Copper Distribution and Binding Affinity of Size-Fractioned Humic Substances Taken from Paddy Soil and Correlation with Optical Characteristics

Round 1

Reviewer 1 Report

Discussion of the effect of size of humic substances for the binding affinity is quite interesting.

I have few minor comments below.

1.  In line104, you should describe the detail of the measurement method of total Cu concentration, or add reference.  Did you measure the diluted aqua regia by ICP-MS? I could not understand how did you determine the Cu concentration.

2. In line 202, the calculated value of AEOM-OC content/TOC also have error, since the AEOM concentration and TOC has error.

3. In line 246, author said Cu and OC had a similar trend, however, there is a difference from my point. The percentage of M-B in Cu is quite lager than that of M-E, while percentages in OC is similar between M-B to M-E.  This tendency is clearly showed even in Figure 2a-2b. If author think they are similar, additional explanation may be required. 

Author Response

Reviewer #1

Comments and Suggestions for Authors

Discussion of the effect of size of humic substances for the binding affinity is quite interesting. I have few minor comments below.

Comment #1-1:

In line104, you should describe the detail of the measurement method of total Cu concentration, or add reference. Did you measure the diluted aqua regia by ICP-MS? I could not understand how did you determine the Cu concentration.

Response #1-1:

Thank you for the comment and suggestion.

The Cu concentrations of aqua regia digestion solution were determined by flame atomic absorption spectrometry (Hitachi, Z-2300).

Line 122 has been modified to "The total Cu concentrations were analyzed with an aqua regia digestion method and measured by flame atomic absorption spectrometry (Hitachi, Z-2300). Briefly, three g of soil was added to a 30 mL aqua regia solution; after two h digestion at 180 °C and 16 h settling, the solution was measured at 100 mL to determine Cu concentrations."

Comment #1-2:

In line 202, the calculated value of AEOM-OC content/TOC also have error, since the AEOM concentration and TOC has error.

Response #1-2:

Thank you for the comment.

In Hsieh et al. (2019) study, they used alkaline solution (NaOH) to extract soil organic matter (AEOM). The measured AEOM solution DOC concentration was 0.68±0.25 g/kg and total soil organic carbon (TOC) was 18.1±1.9 g/kg based on soil mass. Therefore, the AEOM-OC content/TOC (0.68/18.1) ratio was 3.70%.

Line 221, the revised manuscript for the AEOM DOC concentration has been modified. "Hsieh et al. [9] reported the average AEOM DOC concentration was 0.68±0.25 g kg-1 and the TOC concentration was 18.1±1.9 g kg-1 based on soil mass. Therefore, the AEOM-OC content/TOC ratio (0.68/18.1) was 3.70%."

Comment #1-3

In line 246, author said Cu and OC had a similar trend, however, there is a difference from my point. The percentage of M-B in Cu is quite lager than that of M-E, while percentages in OC is similar between M-B to M-E. This tendency is clearly showed even in Figure 2a-2b. If author think they are similar, additional explanation may be required. 

Response #1-3:

Thank you for the comment and suggestion.

In the original manuscript, we mentioned that "Cu and OC had a similar trend for the Cu and OC mass distribution in the fractioned AEOM solution." We pointed out that the highest and second-order high percentages were M-B and M-E fractions for both Cu and OC. However, we agree with the reviewer's comment that the percentage of M-B (66.7%) in Cu is relatively larger than that of M-E (14.1%), while percentages in OC are similar between M-B (40.3%) to M-E (38.4%). We recognize the description in the original manuscript may cause misunderstanding.

Line 267 has been changed to "Cu had a much higher percentage in the M-B fraction than other fractions, but OC had a higher percentage for M-B and M-E fractions than the other fractions."

Author Response File: Author Response.pdf

Reviewer 2 Report

The significance of this study and scientific hypothesis as well as its creativity are not clearly demonstrated in the introduction section. The authors should provide a clearly description of the defects in the existing researches and how this study deal with these problem.

 There are few explanations of the rationale for the sampling design. it is suggestion that an explanation of why the soil samples were taken from ten paddy field should be provided.

The Results and Discussion section should be rewritten. It is recommended to analyze your own test results or data first, and then compare them with the literature or discuss the reason. You should ensure that the data in the tables are consistent with those cited in the relevant places in the text, and data have been calculated correctly. In the current version of the manuscript, it is difficult to clearly see what is your own experimental data. Especially in subsections 3.1, 3.2 and 3.3, the data described in the text is difficult to correspond to table 1, table 2 and table 3. For example, where are the supporting data of your description of ‘The FI values of bulk AEOM were 1.32 - 1.86’, ‘The BIX values of bulk AEOM were 0.66 - 1.16’, and ‘The AEOM-OC content/TOC ratio was 6.59±4.12%’.

All abbreviations and value form used in the table must be explained in a footnote to the table. 

Author Response

Reviewer #2:

Comments and Suggestions for Authors

Comment #2-1:

The significance of this study and scientific hypothesis as well as its creativity are not clearly demonstrated in the introduction section. The authors should provide a clearly description of the defects in the existing researches and how this study deal with these problem.

Response #2-1:

Thank you for the comment and suggestion.

We agree with the reviewer's comment and suggestion. The significance of this study, scientific hypothesis, and creativity must be clearly presented in the introduction.

The Introduction Section has been modified as follows.

1. Introduction

Soil organic matter (SOM) is one of many important components of soil [1-4]. SOM and fine particles are major components that are associated with heavy metals (HMs) in soil [4-6]. The alkaline and water-extracted organic matter (AEOM/WEOM) of SOM are the main components that bind to HMs [3-7]. AEOM/WEOM comprises phenolic, carboxylic, and hydroxyl functional groups, which have a strong ability to bind HMs [5,8]. The AEOM has a wide range of molecular weight, but the various size-fractioned AEOMs may have different chemical compositions. The binding behavior with HMs is different [9]. The complexation of HMs and AEOM influences the HMs mobility, biotoxicity, and fate in the soil environment [3-6,8,10-13].

The molecular weights and composition of AEOM are greatly influenced by the biogeochemical processes and the source of the organic matter in the soil environment [1,3,8,14]. The size-fractioned AEOM could play a critical role in distinguishing and simplifying the HMs binding behavior [8,9,14-16]. The low molecular weight dissolved organic matter (DOM) and AEOM (< 1 kDa) still contain various HM species (binding with organic acid and free metal ion) [9,16-18]. The low molecular weight DOM/AEOM have a binding capacity to HMs and hydrophobic organic compound [9,14,19,20].

UV-Vis and fluorescence spectroscopy is rapid, non-destructive spectral methodology. These sensitive detection methods are widely used to determine chemical compositions and structures of various DOM/AEOM [21-27]. UV-Vis indicator SUVA₂₅₄ shows DOM/AEOM aromaticity [23,24,28]. The fluorescence index (FI) is relative to the contribution of terrestrial source [21,23]. The biological index (BIX) is relative to the contribution of autochthonous sources [23,26].

The dissolved organic carbon (DOC) concentration cannot fully predict the mobility potential and binding strength of HMs. To understand the HM-AEOM binding behavior, the AEOM chemical composition and structure needs to be included [6,8,11]. The HMs and DOM binding affinity, [Me]/[DOC] ratio, is a useful parameter and has been used to investigate the preferences, distribution, bioavailability, and mobility potential of HMs binding with DOM/AEOM [9-11,16,29-33]. Moreover, the HM-AEOM binding affinity in various molecular weight AEOM is also an important factor to understand the HMs binding behavior. Amery et al. [10,11] studied the Cu and DOM binding affinity [Cu]/[DOC] ratios with many soil solutions (lysimeter leachate, soil pore water, water and CaCl₂ extracted soil solutions). The ratios had a significantly positive correlation with SUVA₂₅₄. However, the studies were lacking the molecular weight effect on the ratio difference in the soil solutions. In aquatic environment, the [Cu]/[DOC] ratios of bulk DOM had been reported by some researchers [16,29,30,32,33]. The ratios were positively correlated with aromaticity for natural water DOM [29,32]. However, when DOM was affected by anthropogenic sources, such as wastewater effluent input, the ratios had a weak correlation with DOM aromaticity. In addition, the ratios were affected by treatment processes [16,34], molecular weight [16,30,34], and the DOM composition [30]. Hsieh et al. [9] studied [Ni, Cd]/[DOC] ratios distribution in soil AEOM. The results showed that Ni and Cd had a different binding affinity with size-fractioned AEOM solutions. Cd preferred binding with low molecular weight AEOM, but Ni favored binding with high molecular weight AEOM. The type of HM, DOM chemical properties, and the DOM molecular weight affected the HM-OM binding affinity. The Cu binding affinity to various molecular weight soil organic matter needs more study in order to assess the Cu biotoxicity and mobility potential in soil.

The binding strength of HMs-OM in the soil is essential in affecting the biotoxicity and bioavailability of heavy metals in agricultural soil. Although previous studies have used sequence extraction to investigate the chemical formation of heavy metals in soil, the method cannot fully provide the heavy metal and organic matter binding strength. Therefore, an extracted soil organic matter was separated into size fractions to investigate size fractioned [Me]/[DOC] ratios. That is an excellent surrogate indicator for understanding heavy metals' binding strength and preference, distribution, bioavailability, and mobility potential in soil environments [9-11,16,29 -33]. At the same time, using sensitive and rapid optical indicators to understand the chemical composition and structure of the extracted organic matter can simplify the analysis of the finding dominant factors to influence HMs-OM binding affinity. In addition, size-fractioned AEOM can differentiate the binding capacity of HM. Unfortunately, extraction of soil organic matter and separation into size-fractioned dissolved organic matter to investigate HMs-OM binding affinity is lacking in the paddy field study.

This study investigated Cu distribution and binding affinity with size-fractioned alkaline-extracted soil organic matter. UV-Vis and fluorescence indicators (SUVA₂₅₄, BIX, and FI) were used to investigate the chemical properties of size-fractioned AEOM. In addition, the correlation method was used to examine the dominant factors of Cu and AEOM binding affinity, [Cu]/[DOC] ratio, in terms of the AEOM optical indicators.

Comment #2-2

There are few explanations of the rationale for the sampling design. it is suggestion that an explanation of why the soil samples were taken from ten paddy field should be provided.

Response #2-2:

Thank you for the suggestion.

The major rice production areas in Taiwan are in the center, east, and south of Taiwan. In the present study, the sampling site was divided into five sections of Taiwan rice production areas: center, south center, south, east north, and east south. The soil samples were taken from these sections and each section selected two paddy fields. The sampling sites are shown in Figure 1.

Line 105 has been modified as "In Taiwan, the major rice production areas are in the center, east and south parts of Taiwan. Therefore, the major rice production area was divided into five regions of Taiwan: center, south center, south, east north, and east south. The soil samples were taken from these areas with two sampling sites. Ten paddy fields were selected from the five regions for the present study, and the site's location is shown in Figure 1."

Comment #2-3

The Results and Discussion section should be rewritten. It is recommended to analyze your own test results or data first, and then compare them with the literature or discuss the reason. You should ensure that the data in the tables are consistent with those cited in the relevant places in the text, and data have been calculated correctly. In the current version of the manuscript, it is difficult to clearly see what is your own experimental data. Especially in subsections 3.1, 3.2 and 3.3, the data described in the text is difficult to correspond to table 1, table 2 and table 3. For example, where are the supporting data of your description of 'The FI values of bulk AEOM were 1.32 - 1.86', 'The BIX values of bulk AEOM were 0.66 - 1.16', and 'The AEOM-OC content/TOC ratio was 6.59±4.12%'.

Response #2-3:

Thank you for the comments and suggestions.

We agree with the reviewer's suggestions that the presentation of the result and discussion follow the order to describe our test results and data, then compare them with the literature or discuss the reason.

Because the AEOM in the ten sites had significantly different chemical properties, in the original manuscript, we used the optical indices' mean and standard deviation values for each size-fractioned AEOM in Table 1. However, due to variation in the ten sites and to fully present the data in the present study, we added the data ranges for bulk and size-fractioned AEOM and total values in Table 1. Therefore, the data of the FI values of bulk AEOM were 1.32 - 1.86, and the BIX values of the bulk AEOM were 0.66 - 1.16 as shown in Table 1. Table 2 lists the average and standard deviation of total Cu, AEOM-Cu, ratios of AEOM-Cu to total Cu, TOC, AEOM-DOC, and ratios of AEOM-DOC content to TOC for each site. In addition, Table 2 added total values of average, standard deviation, minimum, and maximum. Hence, the AEOM-DOC content/TOC ratio was 6.59±4.12%, presented in Table 2. In addition, Table 3 has added the total average, standard, and range of AEOM Cu and DOC concentrations.

Subsections 3.1, 3.2, and 3.3 have been modified as follows.

3.1. Optical indicators

Table 1 lists the values of SUVA₂₅₄, BIX, and FI for bulk and size-fractioned AEOM solutions. The bulk AEOM optical indicators were within the reported values range in soil solution. ^{[2,9-11,35,36,43]}

The SUVA₂₅₄ is positively correlated with the aromatic content [27,28]. The SUVA₂₅₄ of bulk AEOM ranged from 0.30 to 7.92 L mg-C^-1 m^-1 with a 26-fold variation. The bulk and high molecular weight AEOM (1 kDa - 0.45 μm, HMW, n= 90), SUVA₂₅₄ value 3.67 ± 2.22 L mg-C^-1 m^-1 was significantly higher than the low molecular weight AEOM SUVA₂₅₄ values 1.76±0.95 L mg-C^-1 m^-1(< 1 kDa, LMW, n=60, p < 0.001). The HMW AEOM solutions contained more hydrophobic and aromatic compounds than the LMW AEOM solutions [27]. Previous studies reported SUVA₂₅₄ values ranged from 0.26 to 6.30 L mg-C^-1 m^-1 in DOM/AEOM which is comparable to SUVA₂₅₄ values of the present study [2,9-11,35,36,43].

The FI is an indicator relative to terrestrial sources [21,23]. A high FI value indicates low terrestrial source contribution. In the present study, the FI values of bulk AEOM were 1.32 - 1.86. HMW and the LMW AEOM solutions had FI values of 1.48±0.11 and 1.65±0.13, respectively (p < 0.001). HMW AEOM had more terrestrial contribution than the LMW AEOM. Most of the FI values were within the range 1.4 - 1.9, which suggested that the AEOM solutions contained median terrestrial sources [21,23,41]. The FI values in the present study were within FI values, ranging from 1.08 to 2.03 reported in previous DOM/AEOM studies [2,9,35,36,43].

The BIX is an indicator relative to autochthonous origin contribution. In the present study, the BIX values of bulk AEOM were 0.66 - 1.16. HMW and the LMW AEOM solutions BIX values were 0.81±0.18 and 1.01±0.26, respectively. A recently produced DOM of autochthonous origin suggested BIX > 1.0, and an allochthonous origin BIX < 0.6 [21,26]. BIX values suggested that the HMW AEOM solutions had a median allochthonous origin and the LMW AEOM was the autochthonous origin. The bulk BIX values in the present study were comparable to BIX values ranging from 0.43 to 0.96 as reported in previous studies [35,36,43]

The size-fractioned AEOM in the present study showed that fraction M-B (3 - 100 kDa) had the highest SUVA₂₅₄ but the lowest FI and BIX values. In contrast, the fraction M-E (< 0.3 kDa) had the lowest SUVA₂₅₄ but the highest FI and BIX values.

3.2. DOC and Cu concentrations of size-fractioned AEOM

Table 2 lists the DOC and Cu concentrations of the total and the extracted bulk AEOM solutions. The AEOM-OC content ranged from 1.46 to 5.97 g kg^-1 and the total organic carbon (TOC) was 12.5 to 130 g kg^-1 based on the soil mass. The AEOM-DOC content/TOC ratio was 6.59±4.12%. Hsieh et al. [9] reported the average AEOM DOC concentration was 0.68±0.25 g kg^-1 and the TOC was 18.1±1.9 g kg^-1 based on soil mass. Therefore, the AEOM-DOC content/TOC ratio (0.68/18.1) was 3.70%. At room temperature, Fernández-Romero et al. [2] reported that water extraction organic carbon (WEOC) was 80–620 mg kg^-1, and organic matter ranged from 43 - 156 g kg^-1. Therefore, the WEOC/OM ratios were 0.12%-0.40%. Gao et al. [43] reported the DOC concentration of water extracted soil organic matter was 56.1 - 81.1 mg kg^-1, and the TOC was 2.64 - 4.45 g kg^-1. Therefore, the WEOC/TOC ratios were 1.24%-3.07%. In the present study, the alkaline-extracted soil organic carbon from paddy soil was much higher than the water-extracted soil organic carbon and alkaline-extracted soil organic matter from the dry farm soil.

Table 2 shows the total Cu concentrations ranged from 8.99 to 32.30 mg kg^-1 in the soil samples with an average of 20.95±6.19 mg kg^-1, and they are similar to the Cu concentrations in the unpolluted farmland soils [4,9,44-46]. The AEOM Cu concentrations ranged from 2.00 to 8.80 mg kg^-1 with an average of 5.43±1.63 mg kg^-1. The ratios of bulk AEOM Cu to the total Cu concentration ranged from 9.7 to 64.5%, averaging 29.2±14.8%. Hsieh et al. [9] reported the mean AEOM Cu concentration was 1.19 mg kg^-1 and the total Cu concentration was 9.89 mg kg^-1 AEOM Cu to total Cu had a ratio of 12.0%. Matong et al. [4] reported that three agricultural soils were sequentially extracted with acetic, ascorbic, and hydrogen peroxide digestion. The organic matter- and sulfide-binding fractions were 57%–68% for Cu. In the present study, a high extent of Cu and OC was extracted by alkaline solution, and AEOM-Cu/total-Cu and AEOC/TOC ratios were higher than the corresponding ratio extracted with water. This may be explained since the paddy field had high soil organic matter and was readily extracted by alkaline solution compared to dry farmland. Cambier et al. [45] reported that Cu is preferentially combined with soil humic substances. In the aquatic environment, the simulation showed copper had a high percentage of binding with dissolved organic matter [47], which explains the high ratio of AEOM-Cu/total-Cu observed in the present study.

3.3. Cu and OC distribution between size-fractioned AEOM

Table 3 lists each site's DOC and Cu concentrations of bulk and size-fractioned AEOM solutions and lists the average, standard deviation, minimum, and maximum total concentrations. The mass balances of DOC and Cu in the size-fractioned AEOM were calculated with Equation. (2). The average mass balances were 106±16% and 97±18% for Cu and DOC, respectively, which were within a reasonable range of 100±25% [15,48].

In aqueous DOM separation studies, the OC mass balances ranged from 78 to 104% [17,48-50]. In soil and sediment extraction solution separated into size-fractioned solutions, the reported OC mass balances ranged from 80 to 159% [9,14,51,52]. The water-extracted soil organic matter (WEOM) was separated into four size-fractioned WEOMs reported by de Zarruk et al. [51], and the DOC mass balance was 117%. Martin et al. [48] separated lagoon water DOM; the DOC mass balances ranged from 85 to 98%. Wen et al. [50,53] separated seawater into high molecular weight (1 kDa - 0.45 um) and low molecular weight (<1 kDa) DOM. The Cu mass balances ranged from 88 to 106%.

Figures 2a-2b show the average mass percentages of Cu and OC for size-fractioned AEOM in each site. The mass percentages varied among the sampling sites. The quantity order of mass percentages for Cu average values were M-B (66.7%) > M-E (14.1%) > M-A (9.8%) > M-C and M-D (4.7%). The quantity order of mass fractions for total OC values were M-B (40.3%) > M-E (38.4%) > M-A (10.0%) > M-C (6.3%) > M-D (5.0%). Cu had a much higher percentage in the M-B fraction than other fractions, but OC had a higher percentage for M-B and M-E fractions than the other fractions.

Molecular weight at 1 kDa for DOM/AEOM is used to distinguish between the high and low molecular weights of DOM/AEOM. The high molecular weight fractions (> 1 kDa) were 81.2% and 56.6% for Cu and OC, respectively, which suggested Cu favored binding to high molecular weight AEOM.

Wang et al. [54] separated soil solutions and water samples into HMW WEOM/DOM (1 kDa–0.45 μm) and LMW WEOM/DOM (<1 kDa). The Cu mass percentages of HMW were 47%, and 59% for soil solution and water samples, respectively. Ilina et al. [47] reported that water-extracted soil solution, lake water, and river water used ultrafiltration separation; the mass percentages of HMW Cu (1 kDa - 0.45 μm) were 80%, 53% and 38%, respectively. In a municipal wastewater treatment plant, Hargreaves et al. [16] reported the HMW DOM (1 kDa - 0.45 μm) averaged 74% for Cu. Hsieh et al. [9] separated soil AEOM into HMW (1 kDa - 0.45 um) and LMW (< 1 kDa) solutions. The OC mass percentage of HMW AEOM was 44%. Dai et al. [49] separated sea water into HMW DOM (1 kDa - 0.45 um) and LMW DOM (< 1 kDa) solutions. The mass for OC and Cu was 8.2 - 30.4% and 20.5 - 39.2%, respectively.

The mass percentages of high and low molecular weights in water DOM and water extracted organic matter varied. The percentages depend on organic matter sources, biogeochemical process, type of metal, the matrix, extraction solvent and method, solid/liquid ratio, and separation method and conditions [15,54,55].

Table 1. The optical values of SUVA₂₅₄, BIX, and FI for bulk and size-fractioned AEOM solutions.

 ^* mesn ± standard deviation,  ^** (minmum, maximum)

Table 2. The DOC and Cu concentrations of total and the extracted bulk AEOM solutions.

^* mean±standard deviation;  ^** (minimum, maximum)

Table 3. Cu and DOC mean and standard deviation concentrations of bulk and size-fractioned AEOM solutions each sites.

^* mean±standard deviation;  ^** (minimum, maximum)

Comment #2-4

All abbreviations and value form used in the table must be explained in a footnote to the table. 

Response #2-4:

Thank you for the suggestion.

All abbreviations and value form used in the table have been explained in a footnote to the table. 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Exactly as you explanation in your response, the AEOM in the ten sites had significantly different chemical properties. However, where is the result of your mathematical statistical analysis? The methods of ‘the correlation analysis and the different tests’ have been introduced in the subsection of the ‘2.5 Statistical analysis’. It is suggested that the results of the within-group or between-group difference test should be supplemented in the corresponding tables.

Line 200 of page 5, What is the function of the ‘(p < 0.001)’?

Line 216 of page 5, ‘mesn’ should be replaced with the ‘mean’.

Table 5, what does these symbol '*’, ‘**’, and ‘***’ mean? It is suggested that them meaning should be explained in a footnote to the table.

Author Response

Please see attachment

Author Response File: Author Response.pdf