Review Reports - Metal Bioaccumulation by Carp and Catfish Cultured in Lake Chapala, and Weekly Intake Assessment

Round 1

Reviewer 1 Report

I add my comments in a separate PDF.

Comments for author File: Comments.pdf

Author Response

Questions to the authors:

I have a question to the Materials section: How many individuals per test species (carp / catfish) were put into the net cage at the beginning of the experiment? Could you include this information? Were both species cultured in the same cage or were there separated cages and replicates? It is not clear from the text.

The number of fish placed on the net cages was: in the lake 200 carps and 1000 catfish and 70 carps and 300 catfish in the control pond. Both species were cultured in the same cage, but during the early stage of the catfish they were kept in a nursery cage into the big cage. At the catfish age of 30 weeks the nursery cage was removed and the catfish were left in contact with the carps. All these information were introduced in the paragraph Line 56-66.

In L 62 a control fish group cultured in lake Jocotepec is mentioned. However, no data or information on the control group is found in the results or discussion section. (Supplementary material? See below.)

Thank you for the constructive observation. New set of graphics were performed and the information of the control group were introduced. See Figures 2, 3, 4, 5, 6 and 7.

Additionally, comments including the control group were introduced to the text on the lines 146-148, 156-157, 164, 175-178, 203-204, 214, 235, 290.

In L 74-75 analysis of water and sediment is mentioned, as well. However, there is no table / figure in the paper with the results. Only some of the data are mentioned in the text without a reference to a previous research. Did authors mean to collect these data (together with the control group – as mentioned above) in supplementary material or should there be reference to previous article?

We appreciate the comment. Alvarado et al (2020) was a previous study and sediment and water information were widely discussed there. We eliminate the water and sediment from the text, see abstract lines 18-19 and metodology lines 90-91. But in contrast, we decided to make reference to Alvarado et al., (2020)[6] in the discussion section when was necessary, see the lines 302 and 338-341.

A question to Discussion: What was the reason of using different age of the fish species?

The reason for the difference of the age between species was the lack of fish at the same age from the reliable suppliers. We were very careful in selecting suppliers who offered fish free from infections and parasites, and at the time for starting the experiment only these fish were found. It was not possible to extend the starting point of the experiment to have 2 set of experiments because of the limited resource.

Detailed comments to the text:

L56: The values (average ± SD) should be expressed in the same number of digits, so far8.10± 0.05 g would be more appropriate. The same problema is further in some tables.

The correction sugested was made, and every number was writen with 2 digits, see Table 2.

The table 1 was left with the number of digits present in the Certificate of the reference material. And the Table 4 the EWI calculation was left with 3 digits in order to get the mínimum number to obtain %PTWI. If we had express EWI with only 2 digits, we would have had cero in %PTWI some times.

L 60: Please adjust the m2 and m3 units using the upper index, as: m² and m³

The correction sugested was made, see lines 62-63

L 64: 50g – please, add a space between the value and unit.

The correction sugested was made, see line 68

L 87: Same digits for average and SD values.

The correction sugested was made, see line 94.

L 88: Please, write the acid formula correctly (with subscript / lower index).

The correction sugested was made, see line 95.

L 90: Space between value and unit (210 °C)

The correction sugested was made, see line 97.

Table 1: The unit formatted in column “Units” are weird and not consistent with the following text. I suggest to make a correction (mg.kg^-1) and use the same form in the following text, or the opposite, replace the unit format in the table with “mg/kg” etc.

Homogenization of the units throghout entire document were performed. The unit mg/kg was used for Cu and Zn, and µg/kg was selected for non-essential metals and water.

Table 2: Values in a given column should be written with the same digits (average and SD);

Thank you for the comment, homogenization of the digits was made on Table 2, and the LCF was left in integers since is a factor.

Make sure that the units are in the same format (mg/kg vs. µg /kg);

Because of the magnitude of the metal concentration, every the Cu and Zn concentration were reported in mg/kg along the paper. On the other hand, every non-essential metal concentration in tissues were reported in µg/kg along the paper. It is very common to use these way because the non-essential metals usually are present in two or three lower magnitude of concentration than essential metals.

There are errors within the text in L 143 – L 211:

1) Except for the text regarding Copper, wrong Figure numbers are cited. (e.g., L 158: Figure 2 shows… should be Figure 3… etc.) This problem repeats until the “Mercury” section.

We are very thankful for the observation, all the mistakes were corrected in the text.See lines 161 to 239.

2) Some of the values in the text are different from the values in Table 2. (e.g., L 150: 6.44-47.8 ppm does not refer to 4.81-66.4 mg/kg in the Table.) More of such differences in the following text. Please, correct them.

Values different from those in Table 2 are in lines 150, 151, 159, 164.

We appreciate your observation. We checked carefuly every number in the table 2 and in the text and the mistakes were corrected. See Table 2 and Lines 158-158, 170-171, 177

L 256: spelling error (“results”)

The error were corrected in line 298

L 262: spelling error (“couldbe”)

The error were corrected in line 304

L: spelling error (“wilde carp”)

The error were corrected in line 327

L 287: Mexican standard (Norma Oficial Mexicana PROY-NOM-127-SSA1-2017…) is cited but it is not involved in the list of references.

We appreciate your observation. The NOM-127-SSA1-1994 has been included and is now in the reference 31.

At the same time, number 44 in the reference list refers to another norm (NOM-033) which is not cited in the text (the authors probably meant to cite it in the materials part?)

Two references about the code of slauther of domestic animals were better included in the text of Institutional Review Board Statement and eliminated from the References.

In Discussion section, there are some references cited by name + year instead of reference number, such as in L 340. All the citations should be in the same style.

Thank you for the comment, the citations by name were homogenized according to the instruction to the authors, using Name +[number of reference].

Table 5 contains some notes (indexes a, b, c) which are not explained. Also the font used in this table is not unique. Please, make the corrections.

Corrections were made, see Table 5 at the bottom in lines 408-411.

As for not all the literature sources are cited via numbers, I kindly advise the authors to go through the references thoroughly and make sure that every source is cited correctly.

Thank you for the observation, the citations by name were homogenized according to the instruction to the authors, when was neccesarry using Name +[number of reference] wer used. The reference as books were corrected.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper investigates five heavy metal concentrations in the muscle and liver of catfish fingerlings and adult carp which were 15 weeks and 30 weeks old at the start of a caged experiment conducted in Lake Chapala in Mexico. The experiment was run for 473 days and fish sampled 10 times over the course of the study (3-5 individuals each). Cages floated off the lake bottom and fish were fed with Winfish-Ziegler 2505 pellets. A control experiment was conducted in a pond in a lakeside community. Weekly metal intake was estimated and compared with provisional tolerable weekly intake. The authors conclude that metal concentrations in fish do not represent a risk for human consumption. On a first read, the paper appears to merit production, but a closer examination reveals a number of flaws which go beyond a major revision. Hence, I am recommending rejection with an opportunity to resubmit. Matters that require addressing are as follows.

The study is not well designed in my opinion. No reason is given for investigating the fish for more than a year when, apart possibly from mercury, there is no reason to assume that metal concentrations would change if the diet remained constant. Temporal data are shown but no statistical tests are performed to determine whether this is just natural variability associated with measuring metals in a small number of fish, with features of fish growth and reproduction over time, and/or changes in their physical and chemical environment. The abstract mentions that the water and sediment were monitored for metals (presumably also basic water quality parameters), but no data are presented.
The rationale for the location of the control lake is not given. I would have thought a better control would have been to have the cages on the bottom of Lake Chapala and/or collecting fish that presumably are naturally living in Chapala Lake. I did not see the pond fish metal data. Are there any data for mercury and other metals in the local fishery? The nature of the fish populations in the lake and their harvesting also is not given.
No statistical tests are performed to investigate whether temporal variability in metals concentrations is significant. Nor are tests performed to compare metal concentrations in liver and fillet and between species. Data are presented as ranges (Table 1); means and standard deviations (or errors) should also be shown. The LCF is based on the average over the 10 observations when the average of the calculations for each sampling period should be calculated. Figures 2-6 either are not showing standard deviations or they are unrealistically small for a sample size of 3-5 fish which presumably were measured individually.
Figures 2-6 are not easily viewed. It would make more sense to plot metal concentrations for a given species on its own graph with two Y axes for the differences in concentration over time. It would be easier to follow temporal patterns that way and I am not sure that they are even real. The use of a small black dot and a small black triangle do not provide for clarity. An open circle would provide more contrast. If this is to appear online, color also could be used.
How does table 3 differ from table 2? Is this the last day of measurement or the time after fish attained the commercial weight; this weight is not given. Again, no standard deviations or error or sample size. Table 3 could be combined with table 4. I see no value to the maximum estimates. At best only the %PTWI column would be needed. It would be an easier read to insert another 6-8 columns into Table 3 or, if too large, have an upper series of rows for the carp and lower row for the catfish or whatever order of presentation is followed for the figure.
Table 5 is poorly organized and should be done by metal, then species and then location. The authors also should proof their references as some of the values seem widely off. I looked up Fierro et al. (reference 42) and see that their figures of metal concentration are in µg/g and this is dry weight. However, their table 2 is mg/kg which is most likely a typographic error missed by these authors in their galley proof. The authors do not show a risk to lead intake and only a small exceedance (2.3) for Cd with quite a different outcome in the manuscript I am reviewing. This points to sloppy reading and synthesis on the authors. They may wish to look at the other data presented in Table 5 of Fierro et al. for additional comparison and then verify that some of their other high values for Cd and Pb are not the result of data entry errors including reading incorrectly from a paper or not mixing wet and dry weight determinations. To the best of my knowledge, daily intake calculations are based on wet weight determinations as that is how food is sold and consumed. The metal data for reference 12 look odd to me with more than 100-fold difference in concentrations Cd and Pb between seasons which is not reflected in similar variability for mercury nor in the current study where river flow is the main source of metal input.
Copper and zinc are essential metals, and no discussion is given on how mineral needs may or may not be met by consuming fish fillet and liver. Would add balance.

In summary, the authors have an interesting data set, but the paper needs a lot of work before it is suitable for publication.

Author Response

We are very grateful to the reviewers for the constructive and useful comments and we are sending the detailed answers of the manuscript ID: applsci-1221374 entitled: Comparison between metal accumulation in catfish and carp cultured in an alkaline lake: Chapala.

We are including one version with the Track changes function.

REVIEWER 2.

The study is not well designed in my opinion. No reason is given for investigating the fish for more than a year when, apart possibly from mercury, there is no reason to assume that metal concentrations would change if the diet remained constant. Temporal data are shown but no statistical tests are performed to determine whether this is just natural variability associated with measuring metals in a small number of fish, with features of fish growth and reproduction over time, and/or changes in their physical and chemical environment. The abstract mentions that the water and sediment were monitored for metals (presumably also basic water quality parameters), but no data are presented.

Thank you for the comment. We have a previous pilot study in catfish and we observed that at least Cu, Zn and Cd concentrations changed even when the food was controlled. Then we decided to follow for one and a half year. The lenght of the time was chosen because it is the age at which the fish are taken for sale. It was very important for us to evaluate mercury in fish from aquaculture, once the Lake Chapala have a historial from mercury in fish. This was exposed in line 393 and the previous Works and references 10, 11 and 38.

In the present work we are focused on the bioaccumulation pattern and food assessment perspective, but we have a previous work, Alvarado et al (2020), where sediment and water information were widely discussed. For these reason we remove water and sediment from the text, see abstract lines 18-19 and metodology lines 90-91. But in contrast, we decided to make reference to Alvarado et al., (2020)[6] in the discussion section when was necessary, see the lines 302 and 338-341.

2. The rationale for the location of the control lake is not given. I would have thought a better control would have been to have the cages on the bottom of Lake Chapala and/or collecting fish that presumably are naturally living in Chapala Lake. I did not see the pond fish metal data. Are there any data for mercury and other metals in the local fishery? The nature of the fish populations in the lake and their harvesting also is not given.

The control group were in an eath pond, not a control lake, this was highlighted in Materials and methods line 66 and in Figure 1. The earth pond allow us to maintain a small group of the same fish, same age at the same weather.

Major emphasis was given to the control group as can be seen in the comments included in the body text on the lines 146-148, 156-157, 164, 175-178, 203-204, 214, 235, 290. Additionally, a new set of graphics were performed and the information of the control group were introduced. See Figures 2, 3, 4, 5, 6 and 7.

Regarding the data for mercury and other metals were mentioned in the line 393 references [10], [11] and [38], and in the table 5, references 10, 11, 37, 38.

Information of the fish harvesting is given in previous work Alvarado et al (2020), and this data do not contribute with the discussion for present work.

3. No statistical tests are performed to investigate whether temporal variability in metals concentrations is significant. Nor are tests performed to compare metal concentrations in liver and fillet and between species. Data are presented as ranges (Table 1); means and standard deviations (or errors) should also be shown. The LCF is based on the average over the 10 observations when the average of the calculations for each sampling period should be calculated. Figures 2-6 either are not showing standard deviations or they are unrealistically small for a sample size of 3-5 fish which presumably were measured individually.

In reference to comparison of metal variability, we evaluate the case of carp in a previous work see Alvarado et al. (2020). In that work we only observe differences in the pattern of the Zn in muscle. As is observed in the graphics, the pattern is changing along the time and present work is an effort to describe it. Because this is a continuous experiment, to make comparison between liver and muscle on/and between species, is not appropriate because is not end-point study.

It is precisely for this reason that the results are presented as a range in the Table 2, because the objective was to make description to a changing data.

The LCF in table 2 was an average of LCF in each point, and was the objective to show a brief summary of the phenomenon.

We thank the reviewer for the suggestion, we have included standard deviations in all the graphs 2-6.

4. Figures 2-6 are not easily viewed. It would make more sense to plot metal concentrations for a given species on its own graph with two Y axes for the differences in concentration over time. It would be easier to follow temporal patterns that way and I am not sure that they are even real. The use of a small black dot and a small black triangle do not provide for clarity. An open circle would provide more contrast. If this is to appear online, color also could be used.

We thank the reviewer for this suggestion. We have modified figures 2-6, including four subplots in each graph. The upper ones (a and b) describe the behavior for the control and experimental runs of the selected metal in the muscle for the carp and the catfish, while the lower ones show the same information for the liver (c and d). We have followed the suggestion to include colors, as well as open and filled markers.

5. How does table 3 differ from table 2? Is this the last day of measurement or the time after fish attained the commercial weight; this weight is not given. Again, no standard deviations or error or sample size. Table 3 could be combined with table 4. I see no value to the maximum estimates. At best only the %PTWI column would be needed. It would be an easier read to insert another 6-8 columns into Table 3 or, if too large, have an upper series of rows for the carp and lower row for the catfish or whatever order of presentation is followed for the figure.

The table 2 and 3 have different purposes, while Table 2 referes to bioaccumulation pattern and show a brief summary of liver concentration capacity, Table 3 refers results of fish in commercial size for EWI and PTWI and food assessment of safety. Table 3 only include sampling 7, 8 and 9 sampling and only from muscle because the people do not eat liver. We prefer showed as different. Moreover, if we could have a little more time we could put the tables together.

6. Table 5 is poorly organized and should be done by metal, then species and then location. The authors also should proof their references as some of the values seem widely off. I looked up Fierro et al. (reference 42) and see that their figures of metal concentration are in µg/g and this is dry weight. However, their table 2 is mg/kg which is most likely a typographic error missed by these authors in their galley proof. The authors do not show a risk to lead intake and only a small exceedance (2.3) for Cd with quite a different outcome in the manuscript I am reviewing. This points to sloppy reading and synthesis on the authors. They may wish to look at the other data presented in Table 5 of Fierro et al. for additional comparison and then verify that some of their other high values for Cd and Pb are not the result of data entry errors including reading incorrectly from a paper or not mixing wet and dry weight determinations. To the best of my knowledge, daily intake calculations are based on wet weight determinations as that is how food is sold and consumed. The metal data for reference 12 look odd to me with more than 100-fold difference in concentrations Cd and Pb between seasons which is not reflected in similar variability for mercury nor in the current study where river flow is the main source of metal input.

The references belonging to table 5 were reviewed and the values needed were adjusted to wet weight. It is worth mentioning that some authors report different body weights and different amounts of consumption for the estimated weekly intake calculation, so in some cases the values as mentioned by the authors were maintained and in others they were re-calculated based on the present study. Table 5 were designed to transform mg/kg in some paper to µg/kg these were previously conducted and the information were added in the footer of the table.

In reference to the results of Fierro et al., they carried out studies in small fish and reported concentrations of the whole fish, it was decided to include this reference to show differences between different species

7.Copper and zinc are essential metals, and no discussion is given on how mineral needs may or may not be met by consuming fish fillet and liver. Would add balance.

Present work is oriented to food assesment on safety and brief mention of the nutritive perpective is mentioned in lines 355-359. To make discussion about Cu and Zn could be very complicated because the sources of nutritive metals are very varied on the diet, but for toxic metals, especially Hg they are scarce.

Reviewer 3 Report

The authors assessed the levels of several heavy metals in 2 species of fish and water as well as in sediments. They evaluated the bioaccumulation of metals (Zn, Cu, Cd, Hg, As, Pb) in fish cultured in a natural environment to calculate the risk of dietary intake of metals in humans. That kind of data is very important both for consumers as well as for the people who decide about food safety.

The work is well documented, and logically organized, it can be published after some minor revision. Specific comments are below.

(1) (Page 1, Line 18-19) 'The pattern of heavy metal accumulation (Cu, Zn, Cd, Hg, Pb, As) in muscle and liver were monitored, as were metal concentrations in water and sediments.' Metal concentrations in water and sediments were not the object of this study. They were described in other works after sampling them once.

(2) (Page 2, Line 60) 'm2' should be replaced by a proper form of 2 (in upper index.

(3) (Page 3, Line 80) 'panoramic' should be replaced by 'general'

(4) (Page 3, analysis of metal) Authors didn't give which part of fish was taken to investigation. It is important because heavy metals are not evenly distributed in fish body.

(5) (Page 3, Line 85) 'the fish food, water, and sediments' - there is not any information about the way of analysis of metals in that mediums. Authors should remove that from this section. Where are the results ??? For sediments and some metals in references 4 and 5, and for water ....?

(6) (Page 3-4) Table 1 - collumn units - please apply the proper units and be consequent if you choose mg/kg or mg kg^-1

(7) (Page 5, Lines 150-159) Please use one way of describing units. Change ppm into mg kg^-1or mg/kg

(8) (Page 8, Fig. 7) It would be nice to be sure which where is the a and b chart

(9) (Page 8, Line 214) remove one dott

(10) (Page 8, Line 218) Mercury content 'Hg 16.45 ± 3.53 μg/kg' in fish food was quite high in that study. Are any standards for heavy metals content in fish food?

(11) (Page 11-12) Table 5 - what is the meaning of a and b indexes in the first collumn and '¹' behind EWI

(12) Depending on fish species the growth rate is very different, so the comparison betweeen investigates species shouldn't be made basing on at the same age (by days).

(13) Check carefully the text of the manuscript for different notation of units like 'Kg' instead of 'kg' as well as for notation of citing the references.

Author Response

Thank you for your detailed comments, there are very helpful. Every question was answered and corrections were made, see below.

We appreciate the comment. Water and sediment were removed from the text, see abstract lines 18-19 and metodology lines 90-91.

(2) (Page 2, Line 60) 'm2' should be replaced by a proper form of 2 (in upper index.

The correction sugested was made, see now lines 62-63.

(3) (Page 3, Line 80) 'panoramic' should be replaced by 'general'

The correction sugested was made, see now line 88.

(4) (Page 3, analysis of metal) Authors didn't give which part of fish was taken to investigation. It is important because heavy metals are not evenly distributed in fish body.

Thank you for the comment, Liver and muscle tissue were removed completely and homogenized individually, except for catfish at the three first sampling where the fish were very small and the same tissues from different fish were placed in the same bag. All these information were introduced in the metodology see lines 78 to 80.

Than you for the comment. Alvarado et al (2020) was a previous study and sediment and water information were widely discussed there. We eliminate the water and sediment from the text, see metodology lines 90-91. But in contrast, we decided to make reference to Alvarado et al., (2020)[6] in the discussion section when was necessary, see the lines 302 and 338-341.

The references 5 (Hansen et al., 1997) and 6 (Alvarado et al., 2020) both includes metals concentration and quality of the water from Chapala. Table 4 from Alvarado et al., 2020 includes additionally a revision of previous works with results of metal concentration in water and sediments performed in Lake Chapala.

(6) (Page 3-4) Table 1 - collumn units - please apply the proper units and be consequent if you choose mg/kg or mg kg^-1

Thank you for the comment, homogenization of the units throghout entire document and of course in the Table 1 was realized. The unit mg/kg was used for Cu and Zn, and µg/kg was selected for non-essential metals and water.

(7) (Page 5, Lines 150-159) Please use one way of describing units. Change ppm into mg kg^-1or mg/kg

Homogenization of the units throghout entire document were performed. The unit mg/kg was used for Cu and Zn, and µg/kg was selected for non-essential metals and water.

(8) (Page 8, Fig. 7) It would be nice to be sure which where is the a and b chart

Thank you for the comment, new set of figures were done and this detail was checked.

(9) (Page 8, Line 214) remove one dott

Thank you for the comment, all the Figure 5 description were change and corrected. Line 221

(10) (Page 8, Line 218) Mercury content 'Hg 16.45 ± 3.53 μg/kg' in fish food was quite high in that study. Are any standards for heavy metals content in fish food?

Thank you for the comment. According with Directive 2002/32/EC, the Hg limits for feedingstuffs produced by the processing of fish or other marine animals is 0.5 mg/kg. The commercial fish foods here evaluated had an average Hg of 0.0165 mg/kg, below the standard. The european standard was incorporated to the reference with the number of 36 and a paragraph with the coment was introduced in the text, see lines 333 to 334.

(11) (Page 11-12) Table 5 - what is the meaning of a and b indexes in the first collumn and '¹' behind EWI

A paragraph with de definition was included at the bottom of Table 5, lines 408-411.

(12) Depending on fish species the growth rate is very different, so the comparison betweeen investigates species shouldn't be made basing on at the same age (by days).

Thank you for your useful comment. A new set of graphics were performed in order to separate the species, see Figures 2, 3, 4, 5, 6 and 7. Additionally the control group were introduced in the text, see lines 146-148, 156-157, 164, 175-178, 203-204, 214, 235, 290.

(13) Check carefully the text of the manuscript for different notation of units like 'Kg' instead of 'kg' as well as for notation of citing the references.

Homogenization of the units throghout entire document were performed. In the same way the citations by name were homogenized according to the instruction to the authors, using Name +[number of reference] when applied.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript can be published. Authors have replied all my questions and made corrections which improved the text.
I would like to thank the authors.

I only saw an error which should be corrected:
L 94: 210 C (the symbol for degree Celsius is not complete - the "ring" is missing)

As for my previous comment on units in different form - I completely understood, that some metals were expressed in mg/kg while the trace metals in µg/kg. The problem was graphical - you had an extra space in the unit (µg /kg) in some lines - and it remains - see Table 2.

Author Response

I only saw an error which should be corrected:

L 94: 210 C (the symbol for degree Celsius is not complete - the "ring" is missing)

Thank you for the comment, the correction were done.

As for my previous comment on units in different form - I completely understood, that some metals were expressed in mg/kg while the trace metals in µg/kg. The problem was graphical - you had an extra space in the unit (µg /kg) in some lines - and it remains - see Table 2.

Thank you so much for the observation, the error was corrected now in Table 3.

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper has a very simple message and that metal concentrations were higher in liver than muscle for fish grown in Lake Chapala and that there was no consumption risk despite the fact that sediments in the lake are contaminated with lead and cadmium. Moreover, metal concentrations in muscle were similar to fish grown in a control pond. Only Cd liver concentrations were higher in the experimental carp, but the converse tended to be observed for lead. The time series data do not add much with uncertainties in when diet changed, numbers of fish analyzed, lack of statistical tests and no environmental data presented to relate trends in fish with trends in water quality for example. Or why seasonal differences would be expected anyway. The fact that the lake is alkaline as mentioned in the title has no bearing to the paper. Why not mention the lake is metal contaminated?

This paper could be shortened and simplified with a clearer message and that is that despite being contaminated with some metals, fish apparently can be reared safely with respect to humans with the primary risk, based on previous studies, with mercury.

Other comments.

The calculations of estimated weekly intake are based on maximum and minimum concentrations of the metals when the mean or median should be used. Intake risk estimates are based on intake over a long period of time and so an average of some type is more realistic. Using a maximum concentration in alarmist and a minimum is unrealistically optimistic.

Line 58. It seems unusual that carp would be adults at 8 months. They take a couple of years at other locations. What about reproduction in the cages?

Line 59. Mesh size of cages not given.

Line 67. When did the change in diet occur? A table is needed of the metal concentrations in the two diets, and this should appear before Table 1 which provides information of reference material.

Line 69. No information is given on growth, mortality, etc.

Line 71. 3-5 individuals are mentioned as being analyzed, but are these individuals of each species? If it is the total for two species and control and experimental then this is hardly rigorous. Were liver and muscle measured for each species and locations at each time and each metal? A lot of figures are missing error bars.

Line 72 should indicate the days in which the catfish were released into the larger and cage and also when diet was changed.

Line 75. “command” and not “cold”

Line 130. The liver concentration factor makes no sense. If the Cu concentration in the liver (48 mg/kg) is divided by the concentration in the muscle (2 mg/kg) than the CF is 24 and there is no need to multiply by 100. Table 2 shows a LCF that clearly was not multiplied by 100. Again, the authors are presenting range data in Table 2 when I requested that data be displayed as means and standard deviations with the sample size (n of fish measured) given). If the reader is interested in temporal variability, they have the figures to examine.

Table 2 in addition to needing data presented as mean and standard deviations for the experimental group (and not ranges) should also show the controls.

Fish show no differences in copper, zinc, arsenic and mercury concentrations between control and experimental and time trends the same. Cadmium is higher in carp liver, but Pb is lower than in carp liver than in experimental.

The sections on estimated weekly intake in Tables 3 and 4 could be combined with the EWI based on means as already mentioned and on fish of consumption size. Data for the control lake also should be shown. Why did the experimental design not include the analyses of market caught fish which had more exposure to the sediments?

Table 5 shows that with the exception mercury in carp in Lake Capala, metals in fish did not exceed percent tolerable weekly intake, <100%. The table is poorly organized and would be clearer if done by metal and then species and lake. Ranges are given for this study and not others. Pb from reference 44 is shown at 6810 µg/kg or16 times higher than Carp in reference 42 but with a lower %PTWI. The actual paper shows a mean concentration of ca. 28 µg/g in Figure 2 and 28 mg/kg (ug/g) in Table 4) versus the 8,520 µg/g shown by the authors with their dry weight conversion.

Author Response

The time series data do not add much with uncertainties in when diet changed, numbers of fish analyzed, lack of statistical tests and no environmental data presented to relate trends in fish with trends in water quality for example. Or why seasonal differences would be expected anyway. The fact that the lake is alkaline as mentioned in the title has no bearing to the paper.

Tank you for the comments, Table 4 was included with F-test to evaluate the tren between experimental and control group. As for the suggestion of the reviewer, the title of the article was changed by: Metal bioaccumulation by carp and catfish cultured in Lake Chapala, and weekly intake assessment.

Why not mention the lake is metal contaminated? ).

Lines 32-38 Introduction mention that the Lake is metal contaminated.

Line 32-38 This river is also the main source of anthropogenic pollution, including heavy metals, because ~3,500 industries pour their treated wastewaters into it [2,3]. Sediments from Lake Chapala are rich in heavy metals, and previous studies have shown that lead (Pb) and cadmium (Cd) are present in the exchangeable fraction of water, and possibly available for absorption by living organisms, including fish [4].

Other comments.

The calculations of estimated weekly intake are based on maximum and minimum concentrations of the metals when the mean or median should be used.

Intake risk estimates are based on intake over a long period of time and so an average of some type is more realistic. Using a maximum concentration in alarmist and a minimum is unrealistically optimistic.

Thank you for your suggestion. New versión of Table 5 was done, the EWI of control group was integrated and only mean concentrations were considered for EWI and %PTWI calculation.

Line 58. It seems unusual that carp would be adults at 8 months. They take a couple of years at other locations. What about reproduction in the cages?

Here in central Mexico is very common to have adult carp at 8 to 10 months. The water temperature in the lake during summer rages from 20 to 23 Celsius, and during Winter could be 17 to 18 Celsius, then is good temperatura for fish growth. We had carps with eggs at age of 12 monts and catfish with eggs at 17 months. In the case of catfish they need nest on floor, like caves, then catfish do not have appropriate conditions for reproduction. The fisherman told us that carp did reproduce.

Line 59. Mesh size of cages not given.

Mesh size of the cages was 1”. The information was added in the Material and methods section.

Line 67. When did the change in diet occur? A table is needed of the metal concentrations in the two diets, and this should appear before Table 1 which provides information of reference material.

The change diet occured during the sixth month. Thank you for the suggestion, a new Table 1 with the metal concentrations of the two diets were introduced.

Line 69. No information is given on growth, mortality, etc.

Fort he carp the mortality in the lake was 3-4% and 1% in the earth pond. The growth mean during the total experiment 1.74 g/day. For the catfish mortality 8% in the lake, and 3% in the control group. The catfish growth mean was 1.27 g/day, the first 6 months they gorwth very slow. These data were not included in the paper due to little contibution on the subject of study.

Line 71. 3-5 individuals are mentioned as being analyzed, but are these individuals of each species? If it is the total for two species and control and experimental then this is hardly rigorous.

Lines 77-78 were resctructured in the text and the information was clarified.

“The collection of fish sample involved taking 3-5 individuals from each specie, and for each experimental group………..”

Were liver and muscle measured for each species and locations at each time and each metal?

Lines 83 to 84 mention the requested information:

“Liver and muscle tissue were removed, weighed, and homogenized individually except for catfish at the three first sampling where the fish were very small and the same tissues from different fish were placed in the same bag.”

A lot of figures are missing error bars.

Due to the scale, in some points the error bars are not appreciated, however each point contains them.

Line 72 should indicate the days in which the catfish were released into the larger and cage and also when diet was changed.

Thank you for the comment, the informatio was integrated.

Line 75. “command” and not “cold”

Thank you for the observation, the correction was made.

Thank you for your comments, the correction was made and the nwe Table 3 was restructured incorporating your comments. Now the LCF was calculated individually and expressed as a range.

Table 2 in addition to needing data presented as mean and standard deviations for the experimental group (and not ranges) should also show the controls.

In order to present the general trend of the control group, the new Table 4 shows that there is no difference in the most of the control and experimental data.

The new table 4 and the figures from 2 to 6 confirm the observation given.

Thank you for the excelente comment, we integrate Tables 3 and 4 in a new one Table 5 that include all your suggestions.

Why did the experimental design not include the analyses of market caught fish which had more exposure to the sediments?

It is very interesting your comment, we had a very limited resource and many other ideas were put on hold. Yes, it is an excellent idea, these time we had to focus only in presented experiment.

Thank you for your suggestion, the table was restructured, only the mean concentration was considered for the EWI and %PTWI calculation. When it was necessary, the dry weight information of the previous references were transformed in wet weight. Only catfish and cyprinids articles were chosen for the comparisson.

Author Response File: Author Response.pdf