Analysis Method of Heavy Metals in Organic Fertilizers

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1		Yujun Shen	--	3453	2022-12-24 04:15:47	\|
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This entry is adapted from the peer-reviewed paper 10.3390/su142416789

Heavy metals in organic fertilizers pose a risk to the agricultural ecosystem. The environmental risk of heavy metals depends not only on the total amount but also on the speciation. Hence, more information on heavy metals speciation in organic fertilizers is needed to avoid adverse effect. The speciation information of heavy metals is usually obtained by the single-extraction method and sequential extraction method. Common heavy metals that have received attention include Cu, Zn, Pb, Cd, Cr, Hg and As.

sequence extraction procedure single-extraction method speciation Tessier

1. Introduction

With the improvement of people's life level, the development of modern agriculture and large-scale farming has become the major trend. As a result, the production of agricultural wastes and livestock and poultry manure has rapidly increased as well. These wastes can be treated by making biomass fuel ^[1] or composting. In China, the total amount of livestock and poultry manure was stable at 3.7 billion tons in 2010–2020 ^[2]. To inhibit harmful pathogenic bacteria and to promote growth, metal element additives, including Cu, Zn and As, were often added to feed. But the absorption rate of these elements in livestock and poultry is very low, and more than 95% of heavy metals will be excreted ^[3]. In the composting process, the organic matter of livestock manure is mineralized and humified, and some of the material is volatilized and lost. So, the concentration of heavy metals often doses be subject to a “relative concentration effect” in composting process, characterized by an increase in the concentration of heavy metals ^[4]. When this livestock manure is applied to the agricultural soil, the heavy metals will migrate and transform into the soil–plant system and will do harm to human health throughout the food chain ^[5]. The bioavailability of heavy metals in organic fertilizers is not only dependent on the total amount of heavy metals but also on their speciation. Thus, the method of obtaining concentrations of heavy metals in various speciation is very significant.

It is generally recognized that the harm of heavy metals depends not only on the total amount of heavy metals but also on their specific chemical speciation and binding states (coprecipitation with minerals, complexation with organic ligands, etc.) ^[6]^[7]^[8]. The significance of speciation determination is to understand the state of heavy metals in the solid environment and to evaluate the environmental risk according to the different proportions of different speciation. Therefore, obtaining the distribution of various speciation of heavy metals is of great significance for understanding the behavior of heavy metals and environmental risk assessment.

2. Heavy Metals in Manure and Organic Fertilizer

Heavy metals in organic fertilizer from livestock and poultry manure is mainly derived from additives. The common heavy metals include Cu, Zn, Pb, Cd, Cr, Hg and As.

Cu, Zn and Cr are the essential trace elements for animals that participate in metabolism and promote the growth and development of livestock and poultry. Cu has low price and high efficiency. Zn can improve the immunity of the body to prevent diarrhea ^[9]. Cr strengthens the function of insulin ^[10]; moreover, Cr can improve the efficiency of feed utilization and save costs. As is an essential trace element for livestock and poultry as well as a toxic element. A right amount of As has a good antibacterial effect, which can significantly enhance disease resistance and speed up metabolism to promote the growth of livestock and poultry. Meanwhile, excessive As can interfere with the normal metabolism of cells, affect the process of respiration and cause cell pathological changes. Pb and Cd are often accompanied in the feed due to different feed additive processes, which are toxic to animals. Excessive levels of Pb and Cd can affect the animals’ nervous systems, inhibit hemoglobin synthesis and impair immune function.

3. Speciation of Heavy Metals in Organic Fertilizers

The total amount of heavy metals in livestock and poultry manure is insufficient to assess their environmental risk because their bioavailability was mainly decided by their speciation in organic fertilizers. There has not been a uniform definition and classification for heavy metals speciation in organic fertilizers so far. It has been pointed out that heavy metals speciation can be classified into valence, chemistry, bound state and structure, according to different perspectives ^[11]. At present, the commonly used chemical extraction procedure is to classify the heavy metals speciation from the perspective of the bound. According to different experimental purpose and operation processes, the extraction procedures of heavy metals speciation in organic fertilizers are classified into two types: single-stage extraction and sequence extraction procedure (SEP).

The single-stage extraction was to obtain the target heavy metals speciation which was get by only one extractant. Organic fertilizers have similar properties to soil, so extraction methods directly from the soil or with slight modifications were always adopted by researchers ^[12]^[13]^[14]^[15]. Extraction agents were dilute acids; natural or synthetic complexing agents, including dilute hydrochloric acid solution; Calcium chloride solution; Diethylenetriaminepentaacetic acid–Triethanolamine (DTPA–TEA) solution, etc.

The heavy metals speciation was distinguished by successive extraction of a series of extractant, is called for the SEP, which of soil heavy metals have been used to extract various speciation of heavy metals in organic fertilizer as well. Tessier’s SEP classified soil heavy metals into five speciation, including exchangeable, bound to carbonates, bound to Fe–Mn oxides, bound to organic matter and residual speciation. The classification was based on different binding modes of heavy metals in soil components ^[16]. The exchangeable fraction of heavy metals was generally considered to be bioavailability; moreover, the fraction of the carbonate-bound, Fe–Mn oxide-bound and organic-bound were potentially bioavailability, while the residue fraction was hard for absorption by plant. This sequence extraction process mimicked the environmental conditions that may exist in the sediment: the exchangeable extraction process simulated ion exchange conditions; the carbonate-bound extraction process simulated acidic conditions; the Fe–Mn oxide-bound extraction process simulated reducing conditions; the organic-bound extraction process simulated oxidizing conditions; and the residue extraction process was the speciation not easily released into the environment under all conditions.

The ”European Community Bureau of Reference Materials” proposed a three-step BCR extraction method ^[17], which divided heavy metals of soils into three speciation: acid-extractable; reducible; and oxidizable speciation. Compared with Tessier’s method, the BCR method is easier to operate, and speciation is more stable, meanwhile the information obtained on the speciation of heavy metals is far less. To check the efficiency of the extraction process and to make self-examination easy, Ure et al. proposed a four-step BCR method, adding the extraction of residue ^[18]. In addition, Amacher classified heavy metals speciation into five speciation: exchangeable; bound to carbonate; bound to oxide; bound to organic; and residue ^[19]. Emmerich classified heavy metals speciation into five speciation: exchangeable; adsorbed; bound to organic; bound to carbonate; and residue speciation ^[20].

4. Extraction Procedure of Heavy Metals in Organic Fertilizers

4.1. Single-Extraction Method of Heavy Metals Speciation in Organic Fertilizers

The single-stage extraction method dissolved a specific speciation directly by an extractant only and was usually used for extracting some specific speciation, such as the active, migrated, bioavailable and plant-available speciation. It is a simple, time-saving and effective way to determine the level of heavy metals contamination in organic fertilizers visually. There were a wide variety of extraction agents, including deionized water, acid, neutral salt, chelating agent ^[21].

(1)Migration speciation ^[22]^[23]

Migration speciation or the soluble complexed speciation presents one of the most migratory speciation of heavy metal. The method used for extract is adding deionized water to the sample and shaking for 16 h.

(2)Acid leachable speciation ^[24]

Acid leachable speciation presents almost the whole fraction of elements that bind to materials by simply absorption. The method used for extract is adding 10 mL 0.5 mol/L HCl into solution. The dry sample is 1g and shaking time is 16 h.

(3)Effective speciation ^[25]

Effective speciation presents one of the most migratory speciation of heavy metal and is the most easily absorbed by plants. The extract agent is Diethylenetriaminepentaacetic acid (DTPA). DTPA is a chelating agent which can form water-soluble complexes with metal ions. For example, the Cu and Zn content in DTPA’s extracts were relevant with the heavy metals in plant roots.

(4)Plant available speciation ^[26]^[27]

Plant available speciation presents the fraction of heavy metals that can be absorbed by plant roots. The method used for extract is adding 0.05 mol/L Ethylenediaminetetraacetic acid (EDTA) to the sample and shaking for 1 h or adding mixed acid, which simulates plant root exudates, to the sample and shaking for 16 h. EDTA could extract heavy metals by dissolution, complexation and adsorption effect. It could form stable water-soluble complexes with heavy metals and is less aggressive in silicates compared with hydrochloric acid. Acetic acid, lactic acid, malic acid and formic acid, which are low molecular organic acids, were dominate in plant root exudates. The application of this acid mixture could mimic the environment in which heavy metals are absorbed by plants and extracted by acidification, chelation and redox reactions. The extraction method using this acid mixture is called rhizosphere-based extraction (REM) and is more realistic than the other method of extracting plant-available speciation of heavy metals.

(5)Bound to humic acid speciation ^[28]

Bound to humic acid speciation presents the complexes of heavy metals with humic acid. Humic acid, which is soluble in alkaline conditions only, could form water-soluble sodium humate with sodium pyrophosphate. Thus, the methods used for extract is adding 0.1 mol/L Na₄P₂O₇ and 0.1 mol/L NaOH to sample and shaking for 24 h.

(6)Leachable speciation ^[27]

Leachable speciation is used to evaluate the leachability of heavy metals. The leachable process of acetic acid extraction was used to simulate the leaching of metals and their coagulation process in sludge samples, also known as toxicity characteristic leaching procedure (TCLP).

(7)Bioavailable speciation ^[27]

Bioavailable speciation is the fraction of heavy metals that can be absorbed by animals. The extraction is adding 0.4 mol/L glycine (pH adjusted to 1.5 by HCl) and shaking for 1 h. Glycine (hydrochloric acid adjusted pH = 1.5) could be made into a synthetic gastric solution for the extraction of bioaccessible heavy metals. This method is also known as the simplified bioaccessibility extraction test (SEBT).

4.2. SEP of Heavy Metals Speciations in Organic Fertilizers

The Sequence extraction procedure is a method to obtain heavy metals speciation information under different extraction conditions, according to the difficulty level from weak to strong in the extraction process by different extractant ^[16]. This method is the mainstream method for heavy metals speciation analysis in soil, sediment and organic fertilizer at this stage.

Tessier’s method ^[16] was proposed by Tessier in 1979 and is the most widely accepted method for the extraction of heavy metals speciation. The method was described as follows: the quantities indicated below refer to 1-g samples (dry weight of the original sample used for the initial extraction), ① Exchangeable. 8 mL, 1 mol/L MgCl₂ (pH = 7) was added and shaken for 1 h at 25 °C; ② Bound to carbonate. 8 mL, 1 mol/L CH₃COONa was shaken for 5 h at pH = 5 (CH₃COOH adjustment) and temperature of 25 °C; ③ Bound to Fe-Mn oxide. 20 mL 0.04 mol/L NH₂OH-HCl (dissolved in 25% CH₃COOH) was intermittently shaken at 95 °C for 6 h. ④ Bound to Organic. Extraction was performed in three steps, first with 3 mL of 0.02 mol/L HNO₃ and 5 mL of 30% H₂O₂ at pH = 2 (adjusted by HNO₃) and temperature of 85 °C for 2 h intermittent shaking; then with 3 mL of 30% H₂O₂ at pH = 2 (adjusted by HNO₃) and temperature of 85 °C for 3 h intermittent shaking; after cooling, finally with 5 mL of 3.2 mol/L CH₃COONH₄ (dissolved in 20% HNO₃), diluted to 20 mL and shaken for 0.5 h at 25 °C; ⑤ Residue. HF-HClO₄ was digested until clarified and clear. After each extraction step, the supernatant by centrifugation was stored for measurement, and the precipitate was washed with 8 mL of deionized water before proceeding to the next step. MgCl₂, CH₃COONa and CH₃COONH₄ released the metal elements mainly by ion exchange, NH₂OH–HCl released the heavy metals adsorbed on the surface and co-precipitated with iron and manganese oxides by reducing action. While H₂O₂, HNO₃, HF and HClO₄ released metal by destroying the soil matrix ^[21]. The Tessier procedure provided an important idea for the extraction of heavy metals speciation and a template for subsequent scholars to improve the method.

To further enhance the accuracy of the BCR extraction process, A. Sahuquillo et al. ^[17] proposed an improved BCR method by changing the NH₂OH–HCl concentration from 0.10 M to 0.50 M and reducing the NH₂OH–HCl solution pH from 2 to 1.5. Meanwhile, the pH adjustment reagent was recommended to use HNO₃ instead of HCl because chlorine could speciation dissolved complexes with heavy metals. The relative standard deviation of the results was reduced and the accuracy was greatly improved. This was because heavy metals are easier to be soluble at pH 1.5, and the low pH (1.5) improved the buffering capacity of the extractant (the pH of the solution varied before and after extraction). Regarding the solid-liquid separation method, the filter paper was added into a centrifuge tube in the next extraction step to avoid the loss of solid in the filtration process. However, it increased the concentration of the extract in step 3 significantly. So, filtration was recommended to separate the solid and liquid phases, while the centrifugation speed was increased from 1500× g to 3000× g. This method changed Tessier’s five-extraction procedure to a three-step extraction procedure that was easier and simpler to handle than the Tessier method.

Exchangeable speciation is the combination of heavy metal and particulate matter through electrostatic adsorption. Bound to carbonate speciation refers to heavy metals binding with carbonate minerals, which is easily destroyed in acidic environment under the influence of pH values. Bound to Iron-manganese oxide speciation refers to the binding heavy metals with clay minerals such as iron-manganese oxides, which is easy to be destroyed under reduction conditions. Bound to organic speciation refers to the combination of heavy metals and organic matter, which is easy to be destroyed under oxidation conditions. Residual state refers to the remaining forms of heavy metals after the removal of the above four forms, which generally exist in the mineral lattice.

5. Factors Affecting the Speciation of Heavy Metals in Organic Fertilizers

5.1. Property of Organic Fertilizer Affecting Heavy Metals Speciation Distribution

Organic matter is an important factor affecting the distribution of heavy metals speciation. Organic matter comes in various speciation, such as dissolved organic matter (DOM), granular organic matter (POM), humus, etc. The surface of organic matter contained abundant functional groups with which heavy metals can react by adsorption and complexation to organic speciation bound. Soluble organic matter will enhance the mobility of heavy metals, while non-soluble organic matter will reduce the mobility of heavy metals. Meanwhile, the accumulation of organic matter during the formation of organic fertilizer can promote the conversion of heavy metals from the exchangeable to the organic-bound speciation ^[29], which reduces the biological effects of heavy metals. At the same time, these strong binding processes lead to a significant increase of heavy metals in the organic binding state when the content of organic matter is high. Also, it may affect the integrity of the organic binding state extraction.

5.2. Comparison of Heavy Metals Speciation between Organic Fertilizers and Soils

Comparing the total amount of heavy metals in soil and organic fertilizer, it can be found that the content of heavy metals, especially Cu and Zn, in organic fertilizer was much higher than that in soil. The total amount of Zn in organic fertilizer was about 30–550 times (maximum and minimum values) higher than that in soil. The total amount of Cu in organic fertilizer was about 5.8–192 times higher than that in soil. The contents of Cd, Cr, Pb and Ni were about 13.38, 11.99, 8.31 and 20.53 times higher than that in soil, respectively. As and Hg content were close between organic fertilizer and soil.

According to the definition of heavy metals speciation by Tessier and BCR method, the acid-soluble of the BCR can be regarded as the sum of the exchangeable and carbonate bound of the Tessier. The reducible of the BCR can be regarded as iron–manganese oxide bound of the Tessier, and the oxidized of the BCR can be regarded as organic bound of the Tessier.

For soil, the proportions of all heavy metals in the residue were much higher, with an absolutely high percentage of more than 85% of residue in total, in nearly half the number of the literature; the Fe–Mn oxide-bound of Zn, Cr, Pb and Ni accounted for a relatively high proportion, about 25% of Fe–Mn oxide-bound in total.

The heavy metals speciation in organic fertilizers were mainly residue and organically bound, and the residue was no longer in a dominant position compared to the soil. Only 9% of statistical literature showed the residue had a certain proportion of more than 85% in each total heavy metal, and the organic-bound accounted for about 28.5% in each. It also could be seen that organic matter content in organic fertilizers was much higher than that in soil, and it was undeniable that the organic-bound had a far higher amount of binding point with heavy metals in, so the organic-bound had a larger proportion of heavy metals accordingly.

Metal re-adsorption and redistribution influenced the distribution of heavy metals speciation. The re-distribution was a natural phenomenon of adsorption and desorption equilibrium, in which the heavy metal ions released were re-adsorbed by the particles. Heavy metal ions released into the liquid can be retained by added nitrilotriacetic acid (NTA) to chelate the heavy metal ion, which can effectively avoid the re-adsorption phenomenon ^[30]. In addition, pretreatment process can also affect the distribution of heavy metals speciation. Fresh preservation, natural air-drying and freeze-drying were three typical pretreatment methods, and they had significant effects on the speciation of Cr, Pb and As in riverine and marine sediments. The speciation, in descending order of affected level, were exchangeable and carbonate-bounds, Fe-Mn oxides, organic-bounds and the residual ^[31]. Fresh samples tended to cause uneven sample mixing and large weighing errors because of the presence of water, so it was not suitable for test. The freeze drying process was done at low temperature and low pressure. It could exclude the influence of water, microorganisms and dust on the samples, compared with natural air drying. But the requirements of the freeze-drying process were strict, so it was not suitable for the treatment of a large number of samples.

6. Conclusions

The extraction procedures of heavy metals speciation in organic fertilizers were classified into two types: single-stage extraction and SEP. There were three main types of heavy metals speciation extraction methods in organic fertilizers: one was the Tessier method and modified Tessier method; the second was the BCR method and modified BCR method; and the third was the other methods. As the different composition between soil and organic fertilizer, the improvements of the modified method of the speciation of heavy metals in organic fertilizer was taken, included reducing the solid-liquid ratio, changing the type and concentration of extractant, pH, extraction time, centrifugation time, centrifugation speed, solid cleaning procedure, etc. In short, the extractant concentration was reduced and/or the extraction time was extended for the exchangeable state. The extraction time was shortened for the carbonate bound state. The extractant concentration was increased or the extraction temperature was decreased for the Fe/Mn oxide bound state, and the extractant concentration was decreased for the organic bound state. Due to the lack of a complete evaluation system, although BCR method has a certain reference material, which is far from enough, it is difficult to compare the advantages and disadvantages of different methods at present.

According to the result of SEP, the heavy metals speciation in organic fertilizers were mainly residue and organically bound, and the residue was no longer in a dominant position compared to the soil. The change probably stems from the differences in the distribution of heavy metals speciation between soils and organic fertilizers mainly due to the difference in organic matter, minerals and pH value. The pH value was low in soil and fluctuated widely, while pH value was high in organic fertilizer. The acidic environment was prone to the activation of heavy metals, but the effect of pH appears weaker compared to the effect of dissolved organic matter on heavy metals in organic fertilizer. More organic matter will greatly increase the content of heavy metals in the organic bound state, so it may affect the completeness of the organic bound state extraction. Organic matter included humus and non-humic organic matter. The dissolved humus was easy to combine with heavy metal ions to make heavy metal ions easy to extract, thus causing heavy metal activation. But the insoluble heavy metals would make heavy metal ions passivated. Furthermore, the content of heavy metals, especially Cu and Zn, in organic fertilizer was much higher than that in soil. With the characteristics of less binding mineral content and more organic matter content, even some metals with high mineral affinity will increase the content of organic binding state.

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