Do Microplastics Affect the Photodegradation of Duloxetine and Its Phototoxicity to Protozoan Spirostomum ambiguum (Müller, 1786) Ehrenberg, 1835?

Round 1

Reviewer 1 Report

A major aim of this study was to determine whether or not four different microplastics that pollute aqueous environments would influence photodegradation of the antidepressant duloxetine by simulated sunlight. While it was found that the microplastics had no effects on duloxetine photodegradation and phototoxicity, the authors employed HPLC-MS/MS analysis to detect 34 putative duloxetine photoproducts. T.E.S.T. toxicity analyses indicated that one-third were toxic, with two being more toxic than duloxetine: 2-methylnaphthalene-1,4-dione (MNPD) and 2 hydroxynaphthalene-1,4-dione (HNPD). The paper submitted by the authors is extremely well written. Experiments are conducted with great precision and care.

The authors should provide references to support the following statement appearing on page 2 of the manuscript. “Firstly, the presence of MPs may lead to the generation of free radicals on their surfaces which could accelerate the photodegradation of the drug and change its direction, leading to the formation of other photodegradation products. Secondly, the suspension of MPs could slow down the photodegradation process by acting as a quenching agent and reducing radiation exposure to DLX. To the best of our knowledge, the impact of MPs on the photodegradation of selective serotonin reuptake inhibitors (SSRIs), including DLX, has not been studied yet.”

What wavelengths of light does duloxetine absorb? How does this overlap with the spectral output afforded by the SunTest apparatus employed in duloxetine photodegradation experiments?

The authors should avoid using the general term “free radical” and instead provide more specific descriptors such as carbon-centered radical, allyl radical, hydroxyl radical, superoxide anion radical, etc.

Table 4 should be revised to include data obtained for the parent compound in dark control reactions, observed vs calculated masses of all photodegradation products, tentative identities when applicable (e.g., MNPD, HNPD, hydroxy-or epoxy-DLX and dihydroxy-DLX ), and whether or not the photoproducts were observed by other research groups.

What chemical mechanisms have been proposed for duloxetine photodegradation in the literature? Are hydroxyl radicals and/or singlet oxygen thought to be involved? Would the mechanisms account for major photooxidation products observed by the authors (MNPD, MNPD, etc.)? If so, how?

Author Response

Reviewer 1. A major aim of this study was to determine whether or not four different microplastics that pollute aqueous environments would influence photodegradation of the antidepressant duloxetine by simulated sunlight. While it was found that the microplastics had no effects on duloxetine photodegradation and phototoxicity, the authors employed HPLC-MS/MS analysis to detect 34 putative duloxetine photoproducts. T.E.S.T. toxicity analyses indicated that one-third were toxic, with two being more toxic than duloxetine: 2-methylnaphthalene-1,4-dione (MNPD) and 2 hydroxynaphthalene-1,4-dione (HNPD). The paper submitted by the authors is extremely well written. Experiments are conducted with great precision and care.

Authors. Thank you very much for appreciating our efforts in preparing the manuscript and for all your comments.

The authors should provide references to support the following statement appearing on page 2 of the manuscript. “Firstly, the presence of MPs may lead to the generation of free radicals on their surfaces which could accelerate the photodegradation of the drug and change its direction, leading to the formation of other photodegradation products. Secondly, the suspension of MPs could slow down the photodegradation process by acting as a quenching agent and reducing radiation exposure to DLX. To the best of our knowledge, the impact of MPs on the photodegradation of selective serotonin reuptake inhibitors (SSRIs), including DLX, has not been studied yet.”

Authors. The references were added: 1) Trawiński, J.; Skibiński, R. Studies on photodegradation process of psychotropic drugs: A review. Environ Sci Pollut Res 2017, 24, 1152-1199. 2) Zhu, K.; Jia, H.; Zhao, S.; Xia, T.; Guo, X.; Wang, T.; Zhu, L. Formation of environmentally persistent free radicals on micro-plastics under light irradiation. Environ Sci Technol 2019, 53, 8177-8186.

What wavelengths of light does duloxetine absorb? How does this overlap with the spectral output afforded by the SunTest apparatus employed in duloxetine photodegradation experiments?

Authors. The DLX absorption spectrum in the wavelength range 200-350 nm was inserted in Table 1. The emission spectrum of the SunTest CPS+ was added in 2.3 (line 126). Due to the fact that the SunTest CPS+ device emits light 300-800 nm, these spectra overlap in a small range (300-320 nm).

The authors should avoid using the general term “free radical” and instead provide more specific descriptors such as carbon-centered radical, allyl radical, hydroxyl radical, superoxide anion radical, etc.

Authors. Thank you very much for this comment. We quoted the expression "free radicals" from works discussing the action of various radicals, hence the general term was used. We agree with the reviewer that precise radical terms should be provided when discussing detailed photochemical transformations.

Table 4 should be revised to include data obtained for the parent compound in dark control reactions, observed vs calculated masses of all photodegradation products, tentative identities when applicable (e.g., MNPD, HNPD, hydroxy-or epoxy-DLX and dihydroxy-DLX ), and whether or not the photoproducts were observed by other research groups.

Authors. Thank you very much for this comment. Table 4 includes only photoproducts, i.e. compounds not present in the control (DLX in the dark). Instead of providing observed vs. calculated masses, we entered calculated masses and delta in ppm in Table 4. This allows the reader to quickly become aware of a potential error in the mass determination. Additionally, we provided the most important peaks from the MS2 spectrum indicating the potential chemical formula of the photoproduct, and the spectral similarity score between measured and theoretical isotope patterns, the high value of which indicates a high probability of the predicted and obtained structure. In the discussion in section 4.2 we discussed which DLX transformation products had been previously identified.

What chemical mechanisms have been proposed for duloxetine photodegradation in the literature? Are hydroxyl radicals and/or singlet oxygen thought to be involved? Would the mechanisms account for major photooxidation products observed by the authors (MNPD, MNPD, etc.)? If so, how?

Authors. Thank you very much for this comment. Understanding the mechanism of DLX photodegradation requires the use of quenching agents that differentiate radicals. This went beyond the purpose of this work but will be implemented in further stages of our project. Previous studies on the photodegradation of DLX do not determine the role of individual radicals, only Santoke et al. (2012) indicated the involvement of the triplet state in the degradation of DLX. Due to very fragmentary literature data on the mechanisms of DLX degradation and our planned future research, we have not discussed this topic in the current publication.

Reviewer 2 Report

The submitted manuscript entitled “Do Microplastics Affect the Photodegradation of Duloxetine 2 and its Phototoxicity to Protozoan Spirostomum ambiguum 3 (Müller, 1786) Ehrenberg, 1835?” raises an extremely important problem related to the growing pollution of the aquatic environment, including pharmaceuticals and microplastic, and the associated threat to living organisms. The assessed manuscript is an example of well-planned and conducted analyzes and research, and obtained results were properly presented.

However, I have a few comments:

Quantitative DLX analysis was performed using the HPLC-PDA method. The authors only provide the concentration range for the standard curve. Has the method been validated?

The initial DLX concentration was 20 mg/L, but a DLX standard curve was constructed in the concentration range of 0.2 - 10 mg/L. How do the authors explain this?

Moreover, in Figure 1 a) The values corresponding to DLX exceed the value of 20 mg/L, i.e. the maximum initial.

Author Response

Reviewer 2. The submitted manuscript entitled “Do Microplastics Affect the Photodegradation of Duloxetine 2 and its Phototoxicity to Protozoan Spirostomum ambiguum 3 (Müller, 1786) Ehrenberg, 1835?” raises an extremely important problem related to the growing pollution of the aquatic environment, including pharmaceuticals and microplastic, and the associated threat to living organisms. The assessed manuscript is an example of well-planned and conducted analyzes and research, and obtained results were properly presented.

Authors. Thank you very much for appreciating our efforts in preparing the manuscript and for all your comments.

However, I have a few comments:

Quantitative DLX analysis was performed using the HPLC-PDA method. The authors only provide the concentration range for the standard curve. Has the method been validated?

The initial DLX concentration was 20 mg/L, but a DLX standard curve was constructed in the concentration range of 0.2 - 10 mg/L. How do the authors explain this?

Moreover, in Figure 1 a) The values corresponding to DLX exceed the value of 20 mg/L, i.e. the maximum initial.

Authors. Thank you very much for the comments. The method for the determination of DLX with HPLC-PDA was validated. Validation parameters have been added to section 2.4:

A standard curve of DLX solutions was linear in the concentration range of 0.2–10 mg L−1 (r2=0.9970). The LOD value was 0.1 mg L⁻¹. The precision of DLX determination deter-mined at a concentration of 1 mg L⁻¹ was 11%, while the accuracy of the determination was 9.5%. Due to the fact that the concentration of unirradiated samples was 20 mg L⁻¹, these samples were diluted twice before determination, and the suspensions were centrifuged at 10,000 x g for 3 min.

Initial DLX concentration values exceeded 20 mg L⁻¹, especially in the case of analyzes of microplastic suspensions. We were unable to explain this situation. The reason may have been the use of a different matrix (Tyrod's medium) than the standard curve (deionized water).