Fluorene-9-bisphenol is anti-oestrogenic and may cause adverse pregnancy outcomes in mice

Bisphenol A (BPA) is used in the production of plastic but has oestrogenic activity. Therefore, BPA substitutes, such as fluorene-9-bisphenol (BHPF), have been introduced for the production of so-called ‘BPA-free' plastics. Here we show that BHPF is released from commercial ‘BPA-free' plastic bottles into drinking water and has anti-oestrogenic effects in mice. We demonstrate that BHPF has anti-oestrogenic activity in vitro and, in an uterotrophic assay in mice, induces low uterine weight, atrophic endometria and causes adverse pregnancy outcomes, even at doses lower than those of BPA for which no observed adverse effect have been reported. Female mice given water containing BHPF released from plastic bottles, have detectable levels of BHPF in serum, low uterine weights and show decreased expressions of oestrogen-responsive genes. We also detect BHPF in the plasma of 7/100 individuals, who regularly drink water from plastic bottles. Our data suggest that BPA substitutes should be tested for anti-oestrogenic activity and call for further study of the toxicological effects of BHPF on human health.


1.
The paper needs a solid edit from a native English speaker for language, grammar, spelling, and usage.

2.
Details of the human population need to be provided, including exclusion criteria, status about the women's menstrual cycles (or use of steroid contraceptives), time of day of collection, and other experimental details.

3.
Were field blanks used for the measures of BHPF in human serum? This is essential and appears to have been omitted.

4.
In the yeast assay, the range of dosages used for BHPF should be extended into the lower range. EDCs are well-established as acting with non-monotonic dose-response curves and sometimes low dose effects are seen in the absence of high dose effects.

5.
In vivo work on mice evaluates crude gross morphological changes that are crude that endocrinologists agree are poor measures of either estrogenic or anti-estrogenic activity. Neither the established uterotrophic assay is, or the author's new anti-uterotrophic assay, is a cutting-edge assay of hormonal actions. Similarly, global gene expression profiling of whole organs is not usually terribly informative, as tissues are highly heterogeneous.

6.
By "intragastric administration" do the authors mean oral gavage, or were they surgically implanted with a feeding tube? This requires clarification. Moreover, gavage is highly stressful and should be avoided.

7.
Justification and clarification of the dose of BHPF needs to be provided. In the antiuterotrophic assay, the authors say administration was 5 mL/kg BW which is not meaningful. For the qPCR work, authors mention 50 mg/kg BW. If this was the dose given in prior studies, it is an unrealistic dose and not relevant to human exposures. Subsequent work on subchronic toxicity uses a range of dosing from 0.4 to 50 mg/kg, still in a high range.

8.
The authors might consider doing the obvious experiment of allowing the mice to drink from water bottles with BHPF, compared to a vehicle water bottle. This would avoid the gavage problem and would use realistic amounts.

9.
Mating studies were conducted with same-treatment males and females. It is important to mate treated animals with non-treated controls.

10.
In Figure 4 legend, clarify that heatmaps are shown relative to the control group.
11. The discussion of NOAEL needs to include work on much lower dosages, showing adverse effects well below the predicted NOAEL.
12. There are other mechanisms of action of bisphenols beyond estrogen signaling that should be discussed.

Reviewer #3 (Remarks to the Author):
General Comments: First, I note that the Editor asked me to pay specific attention to the analytical-chemistry and the statistical aspects of this work. These are certainly most central to my expertise; however, I am also reasonable well qualified to judge the (anti-)estrogenic assays and microarray results. The histopathology and some details of the in vivo studies are the only aspects of the paper that are beyond my comfortable reach. This is a very thorough, compelling, and interesting piece of work. The Authors conducted experiments that span a wide range of disciplines, assembling all the pieces needed to strongly "make their case". Specifically, they show conclusively that: 1) fluorene-9-bisphenol (BHPF) occurs in plastic bottles (using 1H-NMR and 13C-NMR), 2) BHPF leaches from bottles into drinking water (using GC-MS with an in-house synthesized deuterated form of BHPF as an internal standard), 3) BHPF is detected (albeit at low occurrence rate) in serum in the general public (using 100 volunteers), and 4) BHPF is a potent anti-estrogen. To establish the anti-estrogenic mode of action, the Authors use multiple lines of persuasive evidence demonstrating that BHPF: a) blocks the activity of estradiol (on par with a model anti-estrogen) in a well-established yeast assay for estrogenicity, b) fits nicely within the antagonist pocket (but not the agonist pocket) of the estrogen receptor using in silico molecular docking software, c) inhibits relative uterine weight (in a dose-dependent manner) in an optimized in vivo screen for anti-estrogenicity (in similar fashion with a model anti-estrogen), d) selectively down-regulates (in a dose-dependent manner) virtually all of the transcripts that are upregulated by estradiol in mouse microarrays, e) reduces relatively uterine weight in reproductive toxicology in vivo studies (as do model anti-estrogens), and f) impacts tissues similar to model antiestrogens as viewed by histology.
All of these lines of evidence are laid out in a very logical fashion. The Authors use these results to illustrate the larger implications that: a) alternatives to BPA may be as harmful, or more harmful, to the public as BPA, b) thus, the various pressures (regulations, public concerns, etc.) that force companies to replace "hot button" chemicals may be doing more harm than good, c) the topic of anti-estrogens in the environment -much less studied than that of estrogens -deserves more attention from researchers. I have heard each of these larger points a few times before, although each is "fresh" enough that I would consider them relatively novel. Indeed, I cannot recall a single case where these points were made in a more-compelling and thorough manner. I feel strongly that this paper would be of interest to those in the Environmental Science community. On a personal note, I am involved in work that uses chemical monitoring data from waste water treatment plants. Of course, I will abide by the Journal's rules of confidentiality, but I am very curious to know if BHPF could be measured in any of these samples. I do feel that this paper, once published, will likewise impact the thinking of others in this field.
My recommendation is to accept this paper will minor modifications. I have read the scope and criteria for publication for Nature Communications, and I think this paper is well suited. Following are some specific comments that are aimed at improving the presentation (and also some comments regarding the Editor's specific charge to assess the statistical and analytical methodology). 1) With regard to statistics -there really is nothing "fancy" here -which is as it should be. The Authors use 10 replicates, which is about as good as you see with in vivo rodent studies. A wellestablished statistical program is used; differences in classes are assessed with ANOVA using Fisher's as a post-hoc test. In the figures, error bars are generated using standard deviation, which, to their credit, is more conservative than standard-error-of-the-mean error bars (which is often used). If I wanted to be nitpicky, I would point out that Fisher's test is a little less conservative than Tukey's test, but that is really more of a personal preference, and would probably not make any difference. Also, the Authors do not state whether or not they tested the data for normality and heteroscedasticity before applying the ANOVA tests. Strictly speaking, ANOVA is only applicable for data that meet these criteria (although a small degree of non-adherence is well tolerated). Looking at the data, I suspect there are no problems, but it might be worthwhile to close that loop.
2) With regard to analytical measurements -The Author's choices for analyses seem perfectly appropriate. They initially used 1H-NMR and 13C-NMR to confirm that BHPF was leaching from a plastic bottle. BHPF was isolated by fractionating a methanol leachate. NMR is the "gold standard" for identifying (or confirming the identity) of a relatively pure organic chemical. Using both 1H and 13C NMR takes this analysis to a very high level of confidence. My only quibble with this part of the work is that I would have liked to see a Figure with a more explicit comparison of the NMR spectrum of the isolate with that of an authentic standard of BHPF. I feel sure that the Authors have made this comparison for their own sake -indeed, I think the NMR spectrum in Supplementary Information Figure 1A is for the authentic standard. However, this is not clearly stated, and it is not presented in a way that can be compared directly to the NMR spectrum of the isolate in Figure 1.
Once identified, the Authors used GC/MS (with derivatization) to quantify BHPF in drinking water and in human serum. They included a partially deuterated form of BHPF (which was synthesized inhouse) as an internal standard. Again, this is the "gold standard" method for target analysis (and quantification) of an organic chemical whose mass spectrum and GC retention time is known. No qualms here.
3) It seems that there has been some inversions (typos) when referring to Figures and sub-parts of Figures. For example, in the Caption, Figure 7D is described as "dead fetus ...". It seems pretty clear that Figure 7C is actually the dead fetus (and it is referred to as such in the text). In addition, the "order" of Figure 5 and Figure 6 (the actual graphics) are switched (6 appears in the document before 5). And, it appears that some text references to 5/6 are reversed. I am not sure I caught all of these issues, so beware! 4) The microarray results regarding the opposing effects of BHPF and E2 (described from lines 154 -170, and depicted in Figure 4) are very compelling and useful to the argument. However, the more "global" microarray-results discussion in lines 127 -154 is not very useful. While there are a few good points in that section, in my view, the vast majority of the text from line 127 -154 could be omitted or perhaps moved to Supplementary Information. 5) I thought the paper would be more impactful if the Discussion had ended with line 283, which is an effective climactic sentence. The paragraph that follows (line 284-293) is mostly a repetitive summary of the technical findings. I suggest moving any useful thoughts from lines 284-293 to earlier in the Discussion, and closing with the preceding paragraph that ends on line 283.
6) The English linguistics of this paper is quite good; however, some issues will need to be addressed. For example, "drinking water" is called "drink water". I will not list more here, but several other subtle misusages appear.
Finally, I would note that the references seemed appropriate, and that all sections of the manuscript were clear, lucid, and very well written and organized.

Reviewer #4 (Remarks to the Author):
Molecular docking of BHPF Molecular docking was performed using the commercial software Scigress (Ultra Version 3.0.0, Fujitsu. It shows that BHPF can be accommodated into the antagonist pocket of ERα (PDB ID 3ERT), and not in the agonist pocket (PDB ID 1ERE) (Fig. 2c-e). The position of BHPF in the pocket is rather similar to that of OHT observed in the crystal structure. The approach used is a standard one. Considering the stereochemistry of the two ligands the results are convincing and not surprising.  consideration. First, the YES assay has certain weaknesses associated with the mosaic nature of the target receptor. It would strengthen the body of work (which is considerable already) to use a full length human receptor and evaluate separate effects on ERa and ERb. Recognizing that this may be deemed to be outside the scope of this original paper. The description of the data in humans should be perhaps more completely described. First, this should not be described as "general population", but rather volunteers. How were these volunteers identified and selected? What age, sex, etc. Other characteristics? This self-selection process could produce a bias. Second, are the authors confidence that the measurements of BHPF in human serum is not the result of contamination from plastics employed in the procedure? If no field blanks or tests of equipment was performed, the authors ne ed to be very careful about these conclusions. Moreover, there were very few volunteers that exhibited BHPF levels and this should be reflected in the abstract and summaries ("...was detected in a small proportion of volunteers"...). The animal studies appear appropriate. However, it might be useful to describe the methods employed to reduce the risk of bias in data collection. Method of animal assignment, sequence of gavage and animal collection, time of day, etc.
A separate subsection in the supplementary material might be useful for this.

Response:
Thank you very much for your very helpful and constructive comments. To specifically answer these comments, we have divided the "Comment C" into 3 parts and responded the comments point by point, as follows: Comment C-1. Data and methodology. This reviewer cannot evaluate the quality of the chemistry approaches. However, there are potential concerns for consideration. First, the YES assay has certain weaknesses associated with the mosaic nature of the target receptor. It would strengthen the body of work (which is considerable already) to use a full length human receptor and evaluate separate effects on ERa and ERb. Recognizing that this may be deemed to be outside the scope of this original paper.
Response: According to your suggestion, we performed dual-luciferase reporter assays which comprised a full-length human estrogen receptor α or β, and the anti-estrogenicity of BHPF have been demonstrated by the DLR assays. The  2). The IC 50 values of OHT were 8.61 × 10 −9 and 5.19 × 10 −9 M for estrogen receptor α and estrogen receptor β, respectively, which were 12.66 and 14.51 times lower than those of BHTF in the dual-luciferase DLR assays." (page 5, lines 99-106) "Dual-luciferase reporter assays. Dual-luciferase reporter assays were carried out primarily according to the method described in a previous study 39 . In  Comment C-2. The description of the data in humans should be perhaps more completely described. First, this should not be described as "general population", but rather volunteers. How were these volunteers identified and selected? What age, sex, etc. Other characteristics? This self-selection process could produce a bias. Second, are the authors confidence that the measurements of BHPF in human serum is not the result of contamination from plastics employed in the procedure? If no field blanks or tests of equipment was performed, the authors need to be very careful about these conclusions. Moreover, there were very few volunteers that exhibited BHPF levels and this should be reflected in the abstract and summaries ("...was detected in a small proportion of volunteers"...).
Response: According to your suggestion, we replaced the "general population" with "volunteers", added "be detectable in the serum of a small proportion of human volunteers" in the abstract and summaries, and added more clinical information and experimental details on the human volunteers and serum sample measurements in the revised manuscript. The following sentences were added in the revised manuscript: "…who habitually used plastic bottles for drinking water." (page 4, line 86) "In China, college students usually undergo a physical examination before  found that "estrogen receptor-α-antagonist" assay was listed in HTS assays of ToxCast, so it is not sure that the US EPA have not employed an "antagonist mode" in their estrogen receptor HTS assays in ToxCast so far. But we think that your suggestion is very constructive, and according to the suggestion, we added the following sentences in the conclusions of the revised manuscript: "Screening programs for endocrine disruptors have been established in many countries in recent years, but many of these programs have neglected to screen anti-estrogens. The results of this study suggest that anti-estrogenic pollutants, as well as their adverse effects on human health, should be of concern in the future."

Response to Reviewer #2's Comments:
General Comments. This study identified a BPA substitute, BHPF, in plastic water bottles, found that it is detectable in the water through leaching at 60C, and tested its potential actions as an endocrine-disrupting chemicals (EDC), particularly as an anti-estrogen through in vitro and in vivo experiments. They also showed that BHPF is detectable in humans. The identification of EDC activity of replacement chemicals is very important, and the study implicates BHPF as another such chemical, alongside other bisphenols such as BPS, BPF, and others. The major strength of work is making comparisons across all of these different levels. There are also novel aspects of work such as the docking experiment. The weakness is that much of the work is done at a very superficial level, and some essential control groups seem to be missing (unless I have misunderstood some of the methods). Details are provided below.
Response: Thank you very much for your comments and kind suggestions. To increase technical nature of the work, we added dual-luciferase reporter assays which comprised a full-length human estrogen receptor α or β. Dual-luciferase reporter assay is a relatively new research tool. In this study, each experiment has a negative (vehicle) control group and a positive control group. According your comment, we revised the manuscript to make the manuscript more legible.
Critique: Comment 1. The paper needs a solid edit from a native English speaker for language, grammar, spelling, and usage.
Response: According to your comment, two native English language editors had help us to revise the manuscript. This is essential and appears to have been omitted.
Response: Thank you for your comment. Quality control was maintained by analyzing a method blank (calf serum) and two spiked calf serum samples along with every 12 samples. No BHPF was detected in the blank and spiked calf serum samples during the analytical procedures. The blood collection device had been verified to be free of BHPF contamination when used for measurement of serum BHPF in mice. In addition, BHPF was not detected in most serum samples in human volunteers suggested that there is no BHPF contamination during blood collection. According to your suggestions, we added more experimental details on BHPF measurements in the revised manuscript. The following sentences were added in the revised manuscript: Response: Thank you for your comment. In the yeast assay, we had studied lower concentrations of BHPF in preliminary experiments, and no obvious effect was found.
In the dual-luciferase reporter assays, we studied lower concentrations of BHPF and found BHPF took anti-estrogenic effect at lower concentrations. According to your suggestions, we added the results of dual-luciferase reporter assays in Fig. 2 in the revised manuscript, and the following sentences were added in the revised manuscript.
"Similarly, BHPF showed no estrogenic activity but strong anti-estrogenic activities in the dual-luciferase reporter assays (Fig. 2c-2f) Response: Thank you for your comments. As you commented, in vivo work using mice is difficult to use for evaluating the estrogenic or anti-estrogenic activity of chemical sometimes. Many factors, such as body weight, age and strain of mice and experimental procedure, may affect the results during experiment. Fortunately, in this study, some of the authors have focally studied on animal experiments for decades and have rich experiences with the study of mice. The selections of body weight, age and strain of mice, as well as the experimental procedure, were resulted from a lot of preliminary experiments. We agree with you that the uterotrophic assay is not a cutting-edge assay of hormonal actions. But the uterotrophic assay is valuable in evaluating strong estrogenic or anti-estrogenic compounds and BHPF is a strong anti-estrogenic compound. After all, the uterotrophic assay is still the most common used in vivo tool for evaluating estrogenic or anti-estrogenic activity of chemicals and is adopted by OECD (OECD Test No. 440), US EPA (OCSPP Guideline 890.1600), etc. As you commented, global gene expression profiling of whole organs is not usually terribly informative because tissues are highly heterogeneous. In this study, the total RNA from each pool of tissue samples was a mixture from three animals; it is helpful to reduce the bother of heterogeneity.
"The total RNA from each pool of tissue samples (a mixture from three animals) was used for microarray analysis." (page 24, lines 519-520) Comment 6. By "intragastric administration" do the authors mean oral gavage, or were they surgically implanted with a feeding tube? This requires clarification. Moreover, gavage is highly stressful and should be avoided.

References
Response: According to your suggestions, we changed the "intragastric administration" to "oral gavage", and an exposure experiment through drinking water using mice was performed to avoid stress caused by gavage in the revised manuscript. We agree with you that gavage may induce stress. But oral gavage is still widely employed in animal experiments and accepted by standard evaluation procedures of OECD (OECD Test No. 440), US EPA (OCSPP Guideline 890.1600), etc. In addition, it should be pointed out that: 1) the operators for oral gavage are highly skilled in this study and highly skilled operators would greatly reduce the stress on mice; 2) the mice were pregavaged with vehicle before dosing for acclimatization; 3) the mice of control group were treated by gavage in the same way as those of test groups. These measures helped to prevent influences of gavage on experiment.  2) The "5 mL/kg BW" was the volume of vehicle or chemical solutions administered. According to your comment, the original sentence with "5 mL/kg BW" was revised to make it legible. 3) The experiment with 50 mg/kg BW group, as well as a control, was specifically conducted for the expression profiling and Q-RT-PCR works. According to your comment, we added more details about the experiment. 4) According to the suggestion of "experiment of allowing the mice to drink from water bottles with BHPF", we performed exposure experiment through drinking water using mice, and studied the effects of low doses of BHPF relevant to human exposure. The following sentences were added or revised in the revised manuscript:  34% ± 26.99%, 76.62% ± 19.97%, 94.81% ± 34.45%, and 77.89% ± 37.50% that of the control (P > 0.05), respectively (Fig. 8a). The gene expressions of sprr2a and sprr2b in the uteri of the mice were also studied by Q-RT-PCR (Fig. 8b) Response: Thank you for your comment. In this study, in cases where pairing was unsuccessful, females were re-mated with proven males of the same group, except that females in the 1.2 mg/kg TAM group were re-mated with proven males of the control group because all of the females were non-pregnant in the TAM-treated group.

References
This procedure is in reference to the mating procedures of reproduction toxicity Tests of OECD Guideline for Testing of Chemicals (OECD Test No. 416,421,and 422). Comment 11. The discussion of NOAEL needs to include work on much lower dosages, showing adverse effects well below the predicted NOAEL.
Response: It is known that the NOAEL is the highest experimental level of a chemical on animal or human that is without adverse effect under certain exposure condition. In the subchronic and reproductive toxicity tests of this study, mice were given doses of 0.4, 2, 10, and 50 mg/kg bw/3-d BHPF. We think that the dose of 0.4 mg/kg bw/3-d is much lower than the NOAEL (50 or 5 mg/kg bw/d) reported for BPA, so we compared the value with the NOAELs of BPA in discussion.

Comment 12.
There are other mechanisms of action of bisphenols beyond estrogen signaling that should be discussed.
Response: According to your suggestion, we added other mechanisms of action of bisphenols beyond estrogen signaling in the discussion.
"Moreover, it should be noted that BHPF might induce anti-estrogenic effects through mechanisms other than nuclear estrogen receptors, as reported for BPA and some other bisphenols, which can induce adverse effects through mechanisms including the estrogen membrane receptor, estrogen-related receptor gamma, pregnane X receptor, etc 2,16,20-22 . The microarray analysis also showed that some genes involved in biotransformation and estrogen metabolism were up-regulated in the uteri of the BHPF-treated mice (Fig. 4b), and the up-regulation of these genes may facilitate the in utero metabolism of estrogen, thereby suppressing the effect of estrogen on the uterus." (pages 12-13, lines 259-266) Response to Reviewer #3's Comments: General Comments: First, I note that the Editor asked me to pay specific attention to the analytical-chemistry and the statistical aspects of this work.
These are certainly most central to my expertise; however, I am also reasonable well qualified to judge the (anti-)estrogenic assays and microarray results. The histopathology and some details of the in vivo studies are the only aspects of the paper that are beyond my comfortable reach. This is a very thorough, compelling, and interesting piece of work. The Authors conducted experiments that span a wide range of disciplines, assembling all the pieces needed to strongly "make their case". Specifically, they show conclusively that: 1) fluorene-9-bisphenol (BHPF) occurs in plastic bottles All of these lines of evidence are laid out in a very logical fashion. The Authors use these results to illustrate the larger implications that: a) alternatives to BPA may be as harmful, or more harmful, to the public as BPA, b) thus, the various pressures (regulations, public concerns, etc.) that force companies to replace "hot button" chemicals may be doing more harm than good, c) the topic of anti-estrogens in the environment -much less studied than that of estrogens -deserves more attention from researchers. I have heard each of these larger points a few times before, although each is "fresh" enough that I would consider them relatively novel. Indeed, I cannot recall a single case where these points were made in a more-compelling and thorough manner. I feel strongly that this paper would be of interest to those in the Environmental Science community. On a personal note, I am involved in work that uses chemical monitoring data from waste water treatment plants. Of course, I will abide by the Journal's rules of confidentiality, but I am very curious to know if BHPF could be measured in any of these samples. I do feel that this paper, once published, will likewise impact the thinking of others in this field.
My recommendation is to accept this paper will minor modificati ons. I have read the scope and criteria for publication for Nature Communications, and I think this paper is well suited. Following are some specific comments that are aimed at improving the presentation (and also some comments regarding the Editor's specific charge to assess the statistical and analytical methodology).
Response: Thank you very much for your kind and positive comments. We are pleased to write the response to your request about if BHPF could be measured in waste water treatment plants. Although we have not performed measurement of BHPF in waste water, we studied the BHPF pollution in urban surface water in 8 cities of China and an informal water sample from Mississippi River in Minneapolis. We found BHPF could be ubiquitously detected in urban surface water in China. The highest level of BHPF was detected in a water sample collected from a lake in Beijing, which was reached 54.8 ng/L. The level of BHPF was about 20.4 ng/L in the water sample from Mississippi River. So, we think BHPF should be detectable in waste water.
Comment 1. With regard to statistics -there really is nothing "fancy" herewhich is as it should be. The Authors use 10 replicates, which is about as good as you see with in vivo rodent studies. A well-established statistical program is used; differences in classes are assessed with ANOVA using Fisher's as a post-hoc test. In the figures, error bars are generated using standard deviation, which, to their credit, is more conservative than standard-error-of-the-mean error bars (which is often used). If I wanted to be nitpicky, I would point out that Fisher's test is a little less conservative than Tukey's test, but that is really more of a personal preference, and would probably not make any difference.
Also, the Authors do not state whether or not they tested the data for normality and heteroscedasticity before applying the ANOVA tests. Strictly speaking, ANOVA is only applicable for data that meet these criteria (although a small degree of non-adherence is well tolerated). Looking at the data, I suspect there are no problems, but it might be worthwhile to close that loop.

Response:
To test the normality and heteroscedasticity of the data before applying the ANOVA tests, we had performed One-Sample Kolmogorov-Smirnov Test and Test of Homogeneity of Variances prior to running one-way analysis of variance and Fisher's least significant difference tests. The data is normally distributed and the statistical methods used in this study should be appropriate. According to your suggestion, we added the following words "after running the one-sample Kolmogorov-Smirnov test and the test of homogeneity of variances" in the revised manuscript. (page 28, lines 598-599) Comment 2. With regard to analytical measurements -The Author's choices for analyses seem perfectly appropriate. They initially used 1H-NMR and 13C-NMR to confirm that BHPF was leaching from a plastic bottle. BHPF was isolated by fractionating a methanol leachate. NMR is the "gold standard" for identifying (or confirming the identity) of a relatively pure organic chemical.
Using both 1H and 13C NMR takes this analysis to a very high level of confidence. My only quibble with this part of the work is that I would have liked to see a Figure with a more explicit comparison of the NMR spectrum of the isolate with that of an authentic standard of BHPF. I feel sure that the Authors have made this comparison for their own sake -indeed, I think the NMR spectrum in Supplementary Information Figure 1A is for the authentic standard.
However, this is not clearly stated, and it is not presented in a way that can be compared directly to the NMR spectrum of the isolate in Figure 1. Once identified, the Authors used GC/MS (with derivatization) to quantify BHPF in drinking water and in human serum. They included a partially deuterated form of BHPF (which was synthesized in-house) as an internal standard. Again, this is the "gold standard" method for target analysis (and quantification) of an organic chemical whose mass spectrum and GC retention time is known. No qualms here.
Response: Thank you for your comments and suggestions. Because the NMR spectrum was reported previously by Liu et al. (2008), the NMR spectrum of BHPF standard was not shown in this manuscript, but we added the following sentence in the revised manuscript to make it legible according to your suggestion.
"The NMR results were consistent with those for BHPF reported previously 11 ." (page 4, lines 69-70) Comment 3. It seems that there has been some inversions (typos) when referring to Figures and sub-parts of Figures. For example, in the Caption, Figure 7D is described as "dead fetus ...". It seems pretty clear that Figure 7C is actually the dead fetus (and it is referred to as such in the text). In addition, the "order" of Figure 5 and Figure 6 (the actual graphics) are switched (6 appears in the document before 5). And, it appears that some text references to 5/6 are reversed. I am not sure I caught all of these issues, so beware! Response: Thank you for your careful works on review this manuscript. According to your suggestions, we conducted a careful modification of the manuscript, and the errors were corrected. Response: According to your suggestion, the sentence was moved to the end of Discussion.
"Moreover, this study raises questions about the safety of BPA substitutes, and indicates the defect of the current chemical management for the substitution of hazardous chemicals." (page 17, lines 361-363) Comment 6. The English linguistics of this paper is quite good; however, some issues will need to be addressed. For example, "drinking water" is called "drink water". I will not list more here, but several other subtle misusages appear.
Finally, I would note that the references seemed appropri ate, and that all sections of the manuscript were clear, lucid, and very well written and organized.
Response: According to your suggestion, we conducted a careful modification of the manuscript and corrected the errors, and two native English language editors had help us to revise the manuscript.