both regional and national levels
  35. Analysis of trends and variability of climatic parameters in Teff growing belts of Ethiopia
  36. Farmers’ food security in the volcanic area: A case in Mount Merapi, Indonesia
  37. Strategy to improve the sustainability of “porang” (Amorphophallus muelleri Blume) farming in support of the triple export movement policy in Indonesia
  38. Agrarian contracts, relations between agents, and perception on energy crops in the sugarcane supply chain: The Peruvian case
  39. Factors influencing the adoption of conservation agriculture by smallholder farmers in KwaZulu-Natal, South Africa
  40. Meta-analysis of zinc feed additive on enhancement of semen quality, fertility and hatchability performance in breeder chickens
  41. Meta-analysis of the potential of dietary Bacillus spp. in improving growth performance traits in broiler chickens
  42. Biocomposites from agricultural wastes and mycelia of a local mushroom, Lentinus squarrosulus (Mont.) Singer
  43. Cross transferability of barley nuclear SSRs to pearl millet genome provides new molecular tools for genetic analyses and marker assisted selection
  44. Detection of encapsulant addition in butterfly-pea (Clitoria ternatea L.) extract powder using visible–near-infrared spectroscopy and chemometrics analysis
  45. The willingness of farmers to preserve sustainable food agricultural land in Yogyakarta, Indonesia
  46. Transparent conductive far-infrared radiative film based on polyvinyl alcohol with carbon fiber apply in agriculture greenhouse
  47. Grain yield stability of black soybean lines across three agroecosystems in West Java, Indonesia
  48. Forms of land access in the sugarcane agroindustry: A comparison of Brazilian and Peruvian cases
  49. Assessment of the factors contributing to the lack of agricultural mechanization in Jiroft, Iran
  50. Do poor farmers have entrepreneurship skill, intention, and competence? Lessons from transmigration program in rural Gorontalo Province, Indonesia
  51. Communication networks used by smallholder livestock farmers during disease outbreaks: Case study in the Free State, South Africa
  52. Sustainability of Arabica coffee business in West Java, Indonesia: A multidimensional scaling approach
  53. Farmers’ perspectives on the adoption of smart farming technology to support food farming in Aceh Province, Indonesia
  54. Rice yield grown in different fertilizer combination and planting methods: Case study in Buru Island, Indonesia
  55. Paclobutrazol and benzylaminopurine improve potato yield grown under high temperatures in lowland and medium land
  56. Agricultural sciences publication activity in Russia and the impact of the national project “Science.” A bibliometric analysis
  57. Storage conditions and postharvest practices lead to aflatoxin contamination in maize in two counties (Makueni and Baringo) in Kenya
  58. Relationship of potato yield and factors of influence on the background of herbological protection
  59. Biology and life cycle Of Diatraea busckella (Lepidoptera: Crambidae) under simulated altitudinal profile in controlled conditions
  60. Evaluation of combustion characteristics performances and emissions of a diesel engine using diesel and biodiesel fuel blends containing graphene oxide nanoparticles
  61. Effect of various varieties and dosage of potassium fertilizer on growth, yield, and quality of red chili (Capsicum annuum L.)
  62. Review Articles
  63. Germination ecology of three Asteraceae annuals Arctotis hirsuta, Oncosiphon suffruticosum, and Cotula duckittiae in the winter-rainfall region of South Africa: A review
  64. Animal waste antibiotic residues and resistance genes: A review
  65. A brief and comprehensive history of the development and use of feed analysis: A review
  66. The evolving state of food security in Nigeria amidst the COVID-19 pandemic – A review
  67. Short Communication
  68. Response of cannabidiol hemp (Cannabis sativa L.) varieties grown in the southeastern United States to nitrogen fertilization
  69. Special Issue on the International Conference on Multidisciplinary Research – Agrarian Sciences
  70. Special issue on the International Conference on Multidisciplinary Research – Agrarian Sciences: Message from the editor
  71. Maritime pine land use environmental impact evolution in the context of life cycle assessment
  72. Influence of different parameters on the characteristics of hazelnut (var. Grada de Viseu) grown in Portugal
  73. Organic food consumption and eating habit in Morocco, Algeria, and Tunisia during the COVID-19 pandemic lockdown
  74. Customer knowledge and behavior on the use of food refrigerated display cabinets: A Portuguese case
  75. Perceptions and knowledge regarding quality and safety of plastic materials used for food packaging
  76. Understanding the role of media and food labels to disseminate food related information in Lebanon
  77. Liquefaction and chemical composition of walnut shells
  78. Validation of an analytical methodology to determine humic substances using low-volume toxic reagents
  79. Special Issue on the International Conference on Agribusiness and Rural Development – IConARD 2020
  80. Behavioral response of breeder toward development program of Ongole crossbred cattle in Yogyakarta Special Region, Indonesia
  81. Special Issue on the 2nd ICSARD 2020
  82. Perceived attributes driving the adoption of system of rice intensification: The Indonesian farmers’ view
  83. Value-added analysis of Lactobacillus acidophilus cell encapsulation using Eucheuma cottonii by freeze-drying and spray-drying
  84. Investigating the elicited emotion of single-origin chocolate towards sustainable chocolate production in Indonesia
  85. Temperature and duration of vernalization effect on the vegetative growth of garlic (Allium sativum L.) clones in Indonesia
  86. Special Issue on Agriculture, Climate Change, Information Technology, Food and Animal (ACIFAS 2020)
  87. Prediction model for agro-tourism development using adaptive neuro-fuzzy inference system method
  88. Special Issue of International Web Conference on Food Choice and Eating Motivation
  89. Can ingredients and information interventions affect the hedonic level and (emo-sensory) perceptions of the milk chocolate and cocoa drink’s consumers?
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