Female Rat Behavior Effects from Low Levels of Hexavalent Chromium (Cr[VI]) in Drinking Water Evaluated with a Toxic Aging Coin Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper attempts to look at the influence of age in female Sprague-Dawley rats and compared to previous work published on males. The purpose is to determine if aging in geriatric populations of humans (65+) have a greater sensitivity to Cr(VI).  Rats were exposed to 0.05 or 0.1 mg Cr(VI)/L in drinking water for 90 days as these are the maximum contaminant levels from the World Health Organization and U.S. Environmental Protection Agency, respectively.  Rats were exposed to a battery of behavior assays to assess grip strength, locomotor coordination, anxiety, spatial memory, sociability, and social novelty preference. The authors observed age differences in Cr(VI) neurotoxicity, with grip strength, locomotor function and spatial memory in middle-aged females being particularly affected. They concluded there is a need to consider age and sex as variables in toxicology, and to revisit drinking water regulations for Cr(VI). The authors did not discuss the large range of response in each test compared to the differences detected. This requires further investigation as the statistics used becomes rather complicated.

Replication showed a large range but is not discussed. 

 

The range of data for each evaluation exceeds that average differences used to draw conclusions.

Author Response

This paper attempts to look at the influence of age in female Sprague-Dawley rats and compared to previous work published on males. The purpose is to determine if aging in geriatric populations of humans (65+) have a greater sensitivity to Cr(VI).  Rats were exposed to 0.05 or 0.1 mg Cr(VI)/L in drinking water for 90 days as these are the maximum contaminant levels from the World Health Organization and U.S. Environmental Protection Agency, respectively.  Rats were exposed to a battery of behavior assays to assess grip strength, locomotor coordination, anxiety, spatial memory, sociability, and social novelty preference. The authors observed age differences in Cr(VI) neurotoxicity, with grip strength, locomotor function and spatial memory in middle-aged females being particularly affected. They concluded there is a need to consider age and sex as variables in toxicology, and to revisit drinking water regulations for Cr(VI). The authors did not discuss the large range of response in each test compared to the differences detected. This requires further investigation as the statistics used becomes rather complicated.

Replication showed a large range but is not discussed.

The range of data for each evaluation exceeds that average differences used to draw conclusions.

Behavior assays are well known to exhibit a wide range of variance, particularly due to differences in housing, environment, strains, and vendors (to name a few). We ensured consistency with these factors to the best of our ability. This is the first study reported with this design or with Cr(VI) levels this low, hence there is nothing to compare to.  Due to the number of comparisons, the number of assays, it is difficult to discuss the variability as it often changes for each behavior, each study group, and each age. For example, considering Fig 3: Range for Cr(VI) groups only was (A) 10208, 3291, and 3608; and (B) 15732, 5512, and 8079. Range for age comparisons was (C) 1758, 1844, 968, 2466, 602, 2608, 2703, 958, and 1074; and (D) 2256, 1971, 2948, 2489, 3026, 5142, 4471, 5513, 3633. Adding the required text to discuss these nuances would require a large word count without adding significant impacts on the interpretation of the results. To be transparent with our study, we presented data with points representing each individual animal wherever possible, enabling readers to make their own interpretation of the data range.

Reviewer 2 Report

Comments and Suggestions for Authors

Type of manuscript: Article

Title: Female Rat Behavior Effects from Low Levels of Hexavalent Chromium (Cr[VI]) in Drinking Water Evaluated with a Toxic Aging Coin Approach Special Issue: Heavy Metal Toxicity: Environmental and Human Health Risk

 In this manuscript, Vielee et al. evaluated the effect of Cr(VI) administered in drinking water, on female rats through behavioral tests. To do this, they carried out different tests at two different doses and with rats of three different ages. I have some comments for the authors to consider during the revision of the manuscript:

- Abstract, line 29: Please clarify that the maximum contaminant level (MCL) is the highest level that is allowed by the two organizations.

- Abstract, lines 35-36: The authors should remove everything that refers to the results obtained in another manuscript, where they used male rats. The manuscript is very large, with 11 figures and a total of 45 graphs. I strongly advise to remove all the data referring to males. Not only is it difficult to keep track of so much information, but it is not consistent with the proposed title.

- Introduction, line 60: Add a more recent publication.

- Introduction, lines 70-71: “Metals are particularly hazardous environmental pollutants, as they are naturally occurring, many are toxic,” Complete this sentence, and mention also the human-caused contamination.

- Introduction, lines 115-116: This sentence needs to be corrected. Anyone who reads it would assume that the authors are describing a study carried out on male and female rats, which is not true.

- Materials and Methods: As the authors carried out the study with mice aged 3, 7 and 18 months, they need to clarify how they kept them until they reached this age.

- Animals: As this is a study with experimental animals, it is mandatory to include the registration number of the Ethics Committee's approval.

- Animals: Given that the chromium was administered through drinking water, it is worth mentioning whether or not there was any variation in the volume of water drunk compared to the control of the same age. This is because it would certainly have an influence on the dose administered, which could be a cause of error in the conclusions obtained.

- Behavior Assays: The reason for the sequence of tests carried out, which is shown in Table 1, should be explained. In other words, why was the Grip Strength test carried out in weeks 1 and 7 and not, for example, in weeks 4 and 10?

- Behavior Assays, lines 178-231: It is not mentioned how many repetitions were carried out for each test.

- Results: All figure legends should be abbreviated. They contain information that is already in the text and is a result. They should always show the N (number of animals studied in each group) and state that the result shown for each point refers to the mean and the deviation refers to the error. It's too much information for the reader and the essentials are lost.

- Results line 483: Figure 11 is an example. It has 10 graphics and a text of over 500 words. I strongly advise the authors to do a major reformulation. Readers will not read the whole text.

- Discussion/conclusion:

 Given the large number of results presented, I advise the authors to:

- Present the average results obtained in the behavioral tests in a more clear/perceptible way, by constructing tables.

- Not include in this manuscript the results obtained with male rats. The comparison between the sexes can be presented in another publication.

Author Response

In this manuscript, Vielee et al. evaluated the effect of Cr(VI) administered in drinking water, on female rats through behavioral tests. To do this, they carried out different tests at two different doses and with rats of three different ages. I have some comments for the authors to consider during the revision of the manuscript:

- Abstract, line 29: Please clarify that the maximum contaminant level (MCL) is the highest level that is allowed by the two organizations.

Edited as requested.

- Abstract, lines 35-36: The authors should remove everything that refers to the results obtained in another manuscript, where they used male rats. The manuscript is very large, with 11 figures and a total of 45 graphs. I strongly advise to remove all the data referring to males. Not only is it difficult to keep track of so much information, but it is not consistent with the proposed title.

Respectfully, we disagree with the Reviewer here. We understand the Reviewer’s concern regarding the length and breadth of the data presented. Sex differences are a critical consideration in toxicology, and we are concerned such a move would not be accepted for publication as all the data will have been published elsewhere. Furthermore, this would only remove one paragraph from the Results and one from the Discussion.

- Introduction, line 60: Add a more recent publication.

Edited as requested.

- Introduction, lines 70-71: “Metals are particularly hazardous environmental pollutants, as they are naturally occurring, many are toxic,” Complete this sentence, and mention also the human-caused contamination.

Edited as requested.

- Introduction, lines 115-116: This sentence needs to be corrected. Anyone who reads it would assume that the authors are describing a study carried out on male and female rats, which is not true.

Edited as requested.

- Materials and Methods: As the authors carried out the study with mice aged 3, 7 and 18 months, they need to clarify how they kept them until they reached this age.

Edited as requested.

- Animals: As this is a study with experimental animals, it is mandatory to include the registration number of the Ethics Committee's approval.

Information added as requested.

- Animals: Given that the chromium was administered through drinking water, it is worth mentioning whether or not there was any variation in the volume of water drunk compared to the control of the same age. This is because it would certainly have an influence on the dose administered, which could be a cause of error in the conclusions obtained.

Edited as requested.

- Behavior Assays: The reason for the sequence of tests carried out, which is shown in Table 1, should be explained. In other words, why was the Grip Strength test carried out in weeks 1 and 7 and not, for example, in weeks 4 and 10?

Information added as requested.

- Behavior Assays, lines 178-231: It is not mentioned how many repetitions were carried out for each test.

Edited as requested.

- Results: All figure legends should be abbreviated. They contain information that is already in the text and is a result. They should always show the N (number of animals studied in each group) and state that the result shown for each point refers to the mean and the deviation refers to the error. It's too much information for the reader and the essentials are lost.

Edited as requested.

- Results line 483: Figure 11 is an example. It has 10 graphics and a text of over 500 words. I strongly advise the authors to do a major reformulation. Readers will not read the whole text.

Edited as requested.

- Discussion/conclusion:

 Given the large number of results presented, I advise the authors to:

- Present the average results obtained in the behavioral tests in a more clear/perceptible way, by constructing tables.

Added as requested.

- Not include in this manuscript the results obtained with male rats. The comparison between the sexes can be presented in another publication.

Respectfully, we disagree with the Reviewer here. We understand the Reviewer’s concern regarding the length and breadth of the data presented. Sex differences are a critical consideration in toxicology, and we are concerned such a move would not be accepted for publication as all the data will have been published elsewhere. Furthermore, this would only remove one paragraph from the Results and one from the Discussion.