The effect of gamma irradiation on the stability of vitamin D in select finfish species

Jessica S Brown; Patricia R Calvo; Pujita Julakanti; Fatima Mohiuddin; Abdullah Basir Khan; Kemly Julien; Varun Natarajan; Leonardo B Maya; Kaylyn A Keith; Anthony P DeCaprio; Ryan Hollingsworth; Frank Benso; Robert P Smith

doi:10.1371/journal.pone.0332867

. 2025 Sep 26;20(9):e0332867. doi: 10.1371/journal.pone.0332867

The effect of gamma irradiation on the stability of vitamin D in select finfish species

Jessica S Brown ^1,^*, Patricia R Calvo ², Pujita Julakanti ¹, Fatima Mohiuddin ¹, Abdullah Basir Khan ¹, Kemly Julien ¹, Varun Natarajan ¹, Leonardo B Maya ³, Kaylyn A Keith ³, Anthony P DeCaprio ³, Ryan Hollingsworth ⁴, Frank Benso ⁴, Robert P Smith ^5,⁶

Editor: Shafaq Fatima⁷

PMCID: PMC12469152 PMID: 41004494

Abstract

Finfish are an excellent source of dietary vitamin D and additional nutrients. Accordingly, their global consumption has increased. To meet this demand, finfish are now imported across large geographic distances. This poses several challenges, including the potential contamination of finfish with pathogenic bacteria, which can cause food-borne illnesses. Gamma irradiation is a potential solution with a history of reducing bacterial density in foods without compromising nutrients or taste. While some previous work has suggested that vitamin D is stable during irradiation, others have suggested that irradiation may reduce vitamin D. Importantly, these studies have not been completed in finfish tissue of commercialized species, which may offer cross-protection against any reduction in vitamin D. However, this has yet to be evaluated. In this study, three filets each of salmon and trout were dissected into multiple sections and each section was exposed to a different dose of gamma irradiation under both chilled (4°C) or frozen condition (−17°C). Here, we show that the stability of vitamin D during irradiation depends on finfish species and temperature in a dose-dependent manner. Specifically, we found that there was no significant change in vitamin D when trout was irradiated in the chilled or frozen state. Conversely, salmon showed a significant decrease in vitamin D when radiation doses exceeded 0.5 kGy in the chilled state and 2 kGy in the frozen state. Overall, our results indicate that irradiation of finfish may not reduce vitamin D concentration when applied at dosages of 0.5 kGy and 2 kGy or less in chilled and frozen conditions, respectively. Consequently, irradiation may represent a mechanism to increase the safety of consuming finfish while not impacting an important source of dietary vitamin D.

Introduction

Finfish are regarded as an excellent source of many essential nutrients and vitamins, including vitamin D. This is being increasingly recognized by the population as per capita consumption rate of finfish and their products has increased. For example, over the past 30 years, a marked increase in finfish consumption has been noted in the United States. In 2021, Americans consumed an average of 20.5 pounds of finfish and shellfish, representing the highest average consumption weight of seafood reported to date [1]. It is expected that to satisfy the increased demand for finfish, the amount of imported finfish sources from geographically distant regions will increase [2].

Importing finfish from geographically distinct regions poses several challenges, including an increased chance for spoilage and contamination with pathogens [3,4]. Among the many bacteria that can contaminate finfish, Vibrio sp., which contaminates finfish in their natural habitat or during the handling and processing phases, is of increasing concern [3,5]. Indeed, the number of individuals infected with Vibrio sp. has generally increased [6]. Infections from these pathogens can be fatal; approximately 50% of immunocompromised patients with V. vulnificus septicemia die [7]. Widespread outbreaks of V. parahaemolyticus have been associated with the consumption of seafood [5]. Strikingly, 18% of foodborne illnesses in the United States have been attributed to the consumption of finfish contaminated with pathogens [8]. Overall, there is a need to identify, develop, and implement strategies that reduce the incidence of contamination of finfish with Vibrio sp. and additional pathogens that cause foodborne illness.

One strategy that has a proven track record of reducing the density of bacteria [9–12], including Vibrio sp. [13], Listeria sp. [13], and Clostridium sp. [14,15], in foods while maintaining food quality is gamma irradiation. In the irradiation process, ionizing energy is applied to the food, which kills bacteria by causing DNA damage [16,17]. The dose of irradiation required to eliminate bacteria is temperature dependent as multiple studies have shown that higher doses of gamma irradiation are required to inactivate bacteria at subfreezing temperatures [18]. Irradiation can also extend the shelf life of foods [18,19] while maintaining key nutrients and organoleptic properties [20–22]. Multiple countries have implemented the irradiation of finfish to reduce the density of pathogenic bacteria and to increase food safety [17]. Furthermore, irradiating chicken, beef, lettuce, and crustaceans is already commonplace in the United States [23].

While irradiation represents an established strategy that could reduce the incidence of pathogens found in finfish, the objective of this study was not to evaluate the microbial efficacy of irradiation, but rather to assess how radiation dose and temperature affect the stability of vitamin D in finfish tissue of commercial species. This is critical to address, as finfish represents one of the only sources of food-acquired vitamin D. As many people are regarded as vitamin D deficient [24], any loss of dietary vitamin D during irradiation could have a negative health outcome. Previous work performed on whole extracts of finfish [10], vitamin D resuspended in organic solvents [10], and chicken [25] has yielded somewhat conflicting results. On the one hand, some studies show that irradiation does not impact the concentration of vitamin D [26]. On the other, some previous work has shown that irradiation can reduce the concentration of vitamin D [10,27]. However, to date, not a single study has investigated the stability of vitamin D in commercially important, fresh and frozen finfish species. This is important to consider as the composition of the chemical matrix where vitamin D is found may react differently to irradiation and may provide protection against any loss. To ensure practical relevance, we chose to irradiate finfish samples under chilled and frozen conditions that reflect how these products are commonly stored and sold commercially. To address this knowledge gap, we measured the effect of irradiation on vitamin D in two commonly consumed finfish species in both the chilled and frozen states.

Materials and methods

Reagents

Vitamin D3 (cholecalciferol), vitamin D3(6,19,19-d3) solution (1 mg/mL in ethanol, 97 atom % D), 25-hydroxyvitamin D3 (6,19,19-d3) solution (100 μg/mL in ethanol, 97 atom % D) and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD, 97%) were purchased from Sigma-Aldrich (St. Louis, MO). Reagent alcohol, potassium hydroxide, pyrogallol, ethyl ether anhydrous, hexanes, and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Hampton, NH).

Preparation of fish samples and irradiation

Filets of salmon and trout were provided as donations from fish vendors near Gulfport, Mississippi. All fish samples were delivered frozen to Gateway America (Gulfport, MS) for irradiation using a Gray*Star Genesis Cobalt-60 Gamma radiation source (Fig 1). Before irradiation, salmon and trout filets were dissected into four equal pieces (salmon 5.3 ± 0.5 cm x 3.6 ± 0.5 cm, trout 8.9 ± 1.1 cm x 6.4 ± 0.9 cm). Each portion was placed into an irradiation safe bag and irradiated at a different dose under chilled (0°C) and frozen (−17°C) conditions. The temperature was monitored for consistency throughout the irradiation process using Tru-temp industrial dial thermometers. One portion of three dissected fish were irradiated at doses 0, 0.5, 1, and 2 kGy for chilled conditions or doses 0, 2, 3, and 4 kGy for frozen conditions. After irradiation, samples were packaged in large coolers with dry ice and shipped overnight to Nova Southeastern University (Fort Lauderdale, FL). Once received they were transferred to a freezer and stored at −4°C until analysis.

Fig 1 — Samples were stored in the freezer before, during, and after shipping.

Saponification and vitamin D extraction

Samples were thawed overnight in a refrigerator (4°C) and the edible parts were homogenized using a stainless-steel food processor. Approximately 3–5 g of sample was transferred to a 100 mL round bottom flask and spiked with 50 µL vitamin D3(6,19,19-d₃) solution (2 ng/µL) internal standard, 30 mL of 2% ethanolic pyrogallic acid, and 20 mL of 50% potassium hydroxide. The flask was sealed and purged with nitrogen and allowed to stir overnight. Two extractions were performed using 30 mL of an 80:20 hexane:ether mixture and the combined extracts were washed once with 30 mL of distilled water. The solvent was evaporated using a rotary evaporator, reconstituted in 5 mL of hexane, transferred to a 20 mL vial, and evaporated to dryness. The final extract was reconstituted in 1 mL of 70% aqueous acetonitrile. The samples were shipped overnight on ice to Florida International University (Miami, FL) for LC-MS/MS analysis. Prior to analysis, 50 µL of the sample was combined with 25 µL 25-hydroxyvitamin D3 (6,19,19-d₃) solution (2 ng/µL) internal standard and 75 µL of PTAD (1 g/100 mL) derivatizing agent.

LC-MS/MS analysis

LC-MS analysis was performed with an Agilent 1290 LC coupled to a 6460 QQQ-MS. The LC column used was a Zorbax reverse-phase C18 (50 x 3.0 mm, 1.8 µm) at a column temperature of 40°C. The mobile phases were (A) 5 mM ammonium formate in water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid, at a flow rate of 0.4 mL/min with the following gradient: hold 75% B for 3.00 min, then increase to 100% B for 2 minutes, held at 100% B for 0.5 min, and returned to 75% B for a total run time of 6 min. Sample injections were 5 µL. Detection was carried out using multiple reaction monitoring with positive ion electrospray ionization (ESI). For quantification, the transition 560.4 → 280.1 was monitored for vitamin D3-PTAD and the transition 563.4 → 301.0 was monitored for deuterated vitamin D3-PTAD. The concentration of vitamin D3-PTAD was quantified using vitamin D3-d3-PTAD as an internal standard.

Data analysis

Initially, changes in vitamin D content were assessed among the four portions of an individual filet. The amount of vitamin D present in an irradiated portion was compared to the amount in the unirradiated portion (control, 0 kGy) to determine the percent of vitamin D recovered in the remaining three portions following irradiation. Then the percentage of vitamin D recovered from the portions irradiated at the same dose were averaged from the three individual fish filets. Analysis was performed in JMP (version 17, Cary, NC) to determine if there was a significant change in vitamin D content following gamma irradiation when compared to the control. Normality for each finfish species and irradiation temperature was assessed using a Shapiro-Wilk test. All samples were normally distributed (salmon – chilled P = 0.3069; salmon – frozen, P = 0.1907; trout – chilled, P = 0.727; trout – frozen, P = 0.8526). Thus, statistical significance was determined using an ANOVA followed by the Tukey HSD post-hoc test for pairwise comparisons of the doses. A two-way ANOVA to assess the contribution of both finfish species and temperature on the stability of vitamin D as also performed. Here, we only used data from 2 kGy as it was the only consistent irradiation dose used for both temperatures. As this data set was found to be not normally distributaries (Shapiro Wilk; P < 0.001), we log transformed the data to achieve normality. We then performed a two-way ANOVA using R version 4.2.2. A significance level of 0.05 was used throughout to indicate significance.

Results and discussion

Vitamin D was extracted by combining aspects of various previously reported protocols [28–30]. This involved alkaline saponification followed by multiple extractions. A deuterated internal standard (vitamin D3-d3) was used in all samples and calibrators. This has previously been shown to be more reliable than an external calibration curve. Additionally, vitamin D3 was derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to improve ionization and the signal-to-noise ratio [31]. The ratio of vitamin D3 to internal standard was used to quantify the vitamin D3 concentration in all samples. Fig 2 shows an example of a standard calibration curve and a chromatogram for the derivatized standard (vitamin D3-PTAD) and the corresponding mass spectrum for the 560.4 → 280.1 mass transition. The calibration curve gave high linearity (R² = 0.998), and strong responses for Vitamin D3 were observed in all samples. The limit of detection (LOD) and limit of quantitation (LOQ) of vitamin D3 for this method were 3 and 10 ng/mL, respectively.

Fig 2 — (a) Standard calibration curve for the analysis of vitamin D3 ranging from 10-750 ppb spiked with 100 ppm vitamin D3-d3 internal standard. (b) Representative chromatogram for 750ppb standard and (c) mass spectrum for relevant retention time.

The effect of gamma irradiation on finfish species in the chilled state was evaluated (Fig 3). Following irradiation, the amount of vitamin D in chilled salmon decreased by an average of 28% ± 6% for 0.5 kGy, 60% ± 7% for 1 kGy, and 53% ± 5% kGy for 2 kGy. For trout in a chilled state, the amount of vitamin D decreased by an average of 38% ± 10% for 0.5 kGy, 38% ± 29% for 1 kGy, and 62% ± 20% for 2 kGy. Interestingly, there was a species-specific change in the concentration of vitamin D post-irradiation. Salmon had a significant decrease in the quantity of vitamin D at 1 kGy and 2 kGy. However, when trout fillets were irradiated under the same conditions, there was no statistically significant reduction in vitamin D across all doses measured. This suggests that the chemical composition of the matrix in which vitamin D is found has different protective properties for each species; protection is significantly better in trout as compared to salmon.

To further examine this trend, the effect of irradiation on fillets of salmon and trout irradiated in the frozen condition was assessed. Owing to the previously reported protective effect of temperature on the stability of vitamins during irradiation [32], filets were irradiated at higher doses (2 kGy, 3 kGy, and 4kGy) than in the chilled state. Similar to the effect of irradiation in the chilled state, the amount of vitamin D in salmon, but not trout, declined with increasing irradiation dose (Fig 4). Following irradiation, the amount of vitamin D3 in frozen salmon decreased by an average of 36% ± 16% for 2 kGy, 65% ± 26% for 3 kGy, and 94% ± 2% kGy for 4 kGy. For frozen trout, the amount of vitamin D3 decreased by an average of 1% ± 19% for 2 kGy and increased for 3 kGy, and 4 kGy. There were significant decreases in the concentration of vitamin D when salmon were irradiated at 3 kGy and 4 kGy. However, when trout were irradiated, there were no significant differences in the concentration of vitamin D amongst all irradiation doses examined.

Fig 4 — Percent recovery standardized to the unirradiated control (0 kGy). * indicates significantly different from the unirradiated control. Statistics: Salmon: ANOVA: P = 0.0045, Tukey HSD (3 kGy: P = 0.017, 4 kGy: P = 0.0039). Trout: ANOVA: P = 0.59. Average from three different fillets, with error bars = SEM.

We also found a statistically significant interaction between species and temperature (two-way ANOVA, P = 0.006), indicating that the effect of temperature on vitamin D stability during irradiation varies depending on the species. When comparing the concentration of vitamin D recovered at 2 kGy in the fresh and frozen states, but within each species, our data supports the previously noted protective effect of lower temperatures during irradiation [32]. When salmon was irradiated at 2kGy in the chilled state, a significant decrease of 53% of vitamin D was observed. However, there was no significant decrease in vitamin D when irradiated at the same dose in the frozen state; here, 64% of vitamin D remained.

Owing to the importance of vitamin D in human health, we sought to understand how the irradiation of finfish would affect its stability. We found species, temperature, and dose-dependent effects on the stability of vitamin D. Previous work found that sharpfin barracuda irradiated on ice at 5 kGy did not show a significant change in the amount of vitamin D as compared to an unirradiated control [26]. This suggests that a reduction in vitamin D is not observed at high irradiation amounts. However, reduced vitamin D due to irradiation has also been previously reported. Reductions of 5%, 10%, and 15% were observed when vitamin D3 was resuspended in either butter or cod oil and irradiated at 2.5, 5, and 10 kGy, respectively [27]. Knapp and Tappel [10] also found that irradiation at 2 kGy and 21^oC reduced the concentration of vitamin D by 30% when it was resuspended in isooctane. This previous finding was consistent with our observation that when salmon was irradiated at 2 kGy, more vitamin D remained when irradiation occurred in frozen fillets as opposed to chilled fillets. Interestingly, when vitamin D was resuspended in salmon oil and irradiated at 2 kGy and 21^oC [10], there was no apparent loss when irradiated under the same conditions, likely due to the protective effects of the matrix [32]. This finding is similar to our findings that showed reductions in vitamin D in salmon but not trout, which may be owing to differences in the chemical composition of the matrix and any cross-protective qualities. According to the USDA FoodData Central [33], farmed salmon and trout have similar protein content (20.4 g and 19.9 g per 100 g, respectively), but differ notably in fat and water content. Salmon contains more than twice the fat (13.4 g vs. 6.18 g) and less water (64.6 g vs. 73.8 g) than trout. We propose that these compositional differences—along with the presence of skin on one side of the trout fillets versus fully skinned salmon fillets—may help contribute to the observed species-dependent differences observed in vitamin D. How the individual components of the matrix provided cross-protective effects is largely unknown and should be studied in the future. This information could be used to determine which finfish species are likely to show a reduction in vitamin D upon irradiation.

Irradiation is often considered a method to improve food safety without affecting organoleptic properties. However, this must be evaluated for each food product, and the irradiation dose and temperature during irradiation must be considered. Warming during irradiation has been associated with a reduction in organoleptic properties [32]. Increasing irradiation dose is also associated with a reduction in organoleptic properties [34]. Accordingly, lower doses of radiation are often preferred (< 5 kGy), which have been shown to continue to inactivate between 90 and 95% of pathogens [35], thus maintaining food safety. While the organoleptic properties of irradiated finfish were not specifically evaluated in this study, we did observe undesirable changes in color and odor. For example, when our chilled salmon samples were irradiated at doses of 2 kGy, the pink color of the salmon meat turned dull, and the fish emitted a smell akin to cooked fish. Thus, like most previous work, irradiation dose and temperature should be carefully selected to maintain the organoleptic properties of finfish while ensuring a reduction in the density of pathogenic bacteria.

Ultimately, the goal of irradiation is to reduce the quantity of pathogenic bacteria that may contaminate foods to make them safer for consumption. Importantly, the irradiation doses examined in this study, including those that did not significantly reduce vitamin D, are within and above the doses required to lead to significant reductions in pathogens most often associated with finfish, including Vibrio sp. For example, the D₁₀ value (the dose of irradiation required to achieve a 90% reduction in bacterial density) of V. parahaemolyticus when irradiated in saline and on ice ranges from 0.03–0.04 kGy [36]. Similar findings are reported in additional studies for V. parahaemolyticus [37,38,39] and additional Vibrio species [40,41]. Salmonella sp. and Listeria sp., both of which can be introduced during the food handling and processing phases [42–44], are also sensitive to irradiation at these doses [45,46]. For example, the density of L. monocytogenes in Nagli fish (Sillago sihama) was reduced to undetectable levels after irradiation at 3 kGy and subsequent storage for 5 days at ~1^oC. However, L. monocytogenes was detected in the unirradiated control samples [37]. Taken together, irradiation of finfish that are potentially contaminated with the most frequently observed pathogens could potentially reduce the density of such bacteria, improving safety significantly. However, this would have to be evaluated for each finfish species studied herein in a future study.

In conclusion, we found that irradiating select finfish species could reduce the vitamin D concentration in a species, dose, and temperature-dependent manner. Together, these findings suggest that irradiation at appropriate intensities can reduce the pathogenic burden on finfish while maintaining the concentration of vitamin D.

Acknowledgments

We thank McHenry Mauger for his assistance with the statistical analysis of the data.

Data Availability

All raw data associated with the manuscript can be found in figshare (https://doi.org/10.6084/m9.figshare.30128818.v1).

Funding Statement

Robert P Smith received funding from the Seafood Industry Research Fund.

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45.Kamat A, Thomas P. Radiation inactivation of some food-borne pathogens in fish as influenced by fat levels. J Appl Microbiol. 1998;84(4):478–84. doi: 10.1046/j.1365-2672.1998.00369.x [DOI] [PubMed] [Google Scholar]
46.Rajkowski KT. Radiation D10-values on thawed and frozen catfish and tilapia for finfish isolates of Listeria monocytogenes. J Food Prot. 2008;71(11):2278–82. doi: 10.4315/0362-028x-71.11.2278 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0332867.r001

Decision Letter 0

Hari Misra

23 Feb 2025

PONE-D-24-54557

The effect of gamma irradiation on the stability of vitamin D in select finfish species

PLOS ONE

Dear Dr. Smith,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Manuscript was reviewed by a member of editorial board and an external reviewer. Both find it unsuitable for publication in its current form. Findings need revisit in their experimentation, data collection and analysis and that might take much longer time, and also may not reach to a quality of manuscript to convince the conclusion drawn from the work. Authors may submit it some other journals if they wish so.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Kind regards,

Hari S. Misra, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. The focus of this manuscript is on the study of the effect of irradiation on vitamin D in fish, but there is literature reporting that irradiation can affect the structure of the substance. Please add characterization of the structure of vitamin D and assess whether there is an effect of different irradiation doses on the structure of vitamin D and thus the function of the vitamin D.

2. Since irradiation has a certain penetration distance for sterilization, please add a flow chart of the process of actually irradiating a fish product. Because the size of the irradiation dose varies for different thicknesses of irradiated material, the structure of vitamin D may be destroyed in order to obtain a sterilizing dose.

3. Line32-42 The focus of this manuscript should be on the importance of vitamin D and the significance of using gamma radiation. If the effects of Vibrio on fish are to be illustrated, the number of Vibrio should be measured after the method is used.

4. Line43-50 List the current methods of preservation used in the transportation of fish and their advantages and disadvantages, followed by a description of the advantages of the methods used in this manuscript.

5. Line49-50 Use of the method for other articles is accompanied by corresponding literature.

6. Line126-137 Whether the effect of different gamma rays on pure vitamin D should be set.

7. Line200-201 Provide relevant literature that does not alter sensory properties.

8. Line208-209 Appropriate sensory tests including color, odor, etc. should be provided.

9. Line213-227 Will the treatment cause adverse effects and within what range will it not cause adverse effects with references.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

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PLoS One. 2025 Sep 26;20(9):e0332867. doi: 10.1371/journal.pone.0332867.r002

Author response to Decision Letter 1

20 Mar 2025

Dear Dr. Mitra,

While we would like to thank you and the reviewer for evaluating our manuscript, we believe that you and the reviewer have made fundamental errors in your assessment, not abided by the review standards set forth by PLoS One, and have simply asked us ‘to do more work’ that is clearly outside the scope of our manuscript.

PLoS One evaluates based on technical rigor and not novelty. As listed on their website, the criteria for review are the following:

1. The study presents the results of original research. Our work and data are entirely novel as they detail the effects of irradiation on vitamin D in commercially important finfish species.

2. Results reported have not been published elsewhere. Our results have not been published elsewhere. They are entirely novel.

3. Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail. Not a single comment below indicates that the technical aspects of the proposal are flawed. We have applied appropriate statistics, including correcting for multiple comparisons, have sufficiently high replication, our experiments and analyses were performed according to industry standards.

4. Conclusions are presented in an appropriate fashion and are supported by the data. Our conclusion, as indicated in the discussion and abstract, is that irradiation can affect the stability of vitamin D in a dose-dependent, temperature-dependent, and fish species-specific data. This conclusion is well supported by the data presented in Figures 2 and 3, and is supported by a statistical analysis. Further, not a single comment below challenged the fundamental conclusions made in our manuscript.

5. The article is presented in an intelligible fashion and is written in standard English. Our manuscript was written and edited by authors with over 50 publications. Not a single comment below indicates that our manuscript needs to be better written; it only indicates that more work needs to be done for, quite frankly, the sake of asking for more work as it is clearly outside of the scope of the manuscript.

6. The research meets all applicable standards for the ethics of experimentation and research integrity. Not a single comment below indicated any issues with ethics. In fact, we supplied all of our raw data for the reviewers to analyze, including calibration curves (Fig. 1). This far exceeds the standard of the community.

7. The article adheres to appropriate reporting guidelines and community standards for data availability. Not a single comment below indicates that our data is not reported correctly or does not meet the community standard of availability. All of our raw data is indeed included in the manuscript.

As further evidence that both you and reviewer did not follow the standard of review and assessment as set forth by PLoS One, the reviewer indicated that all of the above was fine in their evaluation. Directly from the reviewer report:

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Thus, all of the criteria required for publication in PLoS One have been met. What remains are comments from the reviewer that lack a scientific basis and clarity, are outside the scope of our manuscript, or are so minor in their scope that 10 minutes in a document editing system could fix the issue. Certainly, none of these comments, either alone or as a synthesis, warrants a rejection as we have clearly met the review criteria for PLoS One as indicated by the reviewer themselves in the report above.

Our responses to the reviewer comments are below.

1. The focus of this manuscript is on the study of the effect of irradiation on vitamin D in fish, but there is literature reporting that irradiation can affect the structure of the substance. Please add characterization of the structure of vitamin D and assess whether there is an effect of different irradiation doses on the structure of vitamin D and thus the function of the vitamin D.

This is outside of the scope of our manuscript. The central focus of our manuscript is the stability of vitamin in finfish after irradiation. As clearly indicated in the introduction:

“While irradiation represents an established strategy that could reduce the incidence of

pathogens found in finfish, it remains unclear as to how irradiation impacts the stability of vitamin D in finfish tissue of commercial species.”

This is exactly what we have studied and precisely what we have reported on.

Our central conclusion, as clearly indicated in the abstract is:

“Overall, our results indicate that irradiation of finfish may not reduce vitamin D concentration when applied at dosages of 0.5 kGy and 2 kGy or less in chilled and frozen conditions, respectively.”

And, from our discussion:

“In conclusion, we found that irradiating select finfish species could reduce the vitamin D concentration in a species, dose, and temperature-dependent manner. Together, these findings suggest that irradiation at appropriate intensities can reduce the pathogenic burden on finfish while maintaining the concentration of vitamin D.”

We make no claim on the structure of vitamin D, nor does it matter to any single conclusion made in our manuscript. This comment does not impact the technical rigor or conclusions made in the manuscript and is thus entirely unwarranted and outside the scope of our manuscript.

The reviewer is correct that the effects of ionizing radiation on vitamin D have been previously studied. In fact, it is commonly known that to produce vitamin D, ionizing radiation is required. The steps in this process have been well documented 1. Accordingly, there is no reason to reproduce this data in this manuscript.

Finally, Vitamin D was accurately identified by comparing its retention time to a pure chemical standard and confirming the match through molecular and fragment ion patterns in the mass spectral data. This community standard for measuring vitamin D has been used multiple times before 2-12. We have applied the most rigorous methods to detect vitamin D.

Taken together, this comment is a) outside the scope of the manuscript, b) does not challenge the fundamental analysis or conclusions of the manuscript, and c) provides no value to the manuscript if performed.

We have included both the dosimetry data and a figure showing the irradiation process (Figure 1). This new material can be found in the methods section.

However, the second sentence of the reviewer’s comment is entirely unclear. We have no interest and make no claim on sterilizing doses. That is not a conclusion of our manuscript, nor is it a goal. Determining a sterilizing will have not change the fundamental conclusions of our manuscript, nor will it add anything to the manuscript within its scope.

Further to that, many, if not all, papers that have looked at the stability of vitamins and nutrients in finfish or other matrices do not identify sterilizing dose 13-30. Why? Because it is entirely unnecessary and outside of the scope of this and the work of others.

We also note that sterilizing doses for multiple finfish species have been established elsewhere 31. Thus, this work is superfluous to what has already been produced by the community.

In summary, we added a flow chart and dosimetry data. However, determining a sterilizing dose is completely unnecessary within the scope of the manuscript and will have no fundamental impact on our conclusion or data analysis.

We included this information in our original manuscript to highlight the use of gamma irradiation on fish due to concerns about pathogenic bacteria, Vibrio being chief amongst them. Since numerous studies have already demonstrated that gamma irradiation effectively eliminates Vibrio in fish, we did not repeat these experiments. In light of the reviewer’s comment, we have removed this paragraph.

4. Line 43-50 List the current methods of preservation used in the transportation of fish and their advantages and disadvantages, followed by a description of the advantages of the methods used in this manuscript.

Our primary focus is on vitamin D stability and irradiation of finfish, not on all preservation and transportation methods of finfish; that material belongs in a review article. Adding this information will only muddy the waters in terms of the central focus of our manuscript, will not fundamentally enhance readability, or will not change our analysis or conclusions. In addition, many manuscripts have been published on the irradiation of foods, and the vast majority do not summarize this information as it is superfluous and obscures the central message of these publications 2-12.

5. Line 49-50 Use of the method for other articles is accompanied by corresponding literature.

It is not clear what the reviewer is asking for here. We think that they might be asking for references indicating approval for the irradiation of foods in the United States. We have included these references.

6. Line 126-137 Whether the effect of different gamma rays on pure vitamin D should be set.

This has already been determined previously, as noted in our original manuscript.

“Previous work performed on whole extracts of finfish32, vitamin D resuspended in organic solvents32, and chicken25 has yielded somewhat conflicting results. On the one hand, some studies show that irradiation does not impact the concentration of vitamin D 33. On the other, some previous work has shown that irradiation can reduce the concentration of vitamin D 5,32”

For example, previous work has assessed the stability of pure vitamin D in benzene, ethanol, cod oil, liver oil, and butter 5,32. This synthesis of work has clearly established the effects of irradiation on pure vitamin D. Our work establishes the impact of irradiation on vitamin D in finfish and is entirely different from this previous work.

Therefore, why repeat this work? It would not fundamentally alter our manuscript's technical rigor, conclusions, or anything else. Once again, this is a reviewer comment that asks for more work for the sake of asking for more work. It is entirely irrelevant to our manuscript.

Moreover, nearly all other manuscripts investigating the stability of a vitamin or nutrient in foods do not look at the purified product 13-27. Why? Because, as clearly stated in our introduction, the matrix of the food can determine the stability 34.

“However, to date, not a single study has investigated the stability of vitamin D in fresh, commercially important finfish species. This is important to consider as the composition of the chemical matrix where vitamin D is found may react differently to irradiation and may provide protection against any loss.”

The premise of the above statement has been established in multiple studies and in multiple foods (reviewed in 34), but not commercially important finfish, which we are examining here as demonstrated by our specific questions outlined in the introduction

“While irradiation represents an established strategy that could reduce the incidence of pathogens found in finfish, it remains unclear as to how irradiation impacts the stability of vitamin D in finfish tissue of commercial species.”

Thus, not only is the proposed work entirely outside of the scope of the manuscript, but it is entirely contradictory to the specific question we asked, as indicated in the introduction.

To clarify that this work has already been completed, the introduction now reads:

“Previous work performed on whole extracts of finfish32, purified vitamin D resuspended in organic solvents32, and chicken25 has yielded somewhat conflicting results. On the one hand, some studies show that irradiation does not impact the concentration of vitamin D 33. On the other, some previous work has shown that irradiation can reduce the concentration of vitamin D 5,32”

7. Line 200-201 Provide relevant literature that does not alter sensory properties.

It is not clear what the reviewer is requesting here. However, we think that they are asking to provide a reference about the effects of irradiation on organoleptic properties. While this was discussed in depth and with references in the paragraph, we have removed the paragraph in response to the previous comment.

8. Line 208-209 Appropriate sensory tests including color, odor, etc. should be provided.

This is entirely outside the scope of our manuscript. This suggested work will have absolutely no impact on our technical rigor or fundamental conclusions. Moreover, multiple other studies that have examined the effect of irradiation on nutrients and vitamins do not do this 14,17,18,25-30.

The reviewer is simply asking for more work for the sake of asking for more work. We make absolutely no claim on the organoleptic properties of finfish; simply that we noticed changes and that they need to be evaluated elsewhere and carefully chosen. As indicated in our discussion:

“Irradiation is often considered a method to improve food safety without affecting organoleptic properties. However, this must be evaluated for each food product, and the irradiation dose and temperature during irradiation must be considered. Warming during irradiation has been associated with a reduction in organoleptic properties34. Increasing irradiation dose is also associated with a reduction in organoleptic properties35. Accordingly, lower doses of radiation are often preferred (< 5 kGy), which have been shown to continue to inactivate between 90 and 95% of pathogens36, thus maintaining food safety. While the organoleptic properties of irradiated finfish were not specifically evaluated in this study, we did observe undesirable changes in color and odor. For example, when our chilled salmon samples were irradiated at doses of 2 kGy, the pink color of the salmon meat turned dull, and the fish emitted a smell akin to cooked fish. Thus, like most previous work, irradiation dose and temperature should be carefully selected to maintain the organoleptic properties of finfish while ensuring a reduction in the density of pathogenic bacteria.”

In response to the reviewers' comment, we have removed this entire paragraph.

9. Line 213-227 Will the treatment cause adverse effects and within what range will it not cause adverse effects with references.

It is unclear what the reviewer is requesting. We think that they might be asking about organoleptic properties. Accordingly, we have added information about previous studies that have irradiated finfish within the doses we used and examined organoleptic properties post-irradiation. This can be found in the second last paragraph of the discussion.

In conclusion, the reviewer made multiple fundamental errors in assessing our manuscript. The majority of the comments are outside our paper's scope, do not fundamentally affect the analysis, conclusions, or clarity of our manuscript, are nearly impossible to interpret, and are essentially comments asking for more work for the sake of asking for more work. Simply doing more experiments that do not impact the fundamental technical rigor, significance, or conclusions of a manuscript owing to the whims of a reviewer is not a review criterion established by PLoS One.

Literature Cited

1 Bikle, D. D.

Attachment

Submitted filename: finfish_reponse_to_reviewers.docx

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PLoS One. doi: 10.1371/journal.pone.0332867.r003

Decision Letter 1

Shafaq Fatima

20 Jun 2025

<div>PONE-D-24-54557R1The effect of gamma irradiation on the stability of vitamin D in select finfish speciesPLOS ONE

Dear Dr. Smith

Thank you for submitting your manuscript to PLOS ONE. As editor, After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please submit your revised manuscript by June 30. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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“Robert P Smith received funding from the Seafood Industry Research Fund. However, the funding does not cover the cost of publication. “

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Additional Editor Comments (if provided):

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #2: Abstract

Q.1

What was the duration of the study or trial period during which samples were monitored or stored?

Q.2

How many samples were used per species and per treatment condition (irradiation dose, temperature)?

Introduction

Q.3

Furthermore, irradiating chicken, beef, lettuce, and crustaceans is already commonplace in the United States. Add references (Line 49-50).

Q.4

In 2021, Americans consumed an average of 20.5 pounds of finfish and shellfish, representing the highest average consumption weight reported to date. How much of this consumption is specifically attributable to finfish alone? Is it appropriate to generalize this statistic to support an argument focused only on finfish? (Line 27)

Q.5

Irradiation causes DNA damage to kill bacteria, but how does this vary across different bacterial genera mentioned (e.g., Vibrio vs. Clostridium)? Are there species-specific resistance thresholds to irradiation? (Line 45-46)

Q.6

What is the rationale for testing both chilled and frozen conditions?

Methodology

Q.7

How many individual fish were used per species for the entire experiment? The method only mentions "three dissected fish. (Line 75)

Q.8

Were all pieces taken from the same fish used as replicates for each dose, or were they from different fish? If from the same fish, how do the authors justify the use of pseudo-replication in the statistical analysis?

Q.9

Why were different dose ranges used for chilled (0–2 kGy) and frozen (0–4 kGy) conditions? Is there literature or preliminary data justifying this discrepancy? Add reference (Line 83)

Q.10

How were the chilled (0°C) and frozen (-17°C) conditions monitored and maintained during irradiation? (Line 80)

Q.11

What was the rationale for using vitamin D3-d3 as the internal standard at 50 µL (2 ng/µL)? Was the spike level optimized to fall within the linear range of the LC-MS/MS?

Q.12

Samples were stored at –4°C after irradiation. This is an unusually high "freezer" temperature for sample preservation. Could vitamin D degradation have occurred prior to extraction and analysis? (Line 88)

Q.13

Was the assumption of homoscedasticity (equal variance) checked before applying ANOVA? If heterogeneity of variance was present, why was ANOVA used rather than a non-parametric test or transformation?

Results and Discussion

Q.14

The data are reported as mean ± SEM. How many technical replicates per biological replicate were run to ensure analytical precision?

Q.15

Why does 1 kGy in chilled salmon show a greater loss (60%) than 2 kGy (53%)? (Line 144)

Q.16

Author hypothesized a matrix effect protects vitamin D in trout but not salmon. But no lipidomic, proteomic, or moisture content comparisons made between the two species to support this claim.

Q.17

2 kGy dose in frozen salmon retained more vitamin D than the chilled equivalent, what are the implications of partial thawing or ice crystal formation during irradiation?

Q.18

Authors should perform Two-way ANOVA to compare the effects of species and temperature simultaneously? A full factorial analysis could better reveal interaction effects.

Q.19

Author mentioned that previous work showing no vitamin D loss at 5 kGy in sharpfin barracuda. Could this species-specific protection due to matrix effects, or are methodological differences?

Q.20

Author must assess the microbial load post-irradiation to confirm pathogen reduction. If not, how this manuscript confidently mentioned that irradiation at appropriate intensities can reduce the pathogenic burden on finfish while maintaining the concentration of vitamin D? (Line 230).

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

**********

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PLoS One. 2025 Sep 26;20(9):e0332867. doi: 10.1371/journal.pone.0332867.r004

Author response to Decision Letter 2

2 Jul 2025

We sincerely thank the reviewers for their thoughtful and constructive feedback on our manuscript. We have carefully considered each comment and have revised the manuscript accordingly. Below, we have copied each reviewer comment in full and provided our detailed response directly beneath each one. Changes made to the manuscript are indicated where applicable, and line numbers have been provided to identify the location of each change. Importantly, the line numbers reflect lines in the marked up version of the manuscript, and not the clean version of the manuscript.

Reviewer #2:

Abstract

Q.1 What was the duration of the study or trial period during which samples were monitored or stored?

The research was performed over a nine-month period.

• Irradiation: Late October 2022

• Vitamin D extraction

o Salmon, chilled: Late November to late December 2022

o Salmon, frozen: Late October to late November 2022

o Trout, frozen: January 2023

o Trout, chilled: February to mid-March 2023

• LC-MS analysis

o Salmon, chilled: Late January 2023

o Salmon, frozen: Late January 2023

o Trout, frozen: Late January 2023

o Trout, chilled: Late June 2023

However, we feel as though this timeline is not relevant to the findings or conclusions of our manuscript. Thus, we have not modified the abstract.

Q.2 How many samples were used per species and per treatment condition (irradiation dose, temperature)?

Three filets (or samples) were used for each species and temperature combination (e.g., three filets of chilled salmon), totaling 12 filets for the four combinations. Each filet was dissected into 4 portions, and each portion was irradiated with a different dose of gamma irradiation. We added a clarifying sentence to the abstract (Lines 132-134).

Introduction

Q.3 Furthermore, irradiating chicken, beef, lettuce, and crustaceans is already commonplace in the United States. Add references (Line 49-50).

A reference from the FDAs approval of gamma irradiation was added to the manuscript (Line 179).

Q.4 In 2021, Americans consumed an average of 20.5 pounds of finfish and shellfish, representing the highest average consumption weight reported to date. How much of this consumption is specifically attributable to finfish alone? Is it appropriate to generalize this statistic to support an argument focused only on finfish? (Line 27)

We acknowledge that the reported figure of 20.5 pounds includes both finfish and shellfish; however, we chose to include this statistic because it is a widely reported and an authoritative measure of U.S. seafood consumption trends. While it does not distinguish between finfish and shellfish, it effectively illustrates the broader point that seafood consumption is increasing in the United States.

Q.5 Irradiation causes DNA damage to kill bacteria, but how does this vary across different bacterial genera mentioned (e.g., Vibrio vs. Clostridium)? Are there species-specific resistance thresholds to irradiation? (Line 45-46)

The references included in the manuscript were intended to demonstrate that the use of gamma irradiation to eliminate bacterial pathogens is a well-established and accepted practice. While we recognize that bacterial resistance to irradiation can vary by species and genus, a detailed examination of species-specific resistance thresholds was beyond the scope of our study.

Nevertheless, our primary objective was to evaluate whether gamma irradiation adversely affects the nutritional components of fish, rather than to assess its differential efficacy across bacterial genera. A sentence was added to the introduction to specify this objective (Lines 180-183)

Q.6 What is the rationale for testing both chilled and frozen conditions?

Commercial fish products are sold either fresh (chilled) or frozen and we sought to test the stability of vitamin D for both types of products. A sentence was added to the introduction to explain our choice for irradiating at chilled and frozen conditions (Lines 190-195).

Additionally, it has also been well-established that reducing the temperature of irradiation can reduce the effects of irradiation on changes in nutrition and microbial load. Thus, we chose to examine the impacts of irradiation at these two temperatures. Two sentences were added to the introduction that mentions this occurrence and why we chose two ranges of doses for chilled and frozen samples (Lines 173-175).

Methodology

Q.7 How many individual fish were used per species for the entire experiment? The method only mentions "three dissected fish. (Line 75)

Q.8 Were all pieces taken from the same fish used as replicates for each dose, or were they from different fish? If from the same fish, how do the authors justify the use of pseudo-replication in the statistical analysis?

To account for biological variability among fish samples and to standardize comparisons across irradiation doses, we selected three filets from three different individual fish and dissected each into four portions. Each piece was then subjected to a different irradiation dose. This approach allowed us to monitor relative changes in vitamin D concentration within the same filet, using the non-irradiated portion as the control (set at 100%). By doing so, we minimized inter-sample variability and ensured that observed differences were attributable to irradiation rather than natural variation among individual fish. This has been clarified in the Data Analysis subsection of the Materials and Methods section (Lines 288-291).

Q.9 Why were different dose ranges used for chilled (0–2 kGy) and frozen (0–4 kGy) conditions? Is there literature or preliminary data justifying this discrepancy? Add reference (Line 83)

Previous studies show that gamma irradiation is less effective at killing bacteria at lower temperatures, requiring higher doses at subfreezing conditions [1]. These ranges are also consistent with previous work that has examined the impacts of irradiation on finfish, as well as irradiation doses currently used to treat finfish in international markets. Two sentences were added to the introduction that mentions this occurrence and why we chose two ranges of doses for chilled and frozen samples (Lines 173-175).

Q.10 How were the chilled (0°C) and frozen (-17°C) conditions monitored and maintained during irradiation? (Line 80)

All irradiation was performed at Gateway America, which routinely irradiates food for commercial distribution. During the irradiation process, probes that monitor temperature and dose are inserted amongst the irradiated samples. All irradiation occurs in a large, refrigerated warehouse, which assists to ensure that fluctuations in temperature do not occur. We have clarified this in the Methods section (Lines 226-227)

Q.11 What was the rationale for using vitamin D3-d3 as the internal standard at 50 µL (2 ng/µL)? Was the spike level optimized to fall within the linear range of the LC-MS/MS?

During initial method development, a larger volume of internal standard (IS) was added to each sample. However, we observed that this produced a disproportionately large IS signal relative to the vitamin D3 signal in fish extracts, which could interfere with accurate quantitation. As a result, we optimized the IS spike volume to 50 µL at 2 ng/µL to ensure that the IS concentration was comparable to the analyte concentration in typical samples. This adjustment provided a more balanced signal, minimized the risk of ion suppression or detector saturation, and ensured accurate, reproducible quantitation within the linear dynamic range of the method.

Q.12 Samples were stored at –4°C after irradiation. This is an unusually high "freezer" temperature for sample preservation. Could vitamin D degradation have occurred prior to extraction and analysis? (Line 88)

Previous studies have shown that vitamin D3 is more stable at lower temperatures, with significantly greater stability reported at 4 °C compared to 27 °C [2]. Thus, we are confident that storing our samples at –4 °C, a subfreezing temperature, did not lead to degradation of vitamin D3 prior to extraction and analysis. Although –4 °C is slightly higher than typical ultra-low freezer settings (e.g., –20 °C), it is still below the threshold at which vitamin D degradation has been reported under standard storage conditions.

Q.13 Was the assumption of homoscedasticity (equal variance) checked before applying ANOVA? If heterogeneity of variance was present, why was ANOVA used rather than a non-parametric test or transformation?

As noted in our original submission the distribution of all vitamin D for each finfish species and irradiation temperature was assessed using a Shapiro-Wilk test for normality. Thus, an ANOVA followed by a Tukey HSD is highly appropriate. We have updated our manuscript to include P values for each finfish species and temperature for the Shapiro-Wilk test in the Methods section (Lines 293-296).

Results and Discussion

Q.14 The data are reported as mean ± SEM. How many technical replicates per biological replicate were run to ensure analytical precision?

To ensure analytical precision, randomly selected samples were run in duplicate as technical replicates. This approach allowed us to confirm consistency in measurement across runs without significantly depleting the limited sample volume available for analysis. However, because technical replicates were not consistently run for each sample, we only used biological replicates for our analysis.

Q.15 Why does 1 kGy in chilled salmon show a greater loss (60%) than 2 kGy (53%)? (Line 144)

While it would appear that 1 kGy showed a great loss as compared to 2 kGy, these data points are in fact not different from each other. A two-tailed t-test comparing vitamin D isolated from chilled salmon irradiated at 1kGy and 2kGy did not show significance (P = 0.44). The averages are also well within the error range as noted by the overlapping error bars.

Q.16 Author hypothesized a matrix effect protects vitamin D in trout but not salmon. But no lipidomic, proteomic, or moisture content comparisons made between the two species to support this claim.

Performing lipidomic, proteomic or moisture content analysis is beyond the scope of this study. However, according to the USDA FoodData Central [3], farmed salmon and trout have similar protein content (20.4 g and 19.9 g per 100 g, respectively), but differ notably in fat and water content. Salmon contains more than twice the fat (13.4 g vs. 6.18 g) and less water (64.6 g vs. 73.8 g) than trout. While we did not directly measure these properties in our samples, we proposed that such compositional differences—along with the presence of skin on one side of the trout fillets versus fully skinned salmon fillets—may help explain the species-dependent differences observed in vitamin D stability following irradiation. These details have been added to the Results and Discussion section (Lines 417-422).

Q.17 2 kGy dose in frozen salmon retained more vitamin D than the chilled equivalent, what are the implications of partial thawing or ice crystal formation during irradiation?

We did not observe thawing or ice crystal formation during irradiation, as the temperature was kept constant. It is expected that Vitamin D stability is improved at lower temperatures, which has been previously demonstrated for other compounds during irradiation.

Q.18 Authors should perform Two-way ANOVA to compare the effects of species and temperature simultaneously? A full factorial analysis could better reveal interaction effects.

Unfortunately, we cannot run a two-way ANOVA across our entire data set as only 0 kGy and 2kGy were the only consistently tested irradiation doses tested between both temperatures.

We did, however, perform a two-way ANOVA using the 2 kGy data. Because a Shapiro-Wilk test performed on only the 2kGy indicated that it was not normally distributed (P < 0.0001), we log-transformed the data to achieve normality. We then performed a two way ANOVA considering both species and temperature. Our findings indicate that there is a significant effect of both finfish species and temperature on the stability of vitamin D (P = 0.006). This analysis has been added to the Materials and Methods section (Lines 297-315) and the results included in the Results and Discussion section (Lines 384-386).

Q.19 Author mentioned that previous work showing no vitamin D loss at 5 kGy in sharpfin barracuda. Could this species-specific protection due to matrix effects, or are methodological differences?

It could be either.

The methods used in the article noted above were not well described and simply stated that an 1990 version of the AOAC official method was used. Thus, it is no clear exactly clear what they performed and how it might differ from our extraction and analysis methods. Additionally, the barracuda was sun-dried and had a moisture content of 24.6%, while our samples were raw and had water content of 64.6% for salmon and 73.8% for trout [3].

We also note that it could be a species specific difference due to matrix effects. For example, non-irradiated sharpfin barracuda has 7.09% lipids, 7.09g/100g or lipids. This contrasts to 13.4 g vs. 6.18 g of fat per 100g observed in salmon and trout, respectively [3].

Q.20 Author must assess the microbial load post-irradiation to confirm pathogen reduction. If not, how this manuscript confidently mentioned that irradiation at appropriate intensities can reduce the pathogenic burden on finfish while maintaining the concentration of vitamin D? (Line 230).

It was not the goal of this work to demonstrate the microbial reduction potential of irradiation, as it has been demonstrated extensively in other studies [4-11]. This literature supports that doses much lower than the doses used in this study effectively reduce or eliminate microbial load. This study was solely focused on the effects of irradiation on Vitamin D.

We have modified to statement in the results/discussion section to make it clear that our claim is based on previous literature, and not examined in this manuscript. We have also noted that this should be examine in a future study (Lines 451-456)

References

1. Radomyski T, Murano EA, Olson DG, Murano PS. Elimination of pathogens of significance in food by low-dose irradiation: a review. Journal of food protection. 1994;57(1):73–86.

2. Zareie M, Abbasi A, Faghih S. Influence of storage conditions on the stability of vitamin D3 and kinetic study of the vitamin degradation in fortified canola oil during the storage. Journal of Food Quality. 2021;2021(1):5599140.

3. United States Department of Agriculture ARS. FoodData Central 2019 [cited 2025]. Available from: fdc.nal.usda.gov.

4. Dion P, Charbonneau R, Thibault C. Effect of ionizing dose rate on the radioresistance of some food pathogenic bacteria. Canadian journal of microbiology. 1994;40(5):369–74.

5. Ahmed IO, Alur MD, Kamat AS, Bandekar JR, Thomas P. Influence of processing on the extension of shelf‐life of Nagli‐fish (Sillago sihama) by gamma radiation. International journal of food science & technology. 1997;32(4):325–32.

6. Jakabi M, Gelli DS, Torre JC, Rodas MA, Franco BD, Destro MT, et al. Inactivation by ionizing radiation of Salmonella enteritidis, Salmonella infantis, and Vibrio parahaemolyticus in oysters (Crassostrea brasiliana). Journal of food protection. 2003;66(6):1025–9.

7. Matches J, Liston J. Radiation destruction of Vibrio parahaemolyticus. Journal of Food Science. 1971;36(2):339–40.

8. Ama AA, Hamdy M, Toledo R. Effect of heating, pH and thermoradiation on inactivation of Vibrio vulnificus. Food Microbiology. 1994;11(3):215–27.

9. Torres Z, Kahn G, Vivanco M, Guzman G, Bernuy B. Shelf-life extension and decontamination of fish fillets (Trachurus picturatus murphyi and Mugil cephalus) and shrimp tails (Penaeus vannamei) inoculated with toxigenic Vibrio cholerae O1 El Tor using gamma radiation. Irradiation to control Vibrio infection from consumption of raw seafood and fresh produce I

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PLoS One. doi: 10.1371/journal.pone.0332867.r005

Decision Letter 2

Shafaq Fatima

3 Sep 2025

<div>PONE-D-24-54557R2The effect of gamma irradiation on the stability of vitamin D in select finfish speciesPLOS ONE

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PLoS One. 2025 Sep 26;20(9):e0332867. doi: 10.1371/journal.pone.0332867.r006

Author response to Decision Letter 3

4 Sep 2025

Response to Reviewers

Reviewer/Editor Comment 1

“I recommend a final check to ensure that all references cited in the text are properly included in the Literature Cited section, and that the formatting is consistent throughout.”

Response:

We thank the reviewer for this important suggestion. We undertook a comprehensive review of the reference list and performed the following steps:

1. Cross-check of in-text citations: Every reference cited in the manuscript text was verified against the References section. All in-text citations are now included in the reference list, and no extraneous references remain.

2. Consistency in formatting: References have been reformatted to comply fully with PLOS ONE requirements (Vancouver style):

• All journal titles are now given in full title case (e.g., Journal of Food Protection rather than J Food Prot.).

• Author initials have been standardized without periods (e.g., Smith AB rather than Smith A.B.).

• Page ranges have been expanded (e.g., S391–S395 rather than S391–S5).

• Organizational reports and government documents (e.g., WHO/FAO, USDA, NOAA, CFR) have been reformatted to include full organizational names, place of publication, publisher, year, URL, and access date.

• Book chapters and monographs (e.g., Thayer DW et al. 1991; Diehl JF. 1995) have been revised to include editors, edition, publisher, location, and page ranges.

• Electronic resources (e.g., USDA FoodData Central) now include full URLs and access dates in the proper format ([cited 2025 Apr 18]).

3. Verification of accuracy: We cross-checked all DOIs, PMIDs, and page numbers to ensure accuracy. We also confirmed that no cited articles had been retracted.

These corrections have been applied consistently across all 46 references. We believe this addresses the reviewer’s concern fully.

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PLoS One. doi: 10.1371/journal.pone.0332867.r007

Decision Letter 3

Shafaq Fatima

8 Sep 2025

The effect of gamma irradiation on the stability of vitamin D in select finfish species

PONE-D-24-54557R3

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I as an editor checked the revised version and found that further review is not required. Decision has been made after all references have been cross cheked and improved by the authors. This MS is ready for acceptance.

PLoS One. doi: 10.1371/journal.pone.0332867.r008

Acceptance letter

Shafaq Fatima

PONE-D-24-54557R3

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

All raw data associated with the manuscript can be found in figshare (https://doi.org/10.6084/m9.figshare.30128818.v1).

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PERMALINK

The effect of gamma irradiation on the stability of vitamin D in select finfish species

Jessica S Brown

Patricia R Calvo

Pujita Julakanti

Fatima Mohiuddin

Abdullah Basir Khan

Kemly Julien

Varun Natarajan

Leonardo B Maya

Kaylyn A Keith

Anthony P DeCaprio

Ryan Hollingsworth

Frank Benso

Robert P Smith

Roles

Abstract

Introduction

Materials and methods

Reagents

Preparation of fish samples and irradiation

Fig 1. Process for the gamma irradiation, extraction, and analysis of vitamin D in finfish.

Saponification and vitamin D extraction

LC-MS/MS analysis

Data analysis

Results and discussion

Fig 2. LC/MS/MS results for vitamin D analysis.

Fig 3. The effect of irradiation on the percent recovery of vitamin D in salmon (left) and trout (right) fillets in the chilled state.

Fig 4. The effect of irradiation on the percent recovery of vitamin D in salmon (left) and trout (right) fillets in the frozen state.

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Hari Misra

Roles

Author response to Decision Letter 1

Decision Letter 1

Shafaq Fatima

Roles

Author response to Decision Letter 2

Decision Letter 2

Shafaq Fatima

Roles

Author response to Decision Letter 3

Decision Letter 3

Shafaq Fatima

Roles

Acceptance letter

Shafaq Fatima

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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