Combined Effects of Cadmium and Azithromycin on Soil Nitrification Process

Round 1

Reviewer 1 Report (New Reviewer)

The authors have studied the joint pollution effect of Cd and AZI on soil and their effect on PNR, which is very rare in previous studies. But there are still many problems in each part of the article, and the article still needs major revision:

Title: The article title mentioned Macrolide Antibiotics, but the article only mentioned AZI, there was no other Macrolide Antibiotics, so I think the title was inaccurate.

Abstract: I don't think Statistical methods (ANOVA) and picture format (Box Behnken Design) were needed to be in the abstract

Introduction:  

line 71-86 I think the description of ammonia oxidation process in this paragraph is too detailed and unnecessary. It is suggested to focus on the existing studies demonstrating the inhibitory effect of soil pollution on AOB and AOA bacteria, or the gap of the research field here

line 97-106 Perhaps the description of this reference does not need to be so detailed. Please reduce the content appropriately, quote only the content related to this article, and summarize.

Materials and Methods:

The overall introduction of the experimental design is in a confused order, and it is recommended to introduce the experimental design in the order of operation. Secondly, how does the experiment conducted is unclear.

line 122-131 I don't think 2.1 is a necessary part

Part 2.2, The description of Soil Sampling and Treatment in this part is very poor. How many soil sampling points are there and the number of repetitions collected at each point is not specified, only the collection and preservation methods are described. The experimental design of section 2.5 and Table 4 are recommended to be placed in this section. The treatment time and method of soil samples after adding pollutants are not specified

Table 3, I don't know how variables are combined. Why did the author choose the 17 combinations in Table 4?

Table 2 What does S1-S5 represent? Are they five sampling points?

Part 3.1 Optimization of Incubation Solution is not the subject of this article. It is recommended to delete it. If the article needs this part, it is recommended to put it in the appendix, or simply explain it in the Material and Method section.

Results:

Please mark the figure or table referenced in the text. In addition, please only show the results of your research in the results section. Do not put the comparison with other research results and the interpretation of the results in this section. These should be put in the discussion section, such as line 240-244, 247-248 and 258-260.

The result part of line253-260 and 289-301 should be expressed in the past tense.

In Section 2.3 and Table 2, many soil physical and chemical property indexes were measured, but the results did not include any pictures or tables to express the relationship between these data and PNRs, and there was no appendix. So why are so many indicators measured? Please supplement the corresponding result pictures and tables.

Comments for author File: Comments.docx

Author Response

Responses to Reviewer’s Comments

The authors have studied the joint pollution effect of Cd and AZI on soil and their effect on PNR, which is very rare in previous studies. But there are still many problems in each part of the article, and the article still needs major revision:

Thank you for your scientific contributions. In order to improve the quality of the manuscript, it was revised by considering your comments and suggestions. The changes/revisions have been highlighted in the revised manuscript.

Title: The article title mentioned Macrolide Antibiotics, but the article only mentioned AZI, there was no other Macrolide Antibiotics, so I think the title was inaccurate.

Title of the manuscript revised to “Combined Effects of Cadmium and Azithromycin on Soil Nitrification Process”

Abstract: I don't think Statistical methods (ANOVA) and picture format (Box Behnken Design) were needed to be in the abstract

Related sentences were removed

Introduction:  

line 71-86 I think the description of ammonia oxidation process in this paragraph is too detailed and unnecessary. It is suggested to focus on the existing studies demonstrating the inhibitory effect of soil pollution on AOB and AOA bacteria, or the gap of the research field here

Less necessary sentences in this paragraph were removed. Findings of existing studies are given with the following paragraph.

“Studies in the literature have focused on independent toxic effects of heavy metals on AOB, however, combined toxic effect of heavy metals with various environmental pollutants on soil nitrification process is relatively less reported. Bai et al. [35] experimented the effect of Cd addition on nitrification processes in soil samples taken from a constructed wetland in China.  Incubation experiments were conducted for 15 mg/kg Cd and 100 mg/kg Cd. While no inhibition effect was found for low level of Cd, 100 mg/kg Cd reduced nitrogen mineralization rate from 0.40 mg/kg.d. to 0.21 mg/kg.d. when compared to uncontaminated control soil. Smolders et al. [36] determined PNR of soil samples spiked with 0-200 mg/kg Cd. The PNRs of soil samples were reduced to 50-80% at the highest Cd contamination level. Rosendahl et al. [37] researched the persistence of difloxacin antibiotics in soil, and its effect on soil nitrification potential. It was determined that even difloxacin had long half life (>217 d), its effect on nitrogen turnover was limited. It was concluded that difloxacin formed non-extractible residues in soil resulted in small bio-accessibility.”

line 97-106 Perhaps the description of this reference does not need to be so detailed. Please reduce the content appropriately, quote only the content related to this article, and summarize.

Content of the reference was reduced by deleting experimental details.

Materials and Methods:

The overall introduction of the experimental design is in a confused order, and it is recommended to introduce the experimental design in the order of operation. Secondly, how does the experiment conducted is unclear.

The subtitles of the material method have been rearranged. Experimental design was revised so as to give the operations in true order. Details of the experiment was given with the following revised paragraph.

“As the test principle, taking into account the soil water content, 100 mL of incubation solution was added on 25 g of soil sample. The flasks containing the control soil without contaminant and samples spiked with Cd and AZI were covered with aluminum foil with an open top. Spike concentrations of Cd and AZI were adjusted according to    experimental design given in Table 2. It was placed in a shaking incubator at 26 ^oC, 120 rpm in aerobic conditions. The linearity of NO₂^- formation, which was an indicator of the   formation of AOB, was evaluated by taking samples at the end of each hour. 6 samples were taken in 6 hours within the germination period of nitrifying bacteria. The samples were centrifuged (3000 cycles/2 min) before NO₂^- analysis. PNR inhibition percent of Cd and AZI spiked soil samples were determined by comparing with PNR of control soil samples at the end of incubation period.” 

line 122-131 I don't think 2.1 is a necessary part

2.1. Chemicals and Reagents were removed

Part 2.2, The description of Soil Sampling and Treatment in this part is very poor. How many soil sampling points are there and the number of repetitions collected at each point is not specified, only the collection and preservation methods are described. The experimental design of section 2.5 and Table 4 are recommended to be placed in this section. The treatment time and method of soil samples after adding pollutants are not specified

The experimental design of section 2.5 and Table 4 were given in soil sampling section under a combined subtitle of “2.1 Soil Sampling and Experimental Design”. The following sentences were added to this section to clear sampling method.

“Soil samples were taken from five districts in Konya City (S1, S2, S3, S4, S5). Sampling areas were selected to have different soil physicochemical properties. Three samples were taken from the same district to find the least Cd contaminated soil samples. Besides, three soil samples were taken from each sampling point and homogenized before experiments.”

Table 3, I don't know how variables are combined. Why did the author choose the 17 combinations in Table 4?

The following revised paragraph explains the methodology. The model, proposed by Box Behnkan helps to select the most proper experimental design, so as to avoid unnecessary experiments.

“17 experiment sets were performed for the determination of PNR inhibition. Environmentally relevant concentrations of Cd (1 mg/kg - 21 mg/kg) and AZI (1 mg/kg - 9 mg/kg) were experimented. The second degree polynomial model was used to predict the relationship between the experimental variables and the response (Y). For this, five levels for each variable was experimented. Central level was coded as 0 (mid), low and high levels were coded as -1 and +1, respectively, while low and high control levels were coded as -2 and +2, respectively.”

Table 2 What does S1-S5 represent? Are they five sampling points?

The following sentences were added to 2.1 Soil Sampling and Experimental Design section to clear it.

“Soil samples were taken from five districts in Konya City (S1, S2, S3, S4, S5). Sampling areas were selected to have different soil physicochemical properties….”

Part 3.1 Optimization of Incubation Solution is not the subject of this article. It is recommended to delete it. If the article needs this part, it is recommended to put it in the appendix, or simply explain it in the Material and Method section.

Subtitle was revised to 3.1. Nitrification Potential of Soil Samples

Figure 1 and related explanations were removed. Optimum dose was given with a sentence in materials and methods section.

Results:

Please mark the figure or table referenced in the text. In addition, please only show the results of your research in the results section. Do not put the comparison with other research results and the interpretation of the results in this section. These should be put in the discussion section, such as line 240-244, 247-248 and 258-260.

Figures and tables, referenced in the text were marked in bold now. Interpretation of the results were given only in discussion section. Related references were removed from results section.

The result part of line253-260 and 289-301 should be expressed in the past tense.

Results were given in the past tense.

In Section 2.3 and Table 2, many soil physical and chemical property indexes were measured, but the results did not include any pictures or tables to express the relationship between these data and PNRs, and there was no appendix. So why are so many indicators measured? Please supplement the corresponding result pictures and tables.

Figure 2 was given to show correlations between soil PNRs and soil physichocemical properties. The soil PNRs as functions of soil EC, OM%, CaCO3%, P2O5 (kg/da), K2O (kg/da), Na (mg/kg), Ca (mg/kg), and TN%).

The following paragraphs were added to 3. Results and 4. Discussion sections, respectively.

“Poor correlations were determined for soil EC, CaCO₃, P₂O₅, Na, Ca, and TN values and soil PNR, with 0.42, 0.22, 0.28, 0.26, 0.02, and 0.24 R² values, respectively (Figure 2). A meaningful correlation was determined for soil K₂O and soil PNR, with 0.52 R² value. K is one of the macro nutrients in the soil, effective on soil microbial activity. In this study, the soil sample with 33 kg/da K₂O had 4.52 mg NO₂-N/kg d.m. PNR, while it was 15.38 NO₂-N/kg d.m. for the soil with 143.4 kg/da K₂O.”

“Inorganic salts such as phosphorus, potassium, calcium and magnesium are among the cell building materials of AOB. However, soil physicochemical properties did not show a clear effect on PNR, except for soil pH, OM and K, in this study. Similar results were reported by Rodriquez-Gonzales et al. [53]. While soil pH and cation exchange  capacity affected the toxic effect of clarithromycin on soil bacterial community, effects of soil nutrients was not clear.”

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

The combined effects of heavy metals and antibiotics in soil on nitrification process have been studied a lot, and have attracted widespread attention. The purpose of this manuscript is to study the combined effect of cadmium and azithromycin on soil nitrification, which is relatively less reported. However, the experimental design of this study is relatively simple, and the methods and means are not novel enough. Some specific questions or suggestions are listed below:

1. Line 35-39: How to know the pollution situation of cadmium and azithromycin in agricultural areas around Konya? Please add relevant references.

2. Line 116-117: “Cd and AZI contamination of the selected study area has been demonstrated in previous research studies.” Please add the previous research studies. Are there some relevant references?

3. Why did not measure the content of azithromycin in control soil? We know Cd concentration in control soil from Table 2, but we don’t know the background content of azithromycin.

4. Five kinds of soils were used in the study, in which pH value and the contents of organic matter, total nitrogen, phosphorous and potassium, sodium, calcium were different (Table 2). In addition to the factors that affect the nitrification process discussed in the manuscript, does the difference in carbon to nitrogen ratio, phosphorus content, potassium, sodium and calcium content also have effects on the nitrification process? Please add it in the discussion section.

5. The conclusion is rather empty. The research should give more specific suggestions, for example, the concentration of azithromycin in sludge may inhibit the degree of nitrification and affect the efficiency of nitrogen fertilizer use. What is the relatively quantitative relationship between them? Then, the amount of sewage sludge applied to soil should be determined according to the concentration of antibiotics in the sludge. The relationship between antibiotic concentration and sludge usage should also be established or suggested.

6. If the author can establish a model to describe the relationship between PNR inhibition and AZI, Cd and pH, the article will be more readable and referential.

Author Response

Responses to Reviewer’s Comments

The combined effects of heavy metals and antibiotics in soil on nitrification process have been studied a lot, and have attracted widespread attention. The purpose of this manuscript is to study the combined effect of cadmium and azithromycin on soil nitrification, which is relatively less reported. However, the experimental design of this study is relatively simple, and the methods and means are not novel enough. Some specific questions or suggestions are listed below:

Thank you for your scientific contributions. In order to improve the quality of the manuscript, it was revised by considering your comments and suggestions. The changes/revisions have been highlighted in the revised manuscript.

1. Line 35-39: How to know the pollution situation of cadmium and azithromycin in agricultural areas around Konya? Please add relevant references.

The following paragraph was added to introduction part of revised manuscript so as to explain Cd and AZI contamination in the study area

“Working group of the author of this article conducted a research study to define the effects of long term irrigation of agricultural areas in Konya, Turkey, with untreated municipal wastewater (Aydin et al., 2015). Significant pollution with Cd (8.23-11.6 mg/kg.dm) was determined in the wastewater irrigated soils of the sampling site. Soil Cd contamination was well over the Maximum Admissible Concentrations for Trace   Elements in Agricultural Soils for application of Sewage Sludge Regulation in Turkey, which was given as 1 mg/kg.dm (6 ≤ pH <7) or 1.5 mg/kg.dm pH ≥7 (2010). Digested sewage sludge of urban wastewater treatment plant (WWTP) has been used for soil amendment in the same area. High risk quotient (RQ > 1) was estimated for AZI,  determined nearly 1.5 mg/kg.dm in sewage sludge (Aydin et al., 2022). Questions have been raised about toxic effect of simultaneous occurence of Cd and AZI on soil microbial activity in the region.”

2. Line 116-117: “Cd and AZI contamination of the selected study area has been demonstrated in previous research studies.” Please add the previous research studies. Are there some relevant references?

References; Aydin et al., 2015; Aydin et al., 2022 were given after the related sentence.

3. Why did not measure the content of azithromycin in control soil? We know Cd concentration in control soil from Table 2, but we don’t know the background content of azithromycin.

The main theory of the manuscript depends on the simultaneous occurrence of Cd in soil and occurrence of AZI in sewage sludge used for soil amendment in the area. Therefore, AZI content of the soil samples were not given. The following revised paragraph was given so as to clear the aim of the study;

“In this study, it is aimed to determine the combined toxic effects of Cd and selected antibiotics; AZI on AOB by testing soil PNR. Cd contamination of the soil and AZI content of the sewage sludge used for soil amendment in selected study area have been demonstrated in previous research studies [Aydin et al., 2015; Aydin et al., 2022]. Therefore, it was aimed to determine the toxic effect of simultaneous   occurrence of these two contaminants on soil nitrification potential so as to evaluate the risk of soil amendment with digested sewage sludge for sustainable soil fertility.”

4. Five kinds of soils were used in the study, in which pH value and the contents of organic matter, total nitrogen, phosphorous and potassium, sodium, calcium were different (Table 2). In addition to the factors that affect the nitrification process discussed in the manuscript, does the difference in carbon to nitrogen ratio, phosphorus content, potassium, sodium and calcium content also have effects on the nitrification process? Please add it in the discussion section.

Figure 2 was given to show correlations between soil PNRs and soil physichocemical properties. The soil PNRs as functions of soil EC, OM%, CaCO3%, P2O5 (kg/da), K2O (kg/da), Na (mg/kg), Ca (mg/kg), and TN%)

The following paragraphs were added to 3. Results and 4. Discussion sections, respectively.

“Poor correlations were determined for soil EC, CaCO₃, P₂O₅, Na, Ca, and TN values and soil PNR, with 0.42, 0.22, 0.28, 0.26, 0.02, and 0.24 R² values, respectively (Figure 2). A meaningful correlation was determined for soil K₂O and soil PNR, with 0.52 R² value. K is one of the macro nutrients in the soil, effective on soil microbial activity. In this study, the soil sample with 33 kg/da K₂O had 4.52 mg NO₂-N/kg d.m. PNR, while it was 15.38 NO₂-N/kg d.m. for the soil with 143.4 kg/da K₂O.”

“Inorganic salts such as phosphorus, potassium, calcium and magnesium are among the cell building materials of AOB. However, soil physicochemical properties did not show a clear effect on PNR, except for soil pH, OM and K, in this study. Similar results were reported by Rodriquez-Gonzales et al. [53]. While soil pH and cation exchange  capacity affected the toxic effect of clarithromycin on soil bacterial community, effects of soil nutrients was not clear.”

5. The conclusion is rather empty. The research should give more specific suggestions, for example, the concentration of azithromycin in sludge may inhibit the degree of nitrification and affect the efficiency of nitrogen fertilizer use.What is the relatively quantitative relationship between them? Then, the amount of sewage sludge applied to soil should be determined according to the concentration of antibiotics in the sludge. The relationship between antibiotic concentration and sludge usage should also be established or suggested.

Conclusions section was revised as follows;

“Approximately 140 tons/day treated sewage sludge with 1.5 mg/kg.dm AZI content has been periodically used for soil amendment in Konya City. Farmers could apply various amounts of sewage sludge to their soils with no limitation. Since a significant Cd pollution (8.23-11.6 mg/kg.dm) was also determined in the region, a risk for soil PNR inhibition was clear according to the findings of this study. While the inhibition of PNR was relatively low for soils with neutral pH values, 50% PNR inhibition was determined for environmentally relevant concentrations of Cd and AZI for alkaline soil samples. This means that half of the nitrogen fertilizers applied to the soil could be wasted in alkaline soils. Besides, neither soil has the solely Cd contamination, nor sewage sludge has the only AZI content. Simultaneous occurrence of various heavy metals and pharmaceuticals have been ignored. Pharmaceuticals have not been included in the regulations on the application of sewage sludge to the soil, and the danger of synergistic effects with heavy metals has not been recognized. While the sewage sludge is applied to the soil to benefit from its nutrient content, it may adversely affect nitrogen fertilizer utilization efficieny of the plants. Replicated annual applications may reduce soil fertility. This study aims to draw attention to this issue.”

6. If the author can establish a model to describe the relationship between PNR inhibition and AZI, Cd and pH, the article will be more readable and referential

The second-order quadratic model, proposed by Box Behnkan, was used for interpretation of obtained experimental data. Design-Expert® package, version 11.0.3 (Stat-Ease Inc., MN, USA) software was used for the statistical analysis of obtained data. The data fitted to quadratic model. Model adequacy and the significance for Cd and AZI, at different soil pH were given in Table 5. Model satisfies, since predicted versus experimental data have high correlation as given in Figure 3.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report (New Reviewer)

• The questions I raised were well answered and revised by the authors. 

• I don't have any other suggestions

Reviewer 2 Report (New Reviewer)

The author carefully revised the manuscript according to the review comments. The quality of the manuscript was improved. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Only some minor corrections should be made:

Abstract

Line 14, 20 - 3.782 – 17.642 mg.NO₂/kg.soil.dm, 3.782 mg.NO₂/kg.soil.dm - check dots

Introduction

Line 60,62, 76, 79, 82, 107, 130 - 160 mg/kg.dm, 5.8 mg/kg.dm, 10 mg/kg.dm, mg/kg.dm, 1 mg/kg.dm (6 ≤ pH <7) or 1.5 mg/kg.dm, mg/kg.dm, 4200 μmol/kg.soil.day - check dots

Materials and Methods

Line 130 - CdSO₄.8H₂O – correct the dot

Line 164, 167, 169, 171, 176, 180 - 9546 EN, ISO 10390, ISO 11265,  ASTM D 422-63, ISO 11261, EPA 6010 D - should be in the references

Line 166 - pH-meter (Hach®, USA) - please provide the city

Line 168 - 100 ml -100 mL

Line 169  - Electrical Conductivity (EC) – electrical conductivity (EC), what kind of device ?

Line 172 - FOSS-Kjeltec - manufacturer, city, country

Line 173 - was determined according to Walkley-Black – maybe any reference ???

Line 177 - (CEM, MarsXpress, USA) – city ???

Line 179 - (ICP-OES, Perkin Elmer 5300) – should be (Perkin Elmer 5300, manufacturer, city, country)

Line 194 - NaClO₃ (0,5 mol/L), 28 mL/L KH₂PO₄ (0,2 mol/L), 72 mL/L K₂HPO₄ (0,2 - should be dots

Line 209 - Microsoft Excell + Analyse-it software - manufacturer, city, country

Results

Line 219, 220, 221, 234-237, 244, 245, 303 - (3.782 mg.NO₂/kg.soil.dm) (4,52 mg.NO₂ /kg.soil.dm), (15,515,  mg.NO₂/kg.soil.dm), S4 (15,383 mg.NO₂/kg.soil.dm), and S5 (17,642 mg.NO₂/kg.soil.dm),  0.4 mg.N/kg.day, 2 mg.N/kg.day,  25 μmol/kg.day,  303 μmol/kg.day,  3.782 mg.NO₂/kg.soil.dm,  17,642 mg.NO₂/kg.soil.dm,  3.782 mg.NO₂/kg.soil.dm - check dots

Line 250 - effect …should be affect

Discussion

Line 283 - Toxic effect of the tested contaminants significantly affected by the soil type - …was significantly affected by the soil type

Line 303 - 3.782 mg.NO₂/kg.soil.dm - check dots

Table 1

Cd (mg/kg.dm), (mg/kg.dm) - dots ???

Table 2

Organik Matter - should be Organic Matter

Figure 1, 2 - check dots in description of y-axis

 References

Please correct some minor errors for No. 8, 16 (check doi), 19, 30, 31, 32, 38 (check doi)

Author Response

Thank you for your support and valuable contributions. In order to improve the quality of the manuscript, it was revised by considering your comments and suggestions. The changes/revisions have been highlighted in the revised manuscript. Please see the attachment (Response to your comments).

Author Response File: Author Response.docx

Reviewer 2 Report

General comment

If the subject is interesting, I think that the methodology used and the discussion of data were not appropriate to respond to this question. In addition, the number of replicate is not known, only 4 cases were tested, and must of all no information about the reason of the lower nitrite production was tested.

This work does not respond the the question.

Specific comment

The introduction is not well organized. A paragraph on bacterial processes producing nitrite in soils is missing. On the reverse, the paragraph from line 69 to line 86 is out of purpose. The choice of bacteria used is not justified. The Table 1 is not relevant. What is the meaning of Cd concentration in soils at the scale of a country?

The methodology must be improved. Bacteria activity was only quantifyed through NO2 product but no bacteria disorder was tested. Incubations were conducted under aerobic or anaerobic conditions? Why only NaClO3 concentration can modify the nitrite production? Is the test used is common (ref needed) or correspond to a norm (ref needed)?

Discussion of results is short because of the small number of data and there is no conclusion.

Author Response

Thank you for your valuable scientific contributions. In order to improve the quality of the manuscript, it was revised by considering your comments and suggestions. The changes/revisions have been highlighted in the revised manuscript. Please see the attachment (Response to your comments).

Author Response File: Author Response.docx