Faeces of Capybara (Hydrochoerus hydrochaeris) as a Bioindicator of Contamination in Urban Environments in Central-West Brazil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Introduction

Major correction:

Please add the toxicology of each metalloid (Al, As, Cd, Cr, Co), Cu, Fe, Pb, Mg, Mn, Mo, Ni, P, Se, Zn, for wildlife and humans in the introduction.
Please explain better why you analyzed Mg and P.

Material and Methods

Major correction

Human health risk assessment:
Contact with feces can occur through different routes, and the following are considered in this study: (i) hand-to-mouth contact, which represents dirty hands coming into contact with the mouth, as can happen with children; (ii) hand-to-mouth contact with caregivers’ hands; (iii) object-to-mouth contact; (iv) ingestion of contaminated food and water; and (v) accidental direct ingestion of feces. This hypothesis was also adopted by Kwong et al. (2020) [25] in their study, which considered these routes of contamination or contact with feces, especially for children who may be more exposed due to the habit of playing in contact with the floor.
Was this stage verified? Please explain better.

2.9. Statistical analysis
What was compared with the non-parametric test (Kruskal-Wallis)? Please present the table of this test in the results.
What did you correlate?
Please present the correlations in a table in the results.

A quantity of 550 g of feces from each site was collected using a large 12.5 cm stainless steel spoon and surgical gloves to avoid contamination. Approximately 0.25 g of powder from each excrement sample from different locations was placed in Teflon DAP60®.
3 steps
What number of samples (n) was used for the statistical tests? Please include it in Table 2 for greater clarity in presenting the obtained results.

3. Results

Major correction

3.1. Quantification of macro and microelements in feces
Please replace "macro and microelements" with "Metalloids."

Therefore, one-way ANOVA analysis was used, and there was no difference between the 239 mean element concentrations of the different locations (p = 0.805).
ANOVA was not mentioned in the Material and Methods section. You describe that you used the non-parametric Kruskal-Wallis test? Please make some statistical topics clearer, such as: what was the distribution of the variables (metalloids), which test was used for the comparison between the studied locations. What was actually correlated? These issues are not clear in the manuscript.
Line 245: concentrations of elements quantified
Replace "elements" with "metalloids." Please standardize this term throughout the text, for example in Table 2: "Concentration of macro and microelements quantified in Hydrochoerus hydrochaeris feces (mg/kg)." Please replace "macro and microelements" with "metalloids."

I did not find the results and discussion of the correlations in the manuscript.

Discussion

Major correction

Did you not discuss Figure 2?
The discussion needs to be improved. I advise the authors to discuss the metalloids separately, for a better understanding of the toxicity of each studied metalloid.
Tables 3 and 4 are not in the discussion?

Comments on the Quality of English Language

Dear Editor

The manuscript requires a more comprehensive revision:

• Introduction: The section on the toxicity of the studied metalloids needs to be expanded.
• Materials and Methods: The number of samples is missing, and the statistical analysis is confusing and does not correspond to the results.
• Results: The tables of statistical tests and correlations have not been presented.
• Discussion: The discussion needs to better reflect the obtained results.

Author Response

Please see the attachment

Introduction
Major correction:
Comment 1: Please add the toxicology of each metalloid (Al, As, Cd, Cr, Co), Cu, Fe, Pb,
Mg, Mn, Mo, Ni, P, Se, Zn, for wildlife and humans in the introduction.
Please explain better why you analyzed Mg and P.
Response 1: Thank you. We added the toxicology of metalloids in the introduction.
Some plants present such elements in their mineral profile. Since this is the first study
with the aim of quantifying elements in capybara faeces, we also chose Mg and P, thus
providing values for future studies.

Where it said:
Therefore, it is possible to monitor the health of mammals and infer the level of
environmental pollution through animal excrement [15-19], since is an important source
of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore,
there is the possibility that faeces can cause impacts on human health, as they can
contaminate the water used to supply the population [9]. 

Now it says:
Therefore, it is possible to monitor the health of mammals and infer the level of
environmental pollution through animal excrement [20–24], since it is an important source of
heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, it is possible
that contaminated faeces can cause impacts on human health, as they can pollute the water used
for population supply [9]. In China, for example, the intensive production of poultry and pigs
was considered one of the main environmental contaminants due to the heavy metals presence
in faeces [25–27].
Heavy metals present in soil, such as Cr, Cu, and Zn, can cause health risks to humans,
such as neurological disorders, headaches, and liver disease [28,29]. Excessive Cu intake
promotes liver damage and gastric problems, while Zn can affect high-density lipoprotein
concentrations and alter the immune system [30]. In addition, high doses of Al, As, Cd, Cr, Ni,
and Pb are known to cause cancer [10,31], and Pb and Mn can lead to Alzheimer's disease and
manganism [32]. Furthermore, the ingestion of food contaminated with multiple metals, such as
Cd and Pb, can promote renal dysfunction [33]. 

 

Material and Methods
Major correction
2.4. Human health risk assessment
Contact with feces can occur through different routes, and the following are considered in this study: (i) hand-to-mouth contact, which represents dirty hands coming into contact with the mouth, as can happen with children; (ii) hand-to-mouth contact with caregivers’ hands; (iii) object-to-mouth contact; (iv) ingestion of contaminated food and
water; and (v) accidental direct ingestion of feces. This hypothesis was also adopted by Kwong et al. (2020) [25] in their study, which considered these routes of contamination or contact with feces, especially for children who may be more exposed due to the habit of playing in contact with the floor.
Comment 2: Was this stage verified? Please explain better.

 

Where it said:
Contact with feces can occur through different routes, and the following are considered in this study: (i) hand-to-mouth contact, which represents dirty hands coming into contact with the mouth, as can happen with children; (ii) hand-to-mouth contact with caregivers’ hands; (iii) object-to-mouth contact; (iv) ingestion of contaminated food and water; and (v) accidental direct ingestion of feces.
Now it says:
Contact with faeces can occur through different routes and the following are being considered in this study: (i) hand-to-mouth contact, which represents the contact of dirty hands with the mouth, as can occur with children; hand-to-mouth contact with caregivers' hands; object-mouth contact; ingestion of contaminated food and water; and accidental direct ingestion of faeces; following the hypothesis adopted by Kwong et al. (2020) [35] in their study who considered these routes of contamination or contact with faeces, especially children who may be more exposed due to the habit of playing in contact with the floor (Section 2.5); (ii) particulate inhalation via environmental contamination (Section 2.6); (iii) dermal exposure to metal(loid) contaminants (Section 2.7); (iv) risk analysis due to contact oral, respiratory tract, and dermal contact (Section 2.8).

 

2.9. Statistical analysis
Comment 3: What was compared with the non-parametric test (Kruskal-Wallis)? Please present the table of this test in the results.
What did you correlate?
Please present the correlations in a table in the results.
Response 3: We redid statistics, where:
We used One-way ANOVA to test if each elemental concentration differs in the different sites in faeces samples.
H0: The total amount of detected element is the same in all sampling sites in faeces samples.
H1: The total amount of detected element is different in at least one sampling site in faeces samples.

 

Where it said:
The means and standard deviations of the faeces samples, as well as the statistical tests to verify normal distribution, the Kruskal-Wallis non-parametric test (multiple and independent variables), the Spearman and Kendall correlation test were performed in the Oring 9.1 software (OriginLab’s Origin Corporation Viewer 9.1, Northampton, MA, USA.TM). The significance of the differences between the means for the individual trace element was considered at p < 0.05.
Now it says:
We used One-way ANOVA to test if each elemental concentration differs in the different sampling sites in faeces samples. The significance of the differences between the means for the individual metal(loid) was considered at p < 0.05. We consider that: H0: the total amount of
detected element is the same in all sampling sites in faeces samples; H1: the total amount of detected element is different in at least one sampling site in faeces samples.

 

Comment 4: A quantity of 550 g of feces from each site was collected using a large 12.5 cm stainless steel spoon and surgical gloves to avoid contamination. Approximately 0.25 g of powder from each excrement sample from different locations was placed in Teflon DAP60®.
3 steps
What number of samples (n) was used for the statistical tests? Please include it in Table 2 for greater clarity in presenting the obtained results.
Where it said:
A quantity of 550 g of feces from each site was collected using a large 12.5 cm stainless steel spoon and surgical gloves to avoid contamination. Approximately 0.25 g of powder from each excrement sample from different locations was placed in Teflon DAP60®.

Now it says:
Three fecal samples were collected in a zigzag pattern in locations close to water bodies for each site. Samplings were carried out in September 2021 and samples in direct contact with the soil were avoided. A quantity of 550 g of faeces from each site was collected using a large 12.5 cm stainless steel spoon and surgical gloves to avoid contamination.

 

Where it said:
Table 2. Concentration of macro and microelements quantified in Hydrochoerus hydrochaeris faeces (mg/kg).

Elemento	Parque Ecológico Anhanduí	Parque Estadual Prosa	Parque Ecológico Sóter	Lago do Amor
Todos	124.488 ±0,763	130.443 ± 2.179	139.564 ± 0,669	140.322 ±1.222
Como	0,002760 ± 0,000155	0,004300 ± 0,000267	0,003691 ± 0,00020	0,010041 ± 0,00019
Cd	0,4193 ± 0,008	0,940 ± 0,0407	0,568 ± 0,053	1.042 ± 0,302
Co	1.467 ± 0,130	1.019 ± 0,0399	1.776 ± 0,069	1.946 ± 0,840
Cr	20.275 ± 0,970	17.602 ± 0,827	21.405 ± 0,970	26.866 ± 1.013
Com	29.946 ± 1.296	32.632 ± 0,843	32.511 ± 2.042	50.764 ± 0,853
Fé	300.369 ±0,811	291.713 ±1.998	295.074 ± 2.666	290.366 ±0,033
mg	2.470 ± 0,283	2,08 ± 0,313	1.964 ± 0,043	1.677 ±0,506
homem	180.287 ± 1,98	90.484 ± 1.152	281.015 ± 0,420	291.469 ± 3.250
Mês	1,133 ± 0,107	1.560 ± 0,084	2.233 ± 0,289	3.634 ± 0,164
Não	4.014 ± 0,033	4.197 ± 0,330	4.229 ± 0,230	5.475 ± 0,216
P	ND	ND	ND	ND
Se	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe
Zinco	87.495 ± 1.160	76.320 ± 2.100	68.889 ± 0,829	100.027 ± 1.267
Pb	2.077 ± 0,157	3,3115 ± 0,316	6.016 ± 0,679	8.762 ± 0,282

Note: ND: not determined; < LOD: analyte concentration below the detection limit.
Now it says:
Table 2. Concentration of metalloids quantified in H. hydrochaeris faeces (mg/kg).

 

Metalóide	Parque Ecológico Anhanduí	Parque Estadual Prosa	Parque Ecológico Sóter	Lago do Amor	valor p
Amostra	n = 3	n = 3	n = 3	n = 3
Todos	124,488 a ±0,763	130,443 b ± 2,179	139,564 c ± 0,669	140,322 c ±1,222	<0,0001
Como	0,003 b ± 0,00015	0,004 c ± 0,0003	0,004 c ± 0,0002	0,001 a ± 0,0002	<0,0001
Cd	0,4193 a ± 0,008	0,940 b ± 0,0407	0,568 a,b ± 0,053	1,042 c ± 0,302	0,0033
Co	1,467 b ± 0,130	1,019 a ± 0,0399	1,776 c ± 0,069	1,925 c ± 0,066	<0,0001
Cr	20,275 b ± 0,970	17,602 a ± 0,827	21,405 b ± 0,970	26,866 ± 1,013 c	<0,0001
Com	29,946 a ± 1,296	32,632 a ± 0,843	32,511 a ± 2,042	50,764 b ± 0,853	<0,0001
Fé	300,369 c ±0,811	291,713 a,b ±1,998	295,074 b ± 2,666	290,366 a ± 0,033	0,0004
mg	2,470 ± 0,283	2,08 ± 0,313	1,964 ± 0,043	1,677 ±0,506	0,0969
homem	180,287 b ± 1,98	90,484 a ± 1,152	281,015 c ± 0,420	291,469 d ± 3,250	<0,0001
Mês	1,133 a ± 0,107	1,560 a ± 0,084	2,233 b ± 0,289	3,634 c ± 0,164	<0,0001
Não	4,014 a ± 0,033	4,197 a ± 0,330	4,229 a ± 0,230	5,475 b ± 0,216	0,0002
P	ND	ND	ND	ND	ND
Se	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe	ND
Zinco	87,495 c ± 1,160	76,320 b ± 2,100	68,889 a ± 0,829	100,027 d ± 1,267	<0,0001
Pb	2,077 a ± 0,157	3,3115 b ± 0,316	6,016 c ± 0,679	8,762 d ± 0,282	<0,0001

Note: ND: not determined; < LOD: analyte concentration below the detection limit. Different letters in the same line mean significant differences.

3. Results
Major correction
3.1. Quantification of macro and microelements in feces
Comment 5: Please replace "macro and microelements" with "Metalloids."
Response 5: Thanks. “Macro and microelements” was replaced with “Metal(loid)s”. We cannot use only the term metalloids as it refers to the elements that have intermediate properties between metals and non-metals (Reichelt-Brushett and Batley 2023).
Reichelt-Brushett, A.; Batley, G. Metals and Metalloids. In Marine Pollution – Monitoring, Management and Mitigation; Reichelt-Brushett, A., Ed.; Springer Nature Switzerland: Cham, 2023; pp. 101–127 ISBN 978-3-031-10127-4.

 

 

Comment 6: Therefore, one-way ANOVA analysis was used, and there was no difference between the 239 mean element concentrations of the different locations (p = 0.805).
ANOVA was not mentioned in the Material and Methods section. You describe that you used the non-parametric Kruskal-Wallis test? Please make some statistical topics clearer, such as: what was the distribution of the variables (metalloids), which test was used for the comparison between the studied locations. What was actually correlated? These issues are not clear in the manuscript.
Response 6: We reviewed our statistics section as presented below

Where it said:
The means and standard deviations of the faeces samples, as well as the statistical tests to verify normal distribution, the Kruskal-Wallis non-parametric test (multiple and independent variables), the Spearman and Kendall correlation test were performed in the Oring 9.1 software (OriginLab’s Origin Corporation Viewer 9.1, Northampton, MA, USA.TM). The significance of the differences between the means for the individual trace element was considered at p < 0.05.
Now it says:
We used One-way ANOVA to test if each elemental concentration differs in the different sampling sites in faeces samples. The significance of the differences between the means for the individual metal(loid) was considered at p < 0.05.

 

Where it said:
Therefore, one-way ANOVA analysis was used, and there was no difference between the mean element concentrations of the different locations (p = 0.805). Furthermore, according to the Kruskal-Wallis test, there are no differences between the group medians. In table 2, the chemical element such as P has not been determined, and Se is below the detection limit.
Now it says:
The concentrations of the metal(loid)s Al, As, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, Se, Zn, and Pb quantified in H. hydrochaeris feaces samples from Anhanduí Ecological Park, Prosa State Park, Sóter Ecological Park, and Lago do Amor are presented in Table 2. The concentration values of quantified metal(loid)s showed normal distribution. The chemical element P has not been determined, and Se is below the detection limit. Besides Mg, all other elements had significant difference between collection sites.

 

Where it said:
Table 2. Concentration of macro and microelements quantified in Hydrochoerus hydrochaeris faeces (mg/kg).

Elemento	Parque Ecológico Anhanduí	Parque Estadual Prosa	Parque Ecológico Sóter	Lago do Amor
Todos	124.488 ±0,763	130.443 ± 2.179	139.564 ± 0,669	140.322 ±1.222
Como	0,002760 ± 0,000155	0,004300 ± 0,000267	0,003691 ± 0,00020	0,010041 ± 0,00019
Cd	0,4193 ± 0,008	0,940 ± 0,0407	0,568 ± 0,053	1.042 ± 0,302
Co	1.467 ± 0,130	1.019 ± 0,0399	1.776 ± 0,069	1.946 ± 0,840
Cr	20.275 ± 0,970	17.602 ± 0,827	21.405 ± 0,970	26.866 ± 1.013
Com	29.946 ± 1.296	32.632 ± 0,843	32.511 ± 2.042	50.764 ± 0,853
Fé	300.369 ±0,811	291.713 ±1.998	295.074 ± 2.666	290.366 ±0,033
mg	2.470 ± 0,283	2,08 ± 0,313	1.964 ± 0,043	1.677 ±0,506
homem	180.287 ± 1,98	90.484 ± 1.152	281.015 ± 0,420	291.469 ± 3.250
Mês	1,133 ± 0,107	1.560 ± 0,084	2.233 ± 0,289	3.634 ± 0,164
Não	4.014 ± 0,033	4.197 ± 0,330	4.229 ± 0,230	5.475 ± 0,216
P	ND	ND	ND	ND
Se	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe
Zinco	87.495 ± 1.160	76.320 ± 2.100	68.889 ± 0,829	100.027 ± 1.267
Pb	2.077 ± 0,157	3,3115 ± 0,316	6.016 ± 0,679	8.762 ± 0,282

Note: ND: not determined; < LOD: analyte concentration below the detection limit.
Now it says:
Table 2. Concentration of metalloids quantified in H. hydrochaeris faeces (mg/kg)

 

Metalóide	Parque Ecológico Anhanduí	Parque Estadual Prosa	Parque Ecológico Sóter	Lago do Amor	valor p
Amostra	n = 3	n = 3	n = 3	n = 3
Todos	124,488 a ±0,763	130,443 b ± 2,179	139,564 c ± 0,669	140,322 c ±1,222	<0,0001
Como	0,003 b ± 0,00015	0,004 c ± 0,0003	0,004 c ± 0,0002	0,001 a ± 0,0002	<0,0001
Cd	0,4193 a ± 0,008	0,940 b ± 0,0407	0,568 a,b ± 0,053	1,042 c ± 0,302	0,0033
Co	1,467 b ± 0,130	1,019 a ± 0,0399	1,776 c ± 0,069	1,925 c ± 0,066	<0,0001
Cr	20,275 b ± 0,970	17,602 a ± 0,827	21,405 b ± 0,970	26,866 ± 1,013 c	<0,0001
Com	29,946 a ± 1,296	32,632 a ± 0,843	32,511 a ± 2,042	50,764 b ± 0,853	<0,0001
Fé	300,369 c ±0,811	291,713 a,b ±1,998	295,074 b ± 2,666	290,366 a ± 0,033	0,0004
mg	2,470 ± 0,283	2,08 ± 0,313	1,964 ± 0,043	1,677 ±0,506	0,0969
homem	180,287 b ± 1,98	90,484 a ± 1,152	281,015 c ± 0,420	291,469 d ± 3,250	<0,0001
Mês	1,133 a ± 0,107	1,560 a ± 0,084	2,233 b ± 0,289	3,634 c ± 0,164	<0,0001
Não	4,014 a ± 0,033	4,197 a ± 0,330	4,229 a ± 0,230	5,475 b ± 0,216	0,0002
P	ND	ND	ND	ND	ND
Se	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe	< Nível de detalhe	ND
Zinco	87,495 c ± 1,160	76,320 b ± 2,100	68,889 a ± 0,829	100,027 d ± 1,267	<0,0001
Pb	2,077 a ± 0,157	3,3115 b ± 0,316	6,016 c ± 0,679	8,762 d ± 0,282	<0,0001

Note: ND: not determined;< LOD: analyte concentration below the detection limit. Different letters in the same line mean significant differences

 

 

Where it said:
The descending order of elements within the same group and between groups are not the same. In this case, such differences in concentration values of chemical elements in animal faecescan probably be explained due to the different types of vegetation in differ-ent sites. Individuals of H. hydrochaeris live in environments with riparian vegetation, in open areas and fields [35]. (...)
Now it says:
Except for Mg, metal(loid) content varied between all collection sites. We showed that capybara faeces are bioindicators of pollution and the detection of high concentrations of heavy metal(loid)s is a potential health risk for the population that frequents urban parks. The highest concentrations of metal(loid)s were quantified in samples from a population of capybaras living in a lagoon (Lago do Amor) that receives water from streams that cross the city. Based on the risk assessment, we detected that there is a potential risk to the health of children and adults, mainly due to the presence of Al, As, Cd, Co, Cu, and Mn.
The descending order of metal(loid)s within the same group and between groups are not the same. In this case, such differences in concentration values of metal(loid)s in animal faeces may be explained due to the different types of vegetation in different sites. Individuals of H. hydrochaeris live in environments with riparian vegetation, in open areas and fields [37]. (...)

 

 

 

Comment 7: Line 245: concentrations of elements quantified
Replace "elements" with "metalloids." Please standardize this term throughout the text, for example in Table 2: "Concentration of macro and microelements quantified in Hydrochoerus hydrochaeris feces (mg/kg)." Please replace "macro and microelements" with "metalloids."
Response 7: Thanks. “Macro and microelements” was replaced with “Metal(loid)s”. We cannot use only the term metalloids as it refers to the elements that have intermediate properties between metals and non-metals (Reichelt-Brushett and Batley 2023).
Reichelt-Brushett, A.; Batley, G. Metals and Metalloids. In Marine Pollution – Monitoring, Management and Mitigation; Reichelt-Brushett, A., Ed.; Springer Nature Switzerland: Cham, 2023; pp. 101–127 ISBN 978-3-031-10127-4.
Comment 8: I did not find the results and discussion of the correlations in the manuscript.
Response 8: Thank you for this comment. Now in the revised version, we discuss our results better. Besides, as stated above, we redid statistics, so this time we did not performed correlations. Statistical analysis are presented in Table 2.

 

 

 

Discussão

Discussion
Major correction
Comment 9: Did you not discuss Figure 2?
The discussion needs to be improved. I advise the authors to discuss the metalloids separately, for a better understanding of the toxicity of each studied metalloid.
Response 9: Now we discuss the results in more detail. We also discuss the toxicity of metalloids separately as below:
“Several heavy metals are common in our daily lives, and metalloids such as Al, As, Cd, Cu, Co, Cr, Mo, Ni, and Pb are considered carcinogenic [10]. Aluminium causes neurodegenerative brain pathological changes and changes in cytoskeletal proteins, while cobalt prevents DNA repair [10]. Furthermore, Co and its salts are genotoxic in mammals, mainly due to oxidative damage to DNA. Chromium promotes mutagenesis, RNA polymerase and DNA polymerase arrests, and alters gene expression. Copper is capable of inducing DNA strand breakage and causing base oxidation by oxygen free radicals and hydroxyl radicals, while Manganese inhibits ATP synthesis in brain mitochondria at the glutamate/aspartate exchange site or at the complex II site, subject
to the mitochondrial energy source. Nickel has a wide range of carcinogenic mechanisms that include transcription factors, production of free radicals and controlled expression of specific genes [10].”

Comment 10: Tables 3 and 4 are not in the discussion?
Response 10: The following paragraphs were added into the text (Tables 3-6 were moved to supplemental material):
Our study considered that children and adults put their hands in their mouths, and unintentional ingestion of animal faeces containing metal(loid)s may occur. Nonetheless, for children, the daily doses of metal(loid)s did not surpass the MRLs proposed by the ATSDR for any metal(loid) [44] (Table S1), independent of the type of exposure (acute, intermediate, or chronic). For adults, Cd (intermediate and chronic), Cr (Intermediate and chronic), Cu (acute and chronic), Ni (intermediate and chronic), and Zn (intermediate and chronic) were beyond the MRL accepted levels (Table S2). The MRLs are often used to determine if exposure to contaminated sites may cause adverse effects in a determined window of exposure (acute, intermediate, and chronic) [51]. In this sense, contact with these sites should be restricted.
(...)
Besides the MRL levels, other tools help assess metal(loid) contamination risk. THQ values were below 1 for all individual elements considering children, regardless of the exposure route in all sampling sites, however, for adults, elements such as Cd, Co, and Cu presented THQ above 1 for all sampling sites for oral ingestion, while Mn surpassed THQ of 1 for oral exposure at the sampling points of Anhanduí Ecological Park, Sóter Ecological Park, and Lago do Amor. Besides, Pb oral exposure for adults had THQ above 1 at Prosa State Park, Sóter Ecological Park, and Lago do Amor (Tables S3 and S4). These values contribute to HI above 1, considering the oral exposure route for adults at all sampling sites (Figure 2). No other way of exposure presented HI above 1, and HI was below 1 for children at all sampling sites, independent of the exposure route.

Figure 2. Hazard index considering oral exposure for adults by contact with H. hydrochaeris faeces.
While children may be more susceptible to intoxication considering the higher intake relative to the body weight [57], adults are exposed for longer in the environments, which may explain why THQ and HI were above 1 for adults only.
Comments on the Quality of English Language
Comment 11: Dear EditorThe manuscript requires a more comprehensive revision:
Introduction: The section on the toxicity of the studied metalloids needs to be expanded.
Materials and Methods: The number of samples is missing, and the statistical analysis is confusing and does not correspond to the results.
Results: The tables of statistical tests and correlations have not been presented.
Discussion: The discussion needs to better reflect the obtained results.
Response 11: We appreciate your inputs, and we tried and responded all your suggestions in the review, in specific topics. Briefly: We expanded the toxicity section for the metal(loid)s studied in the article; we performed new statistics, and explained in ‘Material and Methods’ and ‘Discussion’ sections; and the discussion was tailored to results to improve the understanding of their impacts.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments:

The paper is fairly straightforward. My biggest comment relates to the sole focus on metals in feces without discussing any other risks to human health – mostly things like parasite exposures via fecal exposure. It’s fine that you didn’t measure things like parasite loads, but to not mention it anywhere is a miss.

There are also a few sections which could use some thorough editing. The paper is largely written in a passive voice (e.g., ‘a decision was made to’ ) rather than active (‘we decided to’ or ‘we did’); the latter is much more direct and easier on the brain.

Specific comments:

Lines 11-12: I’m not a fecal contamination person, but it seems to me like metals and metalloids are probably not the primary concern with ingesting or inhaling fecal particles – zoonotics, parasites, viruses, and other harmful microbes are. If you agree, and what I said is true, I suggest you recognize this fact much earlier in your paper – as in, this line. Something like: ‘Along with exposure to parasites and other biological disease vectors, animal feces can also contain heavy metal(oids).’

Lines 15-19: samples from a lagoon? Were these water samples? Or is this a sewage lagoon? Do the capybara live in an enclosure or are they free-roaming?

Lines 22-23: Why would someone do this though? Aren’t there already approaches established, like soil and suspended particulate sampling that’d also provide this information?

Line 30: heavy metals and metalloids don’t really fit in this list – the rest are sources and activities; metals come from these sources and activities. You could easily say ‘Contamination of soil and water with heavy metals and metalloids is associated with food production…’

Line 32: how does the presence of heavy metals promote leaching?

Lines 32-33: the link between these statements is not clear – you are missing the step between the soil and metals then being ingested by animals.

Line 34 sounds like you are saying humans in captivity; ‘humans and captive animals’ might be better.

Lines 34-36: this line is probably in the wrong place in this paragraph

Lines 36-37: stating something more concrete would help: metal contamination is routinely observed in animals and humans living in or near contaminated areas.

Line 38: there is a leap here. It’s not clear to me why, based on the previous paragraph, that measuring metals in feces can be used to monitor health of mammals. Especially since the presence of metals in feces is both an indicator of exposure, but also of excretory processes.

Lines 42-44: Again, I suspect the presence of metals is not the only risk associated with animal wastes

Lines 45-52: what are some of the other features of these animals which make them useful for this purpose? Common proximity to humans is one. Another, and I don’t know how to say this delicately, but surely the world’s largest rodent must produce…ample amounts of feces to sample?

Lines 64-65: why would you only care about the aquatic environment here?

Figure 1: I suggest using a satellite imagery basemap for Figure 1C

Line 93: you can probably delete ‘execution of this’ and not lose any information.

Lines 101-102: was the spoon washed between sample collections? What about the gloves?

Line 103: why was only the internal part used? And is there not a technical term for this?

Lines 130-142: ok, so how many LoD and LoQ violations did you have for each metal and each site? And what did you do with the non-detects?

Lines 228-230: what hypotheses were you testing by performing these statistical tests?

Lines 239-240: this test was not described in the statistical analysis section.

Table 2 and Figure 2: I suggest replacing this table and this figure with a more informative figure. While as it stands table 2 can be added to supplemental information, a point/dot plot with error bars annotated with the measured concentrations with each metal in an individual sub-plot (and with unique y-axes) will allow the reader to quickly assess the spatial patterns in the data, but will also not make the matrix plot washed-out by metals with high concentrations, like Fe, Mn, Zn, and Al – using a common scale for all metals obscures the ones with low concentrations. Alternatively, you could log10 transform the plotted scale to better show the variability in the data.

Tables 3, 4, and 5: there is a lot of repeated text within each of the tables. And the tables are very big. I suggest also finding a way to compress this data down into more manageable parts – I also find these sorts of tables presenting quantitative data are impede comprehension. Figures are much better suited for such information.

Lines 342-352: I suggest starting with a brief summary of your results and then finish this summary paragraph with your most impactful/important results. This will then lead nicely into the next paragraph where you can discuss what you think is the most important result and what it means.

Line 357: this is not the only way though

Lines 360-363: this sort of information needs to be included in the methods section

Lines 364-370: this paragraph needs a topic sentence. Something like ‘Other studies have found metal contamination in tissues of capybara.’

Line 372: you can remove ‘it was found that’ and not lose any meaning – as presented in the paper, this is an example of ‘passive voice’ writing. Active voice (direct statements, such as ‘the concentration of Cr measured in capybara excrement was higher than Cr measured in other animals, such as red and roe deer.’ ) is much better for readability.

Line 376: ‘On the other hand’ is colloquial; ‘In contrast’ is better.

Lines 389-399: this probably belongs in the introduction.

Lines 426-435: I also suggest that you mention things like parasite loads and risks for human health. Focus only on metal loads is not a complete picture. Another thing to consider is what advantages measuring feces gives you rather than other media like soil.

Comments on the Quality of English Language

I've already mentioned it before, but there are a few instances of complicated writing in the paper.

Author Response

Please see the attachment

Reviewer 2

 

Comments and Suggestions for Authors

 

General comments:

 

Comment 1: The paper is fairly straightforward. My biggest comment relates to the sole focus on metals in feces without discussing any other risks to human health – mostly things like parasite exposures via fecal exposure. It’s fine that you didn’t measure things like parasite loads, but to not mention it anywhere is a miss.

There are also a few sections which could use some thorough editing. The paper is largely written in a passive voice (e.g., ‘a decision was made to’) rather than active (‘we decided to’ or ‘we did’); the latter is much more direct and easier on the brain.

Response 1: We thank reviewer 2 for this comment. We agree that fecal exposure is a major risk to human health due to its parasitic and zoonotic potential. However, our focus was on the detection and quantification of metalloids in capybara feces. In last years, heavy metal analyses in animal excrement have received attention due to their potential pollution (please see Zhang et al., 2012; Gupta, 2013; Frossard et al., 2019; Hejna et al., 2019). Nevertheless, in the revised manuscript, we also mention other risks of faeces to human health, such as parasite loads and zoonotic potential. Although this was not the focus of our study, we accept its relevance and it is now mentioned in the text.

In addition, we also accept the reviewer’s suggestion and replace the passive voice with the active voice.

 

Frossard, A.; Leite, F.L.G.; Silva, E.L.F.; Carneiro, M.T.W.D.; Júnior, J.L.R.; Gomes, L.C.; Endringer, D.C. The snake Bothrops jararaca (Squamata: Viperidae) is a suitable bioindicator of environmental exposure to cadmium: an experimental study. Ecol. Indic. 2019, 104, 166–171. https://doi.org/ 10.1016/j.ecolind.2019.04.079

Gupta, V. Feces of captive wild mammal use as bio-indicator of heavy metal pollution in urban air. Int. J. Innov. Res. Sci. Eng. Technol. 2013, 2, 2404–2411.

Hejna, M.; Moscatelli, A.; Onelli, E.; Baldi, A.; Pilu, S.; Rossi, L. Evaluation of concentration of heavy metals in animal rearing system. Ital. J. Anim. Sci. 2019, 18, 1372–1384. https://doi.org/ 10.1080/1828051X.2019.1642806

Zhang, F.; Li, Y.; Yang, M.; Li, W. Content of heavy metals in animal feeds and manures from farms of different scales in northeast China. Int. J. Environ. Res. Public. Health. 2012, 9, 2658–2668. https://doi.org/10.3390/ijerph9082658.

 

Specific comments:

 

Comment 2: Lines 11-12: I’m not a fecal contamination person, but it seems to me like metals and metalloids are probably not the primary concern with ingesting or inhaling fecal particles – zoonotics, parasites, viruses, and other harmful microbes are. If you agree, and what I said is true, I suggest you recognize this fact much earlier in your paper – as in, this line. Something like: ‘Along with exposure to parasites and other biological disease vectors, animal feces can also contain heavy metal(oids).’

Response 2: We agree with reviewer 2. In fact, the ingestion and inhalation of faecal particles have zoonotic potential and disease transmission. Despite this, our proposal is to evaluate possible environmental contamination with heavy metal(loid)s from capybara faeces samples. Therefore, we begin the abstract with the sentence suggested by the reviewer 2.

 

Where it said:

Humans can be exposed to heavy metal(loid)s from animal faeces, particularly in communities where they live close to each other. This study quantified metals, metalloids and non-metals in faeces of capybara (Hydrochoerus hydrochaeris) that live in parks in the city of Campo Grande (Brazil). Quantification of elements was obtained after acid digestion and using an inductively coupled plasma optical emission spectrometer.

 

Now it says:

Along with exposure to parasites and other biological disease vectors, animal faeces can also contain heavy metals and metalloids. We quantified metals, metalloids, and non-metals in faeces of capybara (Hydrochoerus hydrochaeris) that live in parks in the city of Campo Grande (Brazil). Quantification of metalloids was obtained after acid digestion and using an inductively coupled plasma optical emission spectrometer

 

Where it said:

Our results are limited only to H. hydrochaeris fecal samples, and we suggest further studies on soil and vegetation analysis and sampling in different seasons. Furthermore, it is also important to consider populations of H. hydrochaeris that live far from crops and urban regions, especially those that reside in Brazilian threatened biomes, such as Cerrado, Pantanal and Atlantic Forest. This way, it will be possible to compare the values of the elements between organs, tissues and excrement. Furthermore, future research should evaluate behaviors related to points of contact with animal faeces; animal fecal contamination with food; cultural animal fecal management behaviors; acute and chronic health risks associated with exposure to animal faeces; and factors that influence the concentrations and excretion rates of pathogens originating from animal faeces.

 

Now it says:

Our results are limited only to H. hydrochaeris fecal samples, and we suggest further studies on soil and vegetation analysis and sampling in different seasons. In addition, it is also important to consider populations of H. hydrochaeris that live far from crops and urban regions, especially those that reside in Brazilian threatened biomes, such as Cerrado, Pantanal, and Atlantic Forest. This way, it will be possible to compare the values of the metalloids between organs, tissues, and excrement.

Furthermore, future research should evaluate behaviors related to points of contact with animal faeces; animal fecal contamination with food; cultural animal fecal management behaviors; acute and chronic health risks associated with exposure to animal faeces; and factors that influence the concentrations and excretion rates of pathogens originating from animal faeces. It is also important to highlight that, for a more complete picture, it is necessary to evaluate the parasite load of capybara populations and the risk they pose to human health, since they are animals with zoonotic potential affected by a wide range of diseases derived from protozoa and metazoa [58–60]. Besides, when considering fecal samples as a tool for biomonitoring, it is possible to detect and quantify metal(loid)s that enter the food chain of wild animals and are capable of bioaccumulation.

 

Comment 3: Lines 15-19: samples from a lagoon? Were these water samples? Or is this a sewage lagoon? Do the capybara live in an enclosure or are they free-roaming?

 

Response 3: Sorry for not being clear. The fecal samples were collected on the banks of a lagoon where a population of free-ranging capybaras live. In the reviewed manuscript version, we hope it is clearer.

 

Where it said:

(iv) Lago do Amor: this is an artificial lagoon, but supplied by the Cabaça and Bandeira streams, which form the Bandeira basin. It is located within the area of the Federal University of Mato Grosso do Sul (UFMS), southern region of the city of Campo Grande (coordinates -20.503133, -54.618797).

 

Now it says:

(iv) Lago do Amor: this is an artificial lagoon, supplied by the Cabaça and Bandeira streams, which form the Bandeira basin. It is located within the area of the Federal University of Mato Grosso do Sul (UFMS), southern region of the city of Campo Grande (coordinates -20.503133, -54.618797). This lagoon is influenced by human activity and receives waste/chemical products brought by streams that cross the city, and the faeces samples were collected on its margins.

 

Comment 4: Lines 22-23: Why would someone do this though? Aren’t there already approaches established, like soil and suspended particulate sampling that’d also provide this information?

 

Response 4: Yes, we agree that soil, water, and particle samplings can also provide such information. However, our objective was to quantify the concentrations of metalloids present only in faeces. We know that capybara diet consists of vegetables, mainly grass (Borges and Colares, 2007; Corriale et al., 2011). Thus, we wanted to verify through faeces whether it is possible for H. hydrochaeris to bioaccumulate metalloids, which in addition to being harmful to the animals themselves can also pose a risk to human health. Furthermore, as previously mentioned, other studies have also evaluated the presence of metalloids in animal faeces (Zhang et al., 2012; Gupta, 2013; Frossard et al., 2019; Hejna et al., 2019). Here, we propose that faeces of capybaras, a synanthropic animal widely distributed in South America, may be an easily accessible material.

 

Borges, L.V.; Colares, I.G. Feeding habits of capybaras (Hydrochoerus hydrochaeris, Linnaeus 1766), in the ecological reserve of Taim (ESEC-Taim) - south of Brazil. Braz. Arch. Biol. Technol. 2007, 50, 409–416. https://doi.org/10.1590/S1516-89132007000300007

Corriale, M.J.; Arias, S.M.; Quintana, R.D. Forage Quality of Plant Species Consumed by Capybaras (Hydrochoerus hydrochaeris) in the Paraná River Delta, Argentina. Rangeland Ecol. Manag. 2011, 64, 257–263. https://doi.org/10.2111/REM-D-10-00024.1

Frossard, A.; Leite, F.L.G.; Silva, E.L.F.; Carneiro, M.T.W.D.; Júnior, J.L.R.; Gomes, L.C.; Endringer, D.C. The snake Bothrops jararaca (Squamata: Viperidae) is a suitable bioindicator of environmental exposure to cadmium: an experimental study. Ecol. Indic. 2019, 104, 166–171. https://doi.org/ 10.1016/j.ecolind.2019.04.079

Gupta, V. Feces of captive wild mammal use as bio-indicator of heavy metal pollution in urban air. Int. J. Innov. Res. Sci. Eng. Technol. 2013, 2, 2404–2411.

Hejna, M.; Moscatelli, A.; Onelli, E.; Baldi, A.; Pilu, S.; Rossi, L. Evaluation of concentration of heavy metals in animal rearing system. Ital. J. Anim. Sci. 2019, 18, 1372–1384. https://doi.org/ 10.1080/1828051X.2019.1642806

Zhang, F.; Li, Y.; Yang, M.; Li, W. Content of heavy metals in animal feeds and manures from farms of different scales in northeast China. Int. J. Environ. Res. Public. Health. 2012, 9, 2658–2668. https://doi.org/10.3390/ijerph9082658.

 

Where it said:

Therefore, it is possible to monitor the health of mammals and infer the level of environmental pollution through animal excrement [15-19], since is an important source of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, there is the possibility that faeces can cause impacts on human health, as they can contaminate the water used to supply the population [9]. In China, for example, the intensive production of poultry and pigs was considered one of the main contaminants of the environment due to the detection of heavy metals in faeces [20-22].

 

Now it says:

The use of sentinels, which are biological indicators, may be used to assess potential health hazards in an environmental set [11] and are a valuable tool for evaluating possible health risks to wildlife and humans. In this sense, using non-invasive tissues, such as faeces, urine, nails, hair, feathers, and spines [18,19] is helpful to screen wildlife compared to traditional methods once they protect animals from stress, pain, and sacrifice. Besides, the samples are easy to acquire, transport, and stock, and allow several measurements in the same individuals [19].

Therefore, it is possible to monitor the health of mammals and infer the level of environmental pollution through animal excrement [17-21], since it is an important source of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, it is possible that contaminated faeces can cause impacts on human health, as they can pollute the water used for population supply [9]. In China, for example, the intensive production of poultry and pigs was considered one of the main environmental contaminants due to the heavy metals presence in faeces [22-24].

 

Comment 5: Line 30: heavy metals and metalloids don’t really fit in this list – the rest are sources and activities; metals come from these sources and activities. You could easily say ‘Contamination of soil and water with heavy metals and metalloids is associated with food production…’

 

AND

 

Comment 6: Line 32: how does the presence of heavy metals promote leaching?

 

AND

 

Comment 7: Lines 32-33: the link between these statements is not clear – you are missing the step between the soil and metals then being ingested by animals.

 

Where it said:

Soil and water contamination is often associated with food production, industrial processes, pesticides and fertilizers production and use, residential sewage pollution, mining, and heavy metals and metalloids [1-3]. The accumulation of heavy metals can not only affect soil fertility and product quality [4], but also promote the migration of metals through leaching and surface runoff [5,6]. Hence, heavy metal residues in animal excrement have received scientific attention in recent years due to potential pollution risks [7-9]. In addition to contaminated soil and water, humans and animals in captivity are also affected by the presence of heavy metals, whose harmful effects exceed bio-recommended limits [10].

 

Now it says:

Contamination of soil and water with heavy metals and metalloids is associated with food production, industrial processes, pesticides and fertilizers production and use, residential sewage pollution, and mining [1–3]. The accumulation of heavy metals, together with leaching and surface runoff [4,5], can promote metal migration and affect soil fertility and product quality [6]. Such metals in soil are incorporated by plants, which in turn are ingested by animals. In addition to contaminated soil and water, humans and captive animals are also affected by the presence of heavy metals, whose harmful effects exceed bio-recommended limits [7]. Hence, heavy metal residues in animal excrement have recently received scientific attention due to potential pollution risks [8–10], and utility in acting as sentinels for environmental pollution [11]. Metal contamination is routinely observed in animals and humans living in or near contaminated areas [12–15].

 

Comment 8: Line 34: sounds like you are saying humans in captivity; ‘humans and captive animals’ might be better.

 

Where it said:

In addition to contaminated soil and water, humans and animals in captivity are also affected by the presence of heavy metals, whose harmful effects exceed bio-recommended limits [10].

 

Now it says:

In addition to contaminated soil and water, humans and captive animals are also affected by the presence of heavy metals, whose harmful effects exceed bio-recommended limits [7].

 

Comment 9: Lines 34-36: this line is probably in the wrong place in this paragraph

Response 9: Thanks for the suggestion. We have relocated this sentence within the paragraph (response from comments 5-7).

 

Comment 10: Lines 36-37: stating something more concrete would help: metal contamination is routinely observed in animals and humans living in or near contaminated areas.

 

Where it said:

Several studies have reported and highlighted results on the concentration of metals in tissues of wild mammals living in or near highly contaminated areas [11-14].

 

Now it says:

Metal contamination is routinely observed in animals and humans living in or near contaminated areas [11-14].

 

Comment 11: Line 38: there is a leap here. It’s not clear to me why, based on the previous paragraph, that measuring metals in feces can be used to monitor health of mammals. Especially since the presence of metals in feces is both an indicator of exposure, but also of excretory processes.

Response 11:  We appreciate this comment. Heavy metal pollution can negatively affect the health and welfare of mammals (Reidinger, 1972; Dollahite et al., 1978; Way and Schroder, 1982; Gupta et al. 2013). In the revised version, we have restructured the introduction and hope that the links between paragraphs are clearer.

 

Dollahite, J.W.; Younger, L.; Crookshank, H.R. Chronic lead poisoning in horses. Am. J. Vet. Res. 1978, 39, 6, 961-964.

Gupta, V. Feces of captive wild mammal use as bio-indicator of heavy metal pollution in urban air. Int. J. Innov. Res. Sci. Eng. Technol. 2013, 2, 2404–2411.

Reidinger Jr., R.F. Factors influencing Arizona bat population levels, Ph.D. Thesis, Univ. Arizona, Tucson, 172, 1972.

Way, C.A.; Schroder, G.D. Accumulation of lead and cadmium in wild population of the commensal rat, Rattus norvegegicus. Arch. Environ. Contam. Toxicol. 1982, 11, 407-417.

 

Where it said:

Therefore, it is possible to monitor the health of mammals and infer the level of environmental pollution through animal excrement [15-19], since is an important source of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, there is the possibility that faeces can cause impacts on human health, as they can contaminate the water used to supply the population [9]. In China, for example, the intensive production of poultry and pigs was considered one of the main contaminants of the environment due to the detection of heavy metals in faeces [20-22].

 

Now it says:

The use of sentinels, which are biological indicators, may be used to assess potential health hazards in an environmental set [11] and are a valuable tool for evaluating possible health risks to wildlife and humans. In this sense, using non-invasive tissues, such as faeces, urine, nails, hair, feather, and spines [18,19] is helpful to screen wildlife compared to traditional methods once they protect animals from stress, pain, and sacrifice. Besides, the samples are easy to acquire, transport, and stock, and allow several measurements in the same individuals [19].

Therefore, it is possible to monitor the health of mammals and infer the level of environmental pollution through animal excrement [17-21], since it is an important source of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, it is possible that contaminated faeces can cause impacts on human health, as they can pollute the water used for population supply [9]. In China, for example, the intensive production of poultry and pigs was considered one of the main environmental contaminants due to the heavy metals presence in faeces [22-24].

 

Comment 12: Lines 42-44: Again, I suspect the presence of metals is not the only risk associated with animal wastes

Response 12: Yes, we agree. However, in this study, we aimed to quantify metal(loid) concentrations in capybara faeces. We recognize the other risks associated with faeces, so to acknowledge the problem we added some information on that, but keeping in mind that the focus of the article is to determine de metal(loid) contamination in capybara faeces, and its possible use as a sentinel/bioindicator for environmental contamination.

 

Where it said:

Our results are limited only to H. hydrochaeris fecal samples, and we suggest further studies on soil and vegetation analysis and sampling in different seasons. Furthermore, it is also important to consider populations of H. hydrochaeris that live far from crops and urban regions, especially those that reside in Brazilian threatened biomes, such as Cerrado, Pantanal and Atlantic Forest. This way, it will be possible to compare the values of the elements between organs, tissues and excrement. Furthermore, future research should evaluate behaviors related to points of contact with animal faeces; animal fecal contamination with food; cultural animal fecal management behaviors; acute and chronic health risks associated with exposure to animal faeces; and factors that influence the concentrations and excretion rates of pathogens originating from animal faeces.

 

Now it says:

Our results are limited only to H. hydrochaeris fecal samples, and we suggest further studies on soil and vegetation analysis and sampling in different seasons. In addition, it is also important to consider populations of H. hydrochaeris that live far from crops and urban regions, especially those that reside in Brazilian threatened biomes, such as Cerrado, Pantanal, and Atlantic Forest. This way, it will be possible to compare the values of the metal(loid)s between organs, tissues, and excrement.

Furthermore, future research should evaluate behaviors related to points of contact with animal faeces; animal fecal contamination with food; cultural animal fecal management behaviors; acute and chronic health risks associated with exposure to animal faeces; and factors that influence the concentrations and excretion rates of pathogens originating from animal faeces. It is also important to highlight that, for a more complete picture, it is necessary to evaluate the parasite load of capybara populations and the risk they pose to human health, since they are animals with zoonotic potential affected by a wide range of diseases derived from protozoa and metazoa [58–60]. Besides, when considering fecal samples as a tool for biomonitoring, it is possible to detect and quantify metal(loid)s that enter the food chain of wild animals and are capable of bioaccumulation.

 

Comment 13: Lines 45-52: what are some of the other features of these animals which make them useful for this purpose? Common proximity to humans is one. Another, and I don’t know how to say this delicately, but surely the world’s largest rodent must produce…ample amounts of feces to sample?

 

Where it said:

Despite investigations using different animal species to determine the effect of contaminants on human health and the environment, some species have not yet been studied to verify whether they have a profile to be good indicators of pollution. In this sense, the capybara Hydrochoerus hydrochaeris Linnaeus, 1766 (Order Rodentia, Family Caviidae) can be an indicator of pollution due to the presence of heavy metal(loid)s in the environment. Although H. hydrochaeris is widely distributed in South America and commonly found in parks and biological reserves in Brazil, there are few studies that monitor the concentration of metals in blood plasma, fur, and excrement [23].

 

Now it says:

Despite investigations using different animal species to determine the effect of contaminants on human health and the environment, some species have not yet been studied to verify whether they have a profile to be good indicators of pollution. In this sense, the capybara Hydrochoerus hydrochaeris Linnaeus, 1766 (Order Rodentia, Family Caviidae) can be an indicator of pollution due to the presence of heavy metal(loid)s in the environment. Although H. hydrochaeris is widely distributed in South America and commonly found in parks and biological reserves in Brazil, few studies monitor the concentration of metals in blood plasma, fur, and excrement [25]. Therefore, characteristics such as large populations, proximity to humans, and the availability of large quantities of faeces (given that it is the world’s largest rodent) make this material viable as an indicator of environmental pollution.

 

Where it said:

Therefore, it is possible to monitor the health of mammals and infer the level of environmental pollution through animal excrement [15-19], since is an important source of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, there is the possibility that faeces can cause impacts on human health, as they can contaminate the water used to supply the population [9]. In China, for example, the intensive production of poultry and pigs was considered one of the main contaminants of the environment due to the detection of heavy metals in faeces [20-22].

 

Now it says:

The use of sentinels, which are biological indicators, may be used to assess potential health hazards in an environmental set [11] and are a valuable tool for evaluating possible health risks to wildlife and humans. In this sense, using non-invasive tissues, such as faeces, urine, nails, hair, feathers, and spines [18,19] is helpful to screen wildlife compared to traditional methods once they protect animals from stress, pain, and sacrifice. Besides, the samples are easy to acquire, transport, and stock, and allow several measurements in the same individuals [19].

Therefore, it is possible to monitor the health of mammals and infer the level of environmental pollution through animal excrement [17-21], since it is an important source of heavy metal(loid)s (Cu, Zn, Cr, Cd, Pb and As) for the environment [7]. Furthermore, it is possible that contaminated faeces can cause impacts on human health, as they can pollute the water used for population supply [9]. In China, for example, the intensive production of poultry and pigs was considered one of the main environmental contaminants due to the heavy metals presence in faeces [22-24].

 

And we kept the paragraph:

Since there are no studies aimed at quantifying heavy metal(loid)s in H. hydrochaeris faeces to propose this animal as an indicator of pollution, it is justifiable to carry out studies for this purpose, especially because it is a non-invasive technique. Given the above, since biological materials are considered bioindicators of pollution due to the presence of metal(loid)s [7] and the use of mammal faeces as bioindicators [22-24,26], we hypothesize that H. hydrochaeris excrement may be bioindicators of heavy metal contamination in wild mammals.

 

Comment 14: Lines 64-65: why would you only care about the aquatic environment here?

Response 14: Sorry about that. Our concern is with aquatic and terrestrial environments, and we removed this section so as not to create doubt for the reader.

 

Comment 15: Figure 1: I suggest using a satellite imagery basemap for Figure 1C

 

Where it said:

Figure 1. Geographic location of Campo Grande/Brazil a Brazilian territory b State of Mato Grosso do Sul (MS), Brazilian Central-West c City of Campo Grande and sampling sites 1 Anhanduí Ecological Park 2 Lago do Amor 3 Sóter Ecological Park 4 Prosa State Park.

Now it says:

Figure 1. Geographic location of Campo Grande/Brazil a. Brazilian territory b. State of Mato Grosso do Sul (MS), Brazilian Central-West c. City of Campo Grande and sampling sites 1. Anhanduí Ecological Park; 2. Lago do Amor; 3. Sóter Ecological Park; and 4. Prosa State Park.

 

Comment 16: Line 93: you can probably delete ‘execution of this’ and not lose any information.

Response 16: Deleted.

 

Comment 17: Lines 101-102: was the spoon washed between sample collections? What about the gloves?

 

Where it said:

A quantity of 550 g of faeces from each site was collected using a large 12.5 cm stainless steel spoon and surgical gloves to avoid contamination. Collections were carried out in the morning and only the internal part of the faeces was collected and stored in a 10x14-80 PE zip lock plastic bag.

 

Now it says:

Three fecal samples were collected in a zigzag pattern in locations close to water bodies for each site. Collections were carried out in September 2021 and samples in direct contact with the soil were avoided. A quantity of 550 g of faeces from each site was collected using a large 12.5 cm stainless steel spoon and surgical gloves to avoid contamination. The stainless steel spoon was washed with ultrapure water and 70% alcohol and surgical gloves were replaced between sample collections. The samples were collected in the morning, using the central faeces in the pile only, then immediately stored in a 10x14-80 PE zip-lock plastic bag.

 

Comment 18: Line 103: why was only the internal part used? And is there not a technical term for this?

Response 18: We only use the central faeces in the pile to avoid contamination of the sample by any external factor (dirt). In addition, elements such as As and Se are volatile. We are not aware of any technical term to refer to the internal part of faecal samples, but we tried to better the understanding of the term by using central faeces in the pile.

 

Comment 19: Lines 130-142: ok, so how many LoD and LoQ violations did you have for each metal and each site? And what did you do with the non-detects?

Response 19:

We just had violations for selenium content (values below LOD), which were discarded. In the same way, phosphorus content was above the instrument detection and we did not consider them, referring to them as not determined (Table 2).

 

Comment 20: Lines 228-230: what hypotheses were you testing by performing these statistical tests?

 

AND

 

Comment 21: Lines 239-240: this test was not described in the statistical analysis section.

Response 20 and 21:

 

1. We used One-way ANOVA to test if each elemental concentration differs in the different sites in faeces samples.

H₀: The total amount of detected element is the same in all sampling sites in faeces samples.

H₁: The total amount of detected element is different in at least one sampling site in faeces samples.

 

Where it said:

The means and standard deviations of the faeces samples, as well as the statistical tests to verify normal distribution, the Kruskal-Wallis non-parametric test (multiple and independent variables), the Spearman and Kendall correlation test were performed in the Oring 9.1 software (OriginLab’s Origin Corporation Viewer 9.1, Northampton, MA, USA.TM). The significance of the differences between the means for the individual trace element was considered at p < 0.05.

 

Now it says:

We used One-way ANOVA to test if each elemental concentration is different in the different sampling sites in faeces samples. The significance of the differences between the means for the individual metal(loid) was considered at p < 0.05.

 

Comment 22: Table 2 and Figure 2: I suggest replacing this table and this figure with a more informative figure. While as it stands table 2 can be added to supplemental information, a point/dot plot with error bars annotated with the measured concentrations with each metal in an individual sub-plot (and with unique y-axes) will allow the reader to quickly assess the spatial patterns in the data, but will also not make the matrix plot washed-out by metals with high concentrations, like Fe, Mn, Zn, and Al – using a common scale for all metals obscures the ones with low concentrations. Alternatively, you could log10 transform the plotted scale to better show the variability in the data.

Response 22: We appreciate this suggestion from the reviewer 2. However, we believe that Table 2 can be maintained. We replaced Figure 2. As an example, Table 1 (Lima et al., 2021), Table 2 (Pour et al., 2024), and Tables 2 and 4 and Figures 3 and 5 (Junior et al., 2024) also present results in this way. Therefore, Table 2 presents the metalloid concentrations of each sampling site in a discriminatory manner, and Figure 2 is a more visual way of presenting the metalloid concentrations of each site.

 

Junior, A.S.A.; Ancel, M.A.P.; Garcia, D.A.Z.; Melo, E.S.P.; Guimarães, R.C.A.; Freitas, K.C.; Bogo, D.; Hiane, P.A.; Vilela, M.L.B.; Nascimento, V.A. Monitoring of Metal(loid)s Using Brachiaria decumbens Stapf Leaves along a Highway Located Close to an Urban Region: Health Risks for Tollbooth Workers. Urban Sci. 2024, 8, 128. https://doi.org/10.3390/urbansci8030128

Lima, N.V.; Arakaki, D.G.; Melo, E.S.P.; Machate, D.J.; Nascimento, V.A. Assessment of Trace Elements Supply in Canned Tuna Fish Commercialized for Human Consumption in Brazil. Int. J. Environ. Res. Public. Health. 2021, 18, 22: 12002. https://doi.org/ 10.3390/ijerph182212002

Pour, E.S.; Yazdi, F.T.; Kahrarianc, M.; Rezazadeh, N. Impacts assessment of municipal and industrial wastewater pollutants on soil invertebrate (Hexapoda; Collembola) biodiversity. Int. J. Environ. Sci. Technol. 2024, 21: 7153-7164. https://doi.org/10.1007/s13762-024-05527-6

 

Comment 23: Tables 3, 4, and 5: there is a lot of repeated text within each of the tables. And the tables are very big. I suggest also finding a way to compress this data down into more manageable parts – I also find these sorts of tables presenting quantitative data are impede comprehension. Figures are much better suited for such information.

Response 23: We agree with this comment. Indeed, the tables present a lot of information. However, we believe it is important to maintain this information, even in table format. Therefore, we have converted tables 3, 4, 5, and 6 to Supplementary Material, now tables S1, S2, S3, and S4, respectively.

 

Comment 24: Lines 342-352: I suggest starting with a brief summary of your results and then finish this summary paragraph with your most impactful/important results. This will then lead nicely into the next paragraph where you can discuss what you think is the most important result and what it means.

Response 24: Thank you for the suggestion. We now begin the discussion with a brief summary of the main results.

 

Where it said:

The descending order of metal(loid)s within the same group and between groups are not the same. In this case, such differences in concentration values of metal(loid)s in animal faeces may be explained due to the different types of vegetation in different sites. Individuals of H. hydrochaeris live in environments with riparian vegetation, in open areas and fields [37]. However, it is a herbivorous animal that feeds on grasses and aquatic vegetation, and can also feed on corn and sugar cane [38].

 

Now it says:

We showed that capybara faeces are bioindicators of pollution and the detection of high concentrations of heavy metal(loid)s is a potential health risk for the population that frequents urban parks. The highest concentrations of metal(loid)s were quantified in samples from a population of capybaras living in a lagoon (Lago do Amor) that receives water from streams that cross the city. Based on the risk assessment, we detected that there is a potential risk to the health of children and adults, mainly due to the presence of Al, As, Cd, Co, Cu, and Mn.

The descending order of metal(loid)s within the same group and between groups are not the same. In this case, such differences in concentration values of metal(loid)s in animal faeces may be explained due to the different types of vegetation in different sites. Individuals of H. hydrochaeris live in environments with riparian vegetation, in open areas and fields [37]. However, it is a herbivorous animal that feeds on grasses and aquatic vegetation, and can also feed on corn and sugar cane [38].

 

Comment 25: Line 357: this is not the only way though

Response 25: Yes, we agree that this is not the only way to assess the health and quality of aquatic and terrestrial environments, but rather a tool that can assist in the assessment of such environments. Therefore, we have included text to make it clearer.

 

Where it said:

Thus, quantifying the bioaccumulation and biomagnification of heavy metals in these mammals can be used to assess the health and quality of aquatic and terrestrial environments [38].

 

Now it says:

Thus, quantifying the bioaccumulation and biomagnification of heavy metals in these mammals can be a tool to assist in assessing the health and quality of aquatic and terrestrial environments [40].

 

Comment 26: Lines 360-363: this sort of information needs to be included in the methods section

Response 26: Thank you for the suggestion. We have added this information to “Materials and Methods” as well, but we have also left it in the “Discussion” section to revisit this topic that we think is important to discuss.

 

Where it said:

(iv) Lago do Amor: this is an artificial lagoon, but supplied by the Cabaça and Bandeira streams, which form the Bandeira basin. It is located within the area of the Federal University of Mato Grosso do Sul (UFMS), southern region of the city of Campo Grande (coordinates -20.503133, -54.618797).

 

Now it says:

(iv) Lago do Amor: this is an artificial lagoon, supplied by the Cabaça and Bandeira streams, which form the Bandeira basin. It is located within the area of the Federal University of Mato Grosso do Sul (UFMS), southern region of the city of Campo Grande (coordinates -20.503133, -54.618797). This lagoon is influenced by human activity and receives waste/chemical products brought by streams that cross the city, and the faeces samples were collected on its margins.

 

Comment 27: Lines 364-370: this paragraph needs a topic sentence. Something like ‘Other studies have found metal contamination in tissues of capybara.’

Response 27: Thanks for this suggestion, and this excerpt has been added to the beginning of the paragraph.

 

Where it said:

In a study carried out with capybaras victims of being run over in Southern Brazil, the presence of Ag, Cd, Cu, Pb, and Zn was found in the liver, kidney, fat and muscle, and is justified by the use of pesticides and herbicides in the region [23]. Such animals are subject to the effects of bioaccumulation of contaminants, through ingestion of plants contaminated by metals, sediments and water. It is worth mentioning that the time of year is a key factor that influences the concentration of elements in the organs of these animals, which can be caused by the availability of food depending on the season [23].

 

Now it says:

Ramm (2015) reported capybara tissue contamination by metals [49]. Capybaras victims of being run over in Southern Brazil, the presence of Ag, Cd, Cu, Pb, and Zn was found in the liver, kidney, fat, and muscle, and is justified by the use of pesticides and herbicides in the region [49]. Such animals are subject to the effects of bioaccumulation of contaminants, through ingestion of plants contaminated by metals, sediments, and water. It is worth mentioning that the time of year is a key factor that influences the concentration of metal(loid)s in the organs of these animals, which can be caused by the availability of food depending on the season [49].

 

Comment 27: Line 372: you can remove ‘it was found that’ and not lose any meaning – as presented in the paper, this is an example of ‘passive voice’ writing. Active voice (direct statements, such as ‘the concentration of Cr measured in capybara excrement was higher than Cr measured in other animals, such as red and roe deer.’ ) is much better for readability.

 

Response 27: We agree with this suggestion and have changed the text for better readability.

 

Comment 28: Line 376: ‘On the other hand’ is colloquial; ‘In contrast’ is better.

Response 28: Thank you. “On the other hand” was replaced with “In contrast” in the manuscript.

 

Comment 29: Lines 389-399: this probably belongs in the introduction.

Response 29: Thank you for this. This passage is now better located in the second paragraph of the introduction.

 

Where it said:

Hence, heavy metal residues in animal excrement have received scientific attention in recent years due to potential pollution risks [7-9]. In addition to contaminated soil and water, humans and animals in captivity are also affected by the presence of heavy metals, whose harmful effects exceed bio-recommended limits [10]. Several studies have reported and highlighted results on the concentration of metals in tissues of wild mammals living in or near highly contaminated areas [11-14].

 

Now it says:

Hence, heavy metal residues in animal excrement have recently received scientific attention due to potential pollution risks [8–10], and utility in acting as sentinels for environmental pollution [11]. Metal contamination is routinely observed in animals and humans living in or near contaminated areas [12–15].

The main sources of fecal pollution of human or animal origin (domestic or wild) that affect urban land surfaces are not specific. They are related to: (i) the lack of sewage treatment; (ii) sewage, septic tank, and tank leaks; (iii) inadequate waste disposal; and (iv) open defecation of humans, and other animals. Such sources are common in developing countries and cause a random dispersion of faeces in urban areas. The ingestion of dust and soil contaminated with fecal material from humans and domestic animals occurs mainly during childhood [16]. Floors or carpets act as collectors for dust that contains dust mites, bacteria, allergens, and faeces from shoes. Hence, ingesting or inhaling this material in urban areas can be a threat to human health [16,17]. From now on, the involuntary ingestion of faeces is referred to as “soil/faeces”.

The use of sentinels, which are biological indicators, may be used to assess potential health hazards in an environmental set [11] and are a valuable tool for evaluating possible health risks to wildlife and humans. In this sense, using non-invasive tissues, such as faeces, urine, nails, hair, feathers, and spines [18,19] is helpful to screen wildlife compared to traditional methods once they protect animals from stress, pain, and sacrifice. Besides, the samples are easy to acquire, transport, and stock, and allow several measurements in the same individuals [19]. (...)

 

Comment 29: Lines 426-435: I also suggest that you mention things like parasite loads and risks for human health. Focus only on metal loads is not a complete picture. Another thing to consider is what advantages measuring feces gives you rather than other media like soil.

Response 29: We agree with reviewer 2. In the reviewed manuscript, we present referenced information about the parasitic potential and the risks this poses to human health. However, we aimed to quantify the concentrations of metalloids present in capybara faeces and prove that such elements enter the food chain of such animals. If we had analyzed other media, such as soil, water or plants, for example, it would not have been possible to prove that metalloids pass through the organism and have the potential for bioaccumulation. Therefore, we have added the following excerpt: “It is also important to highlight that, for a more complete picture, it is necessary to evaluate the parasite load of capybara populations and the risk they pose to human health, since they are animals with zoonotic potential affected by a wide range of diseases derived from protozoa and metazoa (Souza et al., 2021a, b; Uribe et al., 2021). Furthermore, when considering faecal samples as a tool for biomonitoring, it is possible to detect and quantify metalloids that enter the food chain of wild animals and are capable of bioaccumulation.”

 

Where it said:

Our results are limited only to H. hydrochaeris fecal samples, and we suggest further studies on soil and vegetation analysis and sampling in different seasons. Furthermore, it is also important to consider populations of H. hydrochaeris that live far from crops and urban regions, especially those that reside in Brazilian threatened biomes, such as Cerrado, Pantanal and Atlantic Forest. This way, it will be possible to compare the values of the elements between organs, tissues and excrement. Furthermore, future research should evaluate behaviors related to points of contact with animal faeces; animal fecal contamination with food; cultural animal fecal management behaviors; acute and chronic health risks associated with exposure to animal faeces; and factors that influence the concentrations and excretion rates of pathogens originating from animal faeces.

 

Now it says:

Our results are limited only to H. hydrochaeris fecal samples, and we suggest further studies on soil and vegetation analysis and sampling in different seasons. In addition, it is also important to consider populations of H. hydrochaeris that live far from crops and urban regions, especially those that reside in Brazilian threatened biomes, such as Cerrado, Pantanal, and Atlantic Forest. This way, it will be possible to compare the values of the metalloids between organs, tissues, and excrement.

Furthermore, future research should evaluate behaviors related to points of contact with animal faeces; animal fecal contamination with food; cultural animal fecal management behaviors; acute and chronic health risks associated with exposure to animal faeces; and factors that influence the concentrations and excretion rates of pathogens originating from animal faeces. It is also important to highlight that, for a more complete picture, it is necessary to evaluate the parasite load of capybara populations and the risk they pose to human health, since they are animals with zoonotic potential affected by a wide range of diseases derived from protozoa and metazoa [56-58]. Furthermore, when considering faecal samples as a tool for biomonitoring, it is possible to detect and quantify metalloids that enter the food chain of wild animals and are capable of bioaccumulation.

 

Souza, D.S.; Yang, S.G.N.S.; Alves, A.C.A.; Pontes, R.M.P.; Carvalho, C.C.D.; Soares, P.C.; Oliveira, J.B.O. Parasites and health status of free-ranging capybaras (Hydrochoerus hydrochaeris) in the Atlantic Forest and Caatinga biomes of Brazil. Vet. Parasitol. Reg. Stud. Reports. 2021a, 21, 100503. https://doi.org/10.1016/j.vprsr.2020.100503

Souza, S.L.P.; Benatti, H.R.; Luz, H.R.; Costa, F.B.; Pacheco, R.C.; Labruna, M.B. Endoparasites of capybaras (Hydrochoerus hydrochaeris) from anthropized and natural areas of Brazil. Braz. J. Vet. Parasitol. 2021b, 30, e027420. https://doi.org/10.1590/S1984-29612021049

Uribe, M.; Hermosilla, C.; Rodríguez-Durán, A.; Vélez, J.; López-Osorio, S.; Chaparro-Gutiérrez, J.J.; Cortés-Vecino, J.A. Parasites Circulating in Wild Synanthropic Capybaras (Hydrochoerus hydrochaeris): A One Health Approach. Pathogens, 2021, 7, 10: 1152. https://doi.org/10.3390/pathogens10091152. PMID: 34578184; PMCID: PMC8467752.

 

Comments on the Quality of English Language

 

Comment 30: I've already mentioned it before, but there are a few instances of complicated writing in the paper.

Response 30: Thank you for your suggestion. We carefully revised our writing and we hope it is suitable now.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I agree with the corrections and responses provided by the authors, and therefore, I have no further questions.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing the comments