A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; hemato-biochemical assays, histology, oxidant and antioxidant activity and genotoxicity

Hasnain Akmal; Shabbir Ahmad; Muddasir Hassan Abbasi; Farhat Jabeen; Khurram Shahzad

doi:10.1371/journal.pone.0302691

. 2024 May 6;19(5):e0302691. doi: 10.1371/journal.pone.0302691

A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; hemato-biochemical assays, histology, oxidant and antioxidant activity and genotoxicity

Hasnain Akmal ¹, Shabbir Ahmad ¹, Muddasir Hassan Abbasi ¹, Farhat Jabeen ², Khurram Shahzad ^1,^*

Editor: Amel Mohamed El Asely³

PMCID: PMC11073686 PMID: 38709735

Abstract

Parabens are being used as preservatives due to their antifungal and antimicrobial effects. They are emerging as aquatic pollutants due to their excessive use in many products. The purpose of this study was to determine the toxic effect of ethyl paraben (C9H10O3) on the hematobiochemical, histological, oxidative, and anti-oxidant enzymatic and non-enzymatic activity; the study also evaluates the potential of ethyl paraben to cause genotoxicity in Rohu Labeo rohita. A number of 15 fish with an average weight of 35.45±1.34g were placed in each group and exposed to ethyl paraben for 21 days. Three different concentrations of ethyl paraben, i.e., T₁ (2000μg/L), T₂ (4000 μg/L), andT₃ (6000 μg/L) on which fish were exposed as compared to the control T₀ (0.00 μg/L). Blood was used for hematobiochemical and comet assay. Gills, kidneys, and liver were removed for histological alterations. The results showed a significant rise in all hemato-biochemical parameters such as RBCs, WBCs, PLT count, blood sugar, albumin, globulin, and cholesterol. An increase in aspartate aminotransferase (AST) and alanine transaminase (ALT) levels directed the hepatocytic damage. Histological alterations in the liver, gills and kidneys of fish were found. Ethylparaben induces oxidative stress by suppressing antioxidant enzyme activity such as SOD, GSH, CAT and POD. Based on the comet assay, DNA damage was also observed in blood cells, resulting in genotoxicity. Findings from the present study indicate that ethyl paraben induces hemato-biochemical alterations, tissue damage, oxidative stress, and genotoxicity.

Introduction

Aquatic toxicity is a serious health hazard for aquatic ecosystems, including primary producers, microorganisms, fish, and humans, because water bodies contain fertilizers, insecticides, pesticides, and pharmaceutical chemicals that are added via leaching, runoff, and spray drift [1]. Toxic chemicals discharged from anthropogenic activities [2] become part of the aquaculture food chain and cause serious health problems for humans who feed on these contaminated products [3].

To meet the requirements of an increasing population, the use of disinfectants, drugs, preservatives, and antifoulant (biocides) chemicals to remove bacteria and fungi has increased in industries. Biocides in aquatic environments have toxic effects with demonstrated detrimental impacts [3]. Parabens are produced when p-hydroxybenzoic acid is esterified in a catalyst and alcohol [4]. They are biocides and derivatives of esters of p-hydroxybenzoic acid [5]. The antimicrobial and preservative abilities of parabens were first reported in 1924 [6]. For more than 70 years in food, pharmaceutical, and cosmetic industries, it has been widely used as a preservative.

Chemical inertness, low cost, worldwide acceptance, and reduced toxicity are the features of paraben that make it the favorite compound of chemists compared to all other biocides used in food, pharmaceutical, and cosmetic products. Parabens are tasteless, odourless and effectively neutral in PH. They do not harden or discolor cosmetic products [7]. The most common members of the paraben family are methylparaben, ethylparaben, propylparaben, butylparaben, and benzyl paraben. They are used separately and in combination with others. Mainly in the aqueous phase, influent contain (˃97%) and wastewater treatment plants contain 79,600 ng/L concentrations of parabens [8].

Estrogenic effect of parabens on fish was observed by Bjerregaard, Andersen [9]. However, the impact of parabens on fish tissues and hematology has not been evaluated.

Parabens enter the human body via dermal exposure, exhalation, and ingestion [10]. Their negative effects on the body cause diabetes, neurotoxicity, adult obesity, and a reduction in the quality and quantity of human sperm. Parabens have been found in the tumour cells of breast cancer, which indicates that they increase the chances of cancer [11]. These toxic compounds harm endocrine function by interfering with endocrine system [12].

Ethylparaben (C₉H₁₀O₃) is a member of the paraben family with a short chain of alkyl groups. The cell penetrability, stability, and antibacterial activity of parabens increase with the increase in the chain [13]. The increase in the basal and vitellogenin levels in the blood by ethylparaben shows oestrogenic effects [4]. Frederiksen, Taxvig [14] studies on the pregnant rat observed that ethylparaben continues its exposure to the fetus by accumulating in the maternal plasma and placenta of the fetus.

The present study aims to evaluate ethyl paraben’s toxic effects on freshwater fish, Labeo rohita, as the concentration of ethyl paraben in aquatic environments increases day by day, which is harmful to aquatic organisms. Fish is a source of protein and part of the food chain; its consumption after being caught from polluted water resources is harmful to humans.

Materials and methods

Laboratory setting and animal handling

All the experiments were performed in the laboratories of the Department of Zoology and Department of Fisheries, University of Okara, Okara, Pakistan. The standard protocols were followed for the preparation of chemicals and reagents. The ethical research committee (ERC) of the University of Okara, Pakistan, provided ethical approval (UO/ERC/2023/36) for fish handling. The fish were caught from the ponds at Head Balloki, District Kasur, Pakistan and then transported to the laboratories. During the fish’s transport, the standard FAO protocol was followed; thereby no mortality occurred. Before transferring these fish to the experimental tank, they were first acclimatized with 0.1% KMNO₄ solution for 7 days to prevent fungal and microbial attacks. Commercially prepared feed (20% protein) was given to all the fish. Dissolved oxygen (DO) and pH of the water in the aquarium ranged from 6.25±2.01 ppm and 7.2±1.02, respectively. The ammonia and nitrate levels varied between 0.19–0.47 ppm and 0.04–0.14 ppm, respectively. The water temperature varied from 25.4 to 30.2°C in the experimental tanks.

Chemical

Ethylparaben (C₉H₁₀O₃) of 99.9% purity was purchased from MACLIN, China. A standard protocol was followed to make all the reagents of analytic grade.

Experimental design and treatment

Four groups of fish were formed for experimental trial: a control group (T₀) and three treatment groups (T₁, T₂, and T₃) each with a triplicate. 15 fish were placed in each group (n = 15). A stock solution of ethylparaben was prepared using analytic-grade ethanol. LC₅₀ value of ethyl paraben for 48 hours was determined (LC50 = 12000 μg/L) by using the probit analysis method [15]. Fish groups T₀, T₁, T₂, and T₃ were exposed to different concentrations of ethylparaben 0 μg/L, 2000μg/L, 4000μg/L and 6000μg/L respectively, for 21 days. 90% of the water in each glass aquarium was replaced with fresh water every second day.

Haematological and biochemical analysis

The blood was procured by direct heart puncture and from the abdominal vein using disposable syringe and 22-gauge needle (Molnar 1960) [16] (n = 3). The blood was collected in an EDTA tube for hematology and was transpired into mottled red top tubes for biochemical analysis. The haematological parameters, including RBC, hematocrit, haemoglobin, MCH, MCV, and MCHC, were assessed by an automatic haematological analyser (HEMA D6031). The white blood cell count was determined by following Ghaffar, Hussain [17].

For biochemical assay, the blood was centrifuged at 4°C and 1,000 rpm for 10 min to separate plasma. Total protein, albumin, globulin, AST, ALT, total cholesterol, HDL-C and LDL-C were analysed using a chemistry analyzer (AU480, Beckman coulter, US). According to Waterborg [18] method, the protein content of the samples was calculated using bovine serum albumin as standard. Glucose level was determined using the O-Toluidine assay [19].

Histology

The protocol of Barse, Chakrabarti [20] was used for histological determination. At the end of the trial, fish were anesthesized with clove oil (50 μl/L) and dissected. Internal organs such as the liver, kidney, and gills were removed from the fish (n = 3). Samples were stored in 90% alcohol until further processing. They were first fixed in neutral buffered formalin, and then in 70% alcohol in four steps. The tissue was embedded in paraffin after preservation and dehydrated using a progressive alcohol series. 4μm sections were stained with hematoxylin and eosin and viewed using an Olympus BX40 microscope. The photographs were taken with a Nikon (SC 35 type 12) camera with a shutter speed of 30 seconds.

Biochemical assay

ROS assay

The activity of reactive oxygen species (ROS) was observed by following Tvrda [21] with a little modification. Homogenised mixture of tissue was mixed with phosphate buffer (0.1 M, pH 7.4). The cells were solubilized using 2M potassium hydroxide (KOH), centrifuged and determined the absorbance in supernatant between 530 nm and 630 nm.

TBARS

The standard protocol of Yagi and medicine [22] was used to measure the activity of TBARS. The homogenised tissue sample was treated with 0.1% Triton X100. 20 μL,40 μL, 60 μL, and 80 μL solution mixtures were mixed with thiobarbituric acid and then heated at 95°C, centrifuged and determined the absorbance in supernatant at 532nm. Tetrametoxy propane was used as standard.

Catalase

The protocol of Sinha [23] was used to analyse catalase. The reagents were Phosphate buffer (0.1 M, pH 7.0) and 30% H₂O₂ solution. The tissue was homogenised in phosphate buffer. The reaction mixture includes1.95 mL phosphate buffer, 0.1 mL of H₂O₂, and 0.05 mL tissue extract. Change in absorbance was recorded at 240 nm.

Superoxide dismutase

The protocol was according to Beauchamp and Fridovich [24]. For the analysis of superoxide dismutase the reagents were methionine (150mM), riboflavin (24μM), phosphate buffer (0.1M, pH 7.5), nitro blue tetrazolium (840 μM), and Na₂EDTA (1.2 mM). The reaction mixture includes 0.05mL tissue extract, 0.25 mL NBT, 0.25 mL. Na2EDTA, 0.25 mL methionine, 1.95 mL phosphate buffer, and 0.25 mL riboflavin. The extract was pipetted in 4 glass tubes. Four more glass tubes were prepared, adding 0.05 mL of phosphate buffer instead of enzyme extract. Three tubes from each set were then placed on shaker at 25°C in fluorescent light for 15 minutes and the last one was kept in dark at 25°C (reference sample). After the incubation period, the change in the absorbance was measured at 560 nm using respected dark-incubated sample as reference for test samples for each series. The SOD activity was expressed in terms of relative enzyme activity (U/mg protein).

Glutathione

The activity of glutathione (GSH) was evaluated using Carlberg and Mannervik [25]. The activity was observed by measuring NADPH at 340 nm. The reaction mixture includes 16 M oxidised glutathione, 8 mM NADPH, 50 mM MgCl_2, and phosphate buffer (0.1 M, pH 7.5). The reaction is initiated by adding supernatant to the reaction mixture.

Peroxidase

Activity of peroxidase in homogenized mixture was measured by formation of TBARS and quantified as malondialdehyde (MDA) equivalents by following Buege and Aust [26].

Comet assay

The comet assay protocol of [27, 28] has been followed. Slides were examined under a fluorescence microscope at 400X. Comet IV Software, was used for scoring the comet [29].

Statistical analysis

Statistical analysis was done by applying ANOVA to IBM SPSS (Version 21) software at p< 0.05 level of significance. Graphic Prism Version 9.3.1 was used for graphical representation.

Results

Hematology

The results of hematological analysis of L.rohita after exposure to ethyl paraben are shown in (Fig 1). After exposure to a high dose (6000μg/L) of ethylparaben, the concentrations of HGB, WBC, RBCs, HCT, MCV, MCH, MCHC and platelets count increased significantly. When they were exposed to medium dose (4000 μg/L), some of these parameters such as HGB, WBC, MCH, MCHC and MCV showed significant elevation but others like RBCs, HCT, and PLT did not show significant increase. Hematological parameters like, HGB, RBCs, HCT MCH MCHC and PLT after low dose (2000 μg/L) treatment did not show significant increase but in the case of WBCs and MCV this increase was significant.

Biochemical parameters

The lipid profile of L.rohita after being treated with ethylparaben is shown in (Fig 2). After exposure to high (6000μg/L) and medium (4000μg/L) doses of ethylparaben, the lipid profile of L.rohita, such as cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and VLDL cholesterol, showed highly significant increase. Low dose (2000 μg/L) treatment of L.rohita showed that some parameters like cholesterol, LDL cholesterol and HDL cholesterol neither increased nor decreased significantly. VLDL cholesterol showed a significant increase at low doses and triglycerides showed a highly significantly increase.

Results of blood sugar, total protein, albumin, globulin, liver functioning enzymes alanine transaminase (ALT), and aspartate aminotransferase (AST) are shown in Fig 3. All these parameters showed significant increases when exposed to different concentrations of ethyl paraben as compared with control group.

Fig 3 — (a) showing alterations in total protein, (b) showing alterations in globulin, (c) showing alterations in albumin (d) showing alteration in aspartate aminotransferase, (e) showing alterations in alanine transaminase, and (f) showing alterations in blood glucose level between control and all three treated groups (low, medium and high doses). All values are mean ± SD at *p<0*.05 level of significance. Steric (*) shows significant steric (**) shows highly significant.

Oxidative stress and antioxidant response

Fish tissues, liver, kidney and gills were used to evaluate oxidative stress ROS and TBARS (Table 1). The responses of antioxidant enzymes GSH, SOD, CAT, and POD are shown in (Fig 4). The measurements of ROS and TBARS from the liver, gills, and kidney showed a significant increase in these biomarkers after treating L. rohita for 21 days with ethyl-paraben for 2000μg/L, 4000 μg/L, and 6000 μg/L as compared with the control group. The measurements of all anti-oxidant biomarkers, i.e., GSH, SOD, CAT, and POD, showed significant reduced values (p≤ 0:05) in the liver, gills, and kidney.

Table 1. Oxidative stress in the liver, gills and kidney of L.rohita exposed to different concentrations of ethylparaben.

Liver	T0 (Control)	T1 (2000 μg/L)	T2 (4000 μg/L)	T3 (6000 μg/L)
ROS (optical density)	0.3500±0.03606	0.4533±0.0802*	0.830±0.040*	1.02±0.075**
TBARS (nmol/TBARS formed/mg protein/ min)	36.87±0.075	43.65±0.060*	53.13±0.080**	60.12±0.105**
Gills
ROS (optical density)	0.32±0.085	0.44±0.07*	0.54±0.08**	0.61±0.08**
TBARS (nmol/TBARS formed/mg protein/ min)	39.89±0.085	46.76±0.075*	63.65±0.075**	67.34±0.085**
Kidney
ROS (optical density)	0.60±0.085	0.64±0.07	0.72±0.08*	0.81±0.095*
TBARS (nmol/TBARS formed/mg protein/ min)	28.65±0.07	35.12±0.095*	39.62±0.09*	41.39±1.23**

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ROS = Reactive oxygen species; TBARS = Thiobarbituric acid reactive substance

Steric (*) showing significant steric (**) showing highly significant.

Histology

Histology of the gills, liver and kidneys of L. rohita is shown in Figs 5–7. Normal morphological structure i.e. secondary lamellae coated with simple squamous epithelial cells was observed during gill histology of control group (5a). When these fish were exposed to different concentrations of ethylparaben, alterations in histology of gills were observed in treated group (5b-5d) such as lamellar disorganization, fusion of primary lamellae, lamellar aneurysm, hyperplasia of epithelial cells, and bone cell deformities. The normal arrangement of glomeruli in the kidney of L. rohita was observed during the histology of the control group (6a). Histology of the treated group (6b-6d) showed glomerular expansion and absence of Bowman’s capsule, tubular cells with hypertrophied nuclei, Sinusoidal spaces, cluster nuclei formation, Melano-macrophages, and damaged parenchyma cells.

Fig 5 — The images include (a) control group, (b-d) 2000 μg/L, 4000 μg/L, and 6000 μg/L. The gills of the control group showed normal histopathology, whereas the other treated groups showed lamellar disorganization (black arrow), fusion of primary lamellae (red arrow), lamellar aneurysm (yellow arrow), hyperplasia of epithelial cells (white arrow), and bone cell deformities (orange arrow).

Fig 7 — The images include (a) the control group, (b-d) 2000 μg/L, 4000 μg/L, and 6000 μg/L. The liver of the control group showed normal histopathology, whereas the other treated groups showed Pyknotic nuclei (black arrow), eosinophilia granules (red arrow), Sinusoidal spaces (yellow arrow), necrosis (green arrow), and Melano-macrophages (blue arrow).

Fig 6 — The images include (a) the control group, (b-d) 2000 μg/L, 4000 μg/L, and 6000 μg/L. The kidney of the control group showed normal histopathology, whereas the other treated groups showed glomerular expansion and absence of Bowman’s capsule (black arrow), tubular cells with hypertrophied nucleus (red arrow), Sinusoidal spaces (yellow arrow), cluster nuclei formation (green arrow), Melano-macrophages (blue arrow), and damaged parenchyma cells (orange arrow).

Liver histology of the control group (7a) showed no change in the liver of L.rohita. The hepatic parenchyma showed sinusoid-limited hepatocyte plaques. The hepatocytes have polygonal shapes, granular cytoplasm, and rounded nuclei. They were arranged in cord. After exposure to ethylparaben, alterations in the hepatocytes of L.rohita were observed (7b-7d). Histology of the treated groups showed pyknotic nuclei, eosinophilia granules, sinusoidal spaces, necrosis, and melano-macrophages in liver tissues.

Genotoxicity

The blood cells of L.rohita were used for alkaline comet assay to evaluate the potential of ethyl paraben to cause genotoxicity in L.rohita (Fig 8). The results of alkaline comet assay indicate that ethylparaben causes DNA damage in blood cells. This damage increases with the increase in concentrations of ethyl paraben. The percentage of tail DNA observed at 15.69±0.74 after exposure to the high dose as compared with the control group at 0.91±0.55 reveals a significant difference. By increasing concentrations of ethyl paraben in A (0μg/L), B (2000μg/L), C (4000μg/L), and D (6000μg/L), the difference increases significantly. Olive tail moment (OTM) shows substantial variations as well; at the high dose (6000μg/L), it was 8.37±2.45, whereas at the control (0μg/L), it was 0.63±0.11. Tail DNA and olive tail moment (OTM) increase with the increase in the concentration of ethylparaben.

Discussion

Aquatic toxicity has become a major concern due to the high proportion of toxicants being released into aquatic ecosystems that have gone unchecked in the recent years. The majority of these toxicants are carcinogenic and cause anthropogenic effects on humans as they feed on aquatic organisms and plants [30]. Parabens belonging to one of these aquatic toxicants have anthropogenic effects and cause regional and localized pollution problems around the world [31, 32]. These toxicants, such as paraben, cause environmental disasters [33].

Hematological parameters are considered indicators for detecting toxicology. Different toxicants like parabens cause alterations in hematological parameters [34]. These alterations have been considered in response to high oxygen demands and gas transfer during stress conditions. The spleen and liver reactivate erythropoiesis in hypoxic conditions to compensate for increased oxygen demand in peripheral tissue [35]. In the present study, the elevation in the concentrations of HGB, WBC, RBCs, HCT, MCV, MCH, MCHC, and platelet count has been observed after exposure to different concentrations of ethyl paraben to cope with stress conditions. Our results are supported by the study done by Park, Lee [36], which has observed an increased level of RBCs, HGB, Hct, MCV, MCH, and MCHC in rats after exposure to parabens. Similar rises in RBCs, HGB, and Hct in male rates have been observed by İNKAYA, KARABULUT [37]. The higher concentration of WBCs and PLT count after exposure to ethyl paraben has been observed in the present study. Therefore, a similar decrease in the concentration of WBCs and PLT count after exposure has been detected by İNKAYA, KARABULUT [37]. No previous work has been found on ethylparaben exposure to fish. This increase in WBCs is due to the infection and inflammation caused by ethylparaben exposure to the body. During the infection state, the immune system activates, and white blood cell concentration increases [38]. Ethylparaben exposure causes inflection due to which platelet count increases from low to high concentration as compared to the control. This increase in platelet count is due to thrombocytosis—a condition that causes an increase in platelet concentration in the infectious state [39].

Parabens such as ethylparaben, being endocrine disruptors, interfere with normal function in many processes and alter them like the immune system, lipid homeostasis, glucose level, and thyroid functioning [40]. Many hormones, like testosterone and oestrogen, are prepared from lipids. Results of the lipid profile after being exposed to different concentrations of ethyl paraben showed a significant increase in cholesterol, HDL cholesterol, LDL cholesterol, and VLDL cholesterol as compared to the control group. Kim and Chevrier [41] also recorded the same increase in cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and VLDL cholesterol in humans, both male and female. Lipids cannot breakdown in the blood and must be delivered to and from cells via low-density lipoproteins (LDL) and high density lipoproteins (HDL). High density lipoprotein cholesterol (HDL-C) transports cholesterol from the arteries to the liver. As a result, excessive serum cholesterol levels may be caused by hepatic dysfunction [42]. Triglycerides act as a source of immediate and stored energy. Due to high energy demand in stress conditions, the fish showed high triglyceride levels. The current study evaluates the toxic effect of ethyl paraben on hepatic tissues. For liver alanine transaminase (ALT) and aspartate aminotransferase (AST), blood sugar, total protein, albumin, and globulin are good biomarkers for hepatic toxicity. The results of serum biochemical showed that all these parameters were elevated significantly as compared to the control group. These results were supported by the findings of Beriry, Atef [43] and Darbre and Harvey [44]. The increases in liver enzymes occur directly in response to liver toxicity caused by ethylparaben in hepatocytes. The primary function of the liver is to detoxify toxic chemicals and produce antioxidant enzymes such as catalase that break down hydrogen peroxide. That’s why the liver is a primary organ to be damaged due to paraben toxicity [45]. Blood glucose levels also increased after exposure to ethylparaben. Similar increases in blood sugar were recorded by Bellavia, Chiu [46] in pregnant women after they put them at risk for gestational diabetes. No previous study on lipid profile and blood glucose level was found on the fish exposed to ethylparaben in a laboratory or natural environment.

Biochemical alterations are confirmed by histological changes in the current study. In fish, gills are primary organs in contact with water and are involved in osmoregulation, respiration, and excretion. Hence, they remain the primary target for toxicants. Histology of the gills revealed that after exposure to ethylparaben, the gills showed lamellar disorganization, fusion of primary lamellae, lamellar aneurysm, hyperplasia of epithelial cells, and bone cell deformities of secondary lamellae. The alterations in gills, like epithelial lifting, hyperplasia, and fusion of epithelial cells, were also reported by Camargo and Martinez [47]. A similar alteration in histology of the gills after exposure to paraben, as recorded by Beriry, Atef [43] supports our present study.

The kidney, being a vital organ in osmoregulation and excretion, also remains an easy target for toxicants [48]. The most common defects caused by water toxicants are tubule degeneration, dilation of capillaries, and degeneration of parenchyma cells of the kidney Irnidayanti, Fatona [49]. Similar changes came out as results of kidney histology of L. rohita like, glomerular expansion and absence of Bowman’s capsule, tubular cells with hypertrophied nuclei, Sinusoidal spaces, cluster nuclei formation, Melano-macrophages, and damaged parenchyma cells as compared to the control, which showed a normal arrangement of the glomerulus in the kidney of L. rohita.

The primary organ in the body involved in detoxification and biotransformation of toxicants is liver. Therefore, it is one of the most affected organs due to aquatic toxicity [50]. A normal liver has regular shaped hepatocytes, cytoplasmic vacuolation, and a nucleus in a lateral position. The cytoplasm of hepatocytes contains lipids and glycogen inside vacuoles, which are used in normal metabolic functions. During stressful conditions, glycogen inside hepatocytes is depleted because glycogen acts as a reserve for glucose. Due to these large spaces (sinusoidal spaces), hepatocytes are formed [51]. The increase in vacuolization is an indication of metabolic damage due to the toxicity caused by ethyl paraben. The histology of the liver showed pyknotic nuclei, eosinophilia granules, Sinusoidal spaces, necrosis, and Melano-macrophages.

The results of ethylparaben exposure show that ethylparaben causes biochemical alteration and fluctuations in enzymatic activity. Oxidative stress is used to find out the damage caused by ROS (reactive oxygen species) and TBARS (Thiobarbituric acid reactive substances) such as hydroxyl radicals (OH⁻), hydrogen peroxides (H₂O₂), and superoxide (O₂⁻). A large amount of chemicals has toxic effects, such as inducing oxidative stress. The body’s antioxidant defence systems include SOD, catalase, POD, and GSH. Xenobiotics such as ethylparaben suppress antioxidant activity, due to which large number of free radicles accumulate and damage cellular components like DNA, and carbohydrates [52]. Soft tissues of the body, including the liver, kidney and gills are more susceptible to free radicals’ attacks because they are involved in metabolism, excretion etc.

The results of oxidative stress biomarkers such as TBARS and ROS showed that ethyl paraben exposure induces oxidative stress. In tissue damage, the primary biomarker for lipid peroxidation is TBARS assay. TBARS assay is the measurement of MDA (malondialdehyde) [53]. Due to being lipophilic in nature, ethylparaben can easily cross the plasma membrane and increase MDA level and consequently increase ROS. A high level of ROS suppresses the antioxidant system, which works as an antagonistic to oxidative enzymes. Elevated ROS and decreased antioxidant systems change redox potential of plasma membrane and cause oxidative stress in soft tissues such as hepatocytes, renal tissues, and gill filaments. After exposure to ethylparaben, elevated levels of Lipid peroxidase (POD) were recorded in the tissues. Our results are supported by the findings of Shah and Verma [54] and Silva, Serrano [55].

A significantly decreased level of GSH (glutathione) was recorded in fish L.rohita. GSH is an antioxidant and free radical scavenger. Shah and Verma [54] also reported a similar decreased level of GSH after exposure to parabens. Another study by Silva, Serrano [55] shows that parabens exposure significantly increases GSH levels. This suppression of GSH level indicates the high concentration of free radicles. The number of free radicals is greater than the scavenging ability of GSH. The first line of defence against ROS-induced damage is enzymatic anti-oxidants, including catalase, SOD, and GSH [56, 57]. Our results indicate significant decrease in antioxidants such as catalase, SOD, and GSH. The decrease is possibly due to the protein oxidation caused by ethylparaben. Increased Peroxidase levels interact with enzymes and reduce enzyme activity, resulting in modifications in histidine residues and protein crosslink [58]. Similar findings were observed by Silva, Serrano [55] and Shah and Verma [54]. The results of oxidative stress and antioxidant enzymes indicate a reduction in the scavenging activity of antioxidant system on free radicals. Hence, ethylparaben causes oxidative stress in L.rohita.

DNA strand breakage, such as Olive tail moment (OTM) and percentage tail DNA to blood cells, was confirmed by comet assay in L. rohita. The results of the comet assay confirmed that ethyl paraben causes genotoxicity. Genotoxicity caused by parabens in human lymphocytes was previously evaluated by Güzel Bayülken, Ayaz Tüylü [59] which supports our results. Similarly genotoxicity caused by parabens was also evaluated by Martín, Peropadre [60] in the liver cells of monkeys.

Conclusion

In fish, ethylparaben exposure over a longer period of time alters their hemato-biochemistry. As their concentration rises, so does their toxicity. Indicators of ethyl paraben’s harmful effects, primarily on kidney, gills, and liver tissues, were also shown to have undergone histological modification. Ethylparaben causes oxidative stress, and, antioxidant enzyme activity is inhibited when exposed to ethylparaben. A comet assay used on blood cells exposed to ethylparaben also revealed genotoxicity. According to the study’s findings, prolonged exposure to ethyl paraben harms aquatic life and changes its typical anatomical and physiological features, as well as oxidant and antioxidant enzyme activity and DNA damage.

Supporting information

S1 Data. Genotoxicity.

(XLSX)

pone.0302691.s001.xlsx^{(8.8KB, xlsx)}

S2 Data. Hematology and biochemical.

(XLSX)

pone.0302691.s002.xlsx^{(10.5KB, xlsx)}

S3 Data. Oxidative stress.

(XLSX)

pone.0302691.s003.xlsx^{(10.7KB, xlsx)}

Acknowledgments

The current study is a part of PhD research. The manuscript has not been published elsewhere.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0302691.r001

Decision Letter 0

Amel Mohamed El Asely

5 Nov 2023

PONE-D-23-29563A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

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Reviewer #1: The manuscript entitled (A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and Genotoxicity) needs major changes before being published

General comments:

The manuscript needs language editing

The introduction section should be improved

The methodology needs more details

The aim of work not clear

Specific comments

Abstract section

-line 18, insert a full stop after (Products)

-line 19 to 21 rephrase

-the abstract should be informative, the scientific name of fish should be added, number of fish used and their average weight, number of fish groups, number of replicates, and the experimental duration.

The methodology should mention briefly in the abstract.

-Add a space between T0(0.00 µg/L).

-the full name of all abbreviations written in the abstract should be written

- (Due to higher oxidative enzymes activity antioxidant enzymes potential to overcome their stress activity reduced) this sentence should be deleted, no explanations were written in the abstract

-which level of ethyl paraben that produced the results written in the abstract

Keywords: remove the word, aquatic toxicology

Introduction section

-the introduction section lack the information about the toxic impact of paraben on fish tissue, health

Also the residues of such paraben in water bodies, how paraben gain access to the water bodies. The uses of paraben in agriculture practices

-the significance of the studied fish (rohu, Labeo rohita) should be added in the introduction section

Why the authors used this fish as toxicological model

-the aim of work not clear because the problem not clarified well in the introduction section

-add the following references in the introduction section, they will be useful

1- Ahmed, S.A., Ibrahim, R.E., Elshopakey, G.E., Khamis, T., Abdel-Ghany, H.M., Abdelwarith, A.A., Younis, E.M., Davies, S.J., Elabd, H., Elhady, M.J.F., Immunology, S., 2022. Immune-antioxidant trait, growth, splenic cytokines expression, apoptosis, and histopathological alterations of Oreochromis niloticus exposed to sub-lethal copper toxicity and fed thyme and/or basil essential oils enriched diets. 131, 1006-1018.

2- Ibrahim, R.E., El-Houseiny, W., Behairy, A., Mansour, M.F., Abd-Elhakim, Y.M., 2019. Ameliorative effects of Moringa oleifera seeds and leaves on chlorpyrifos-induced growth retardation, immune suppression, oxidative stress, and DNA damage in Oreochromis niloticus. Aquaculture 505, 225-234.

3- Mohamed, W.A., El-Houseiny, W., Ibrahim, R.E., Abd-Elhakim, Y.M., 2019. Palliative effects of zinc sulfate against the immunosuppressive, hepato-and nephrotoxic impacts of nonylphenol in Nile tilapia (Oreochromis niloticus). Aquaculture 504, 227-238.

4- Ibrahim, R.E., Elbealy, M.A., Salem, G.A., Abdelwarith, A.A., Younis, E.M., Wagih, E., Elkady, A.A., Davies, S.J. and Rahman, A.N.A., 2023. Acute mancozeb-fungicide exposure induces neuro-ethology disruption, health disorders, and immune-oxidative dysfunction in Nile tilapia (Oreochromis niloticus). Aquatic Toxicology, 261, p.106630.

5- Ibrahim, Rowida E., Heba I. Ghamry, Saed Ayidh Althobaiti, Daklallah A. Almalki, Medhat S. Shakweer, Mona A. Hassan, Tarek Khamis, Heba M. Abdel-Ghany, and Shaimaa AA Ahmed. "Moringa oleifera and Azadirachta indica Leaves enriched diets mitigate chronic oxyfluorfen toxicity induced immunosuppression through disruption of pro/anti-inflammatory gene pathways, alteration of antioxidant gene expression, and histopathological Alteration in Oreochromis niloticus." Fishes 8, no. 1 (2022): 15.

Material and method section

1-line 91, replace made with assigned

2-number of fish and weight should be mentioned

3-what is the LC50 value for ethyl parapen, why authors evaluated it for 48 hours not for 96 hours

4- What is the relation between the lC50 and the different doses of parapen used in the experiment?

5-line 109, add the dose of clove oil and reference

6-the number of samples should be written for each group in all methodology (blood samples, histological samples, ….etc)

7-the procedures of histological examinations is lack many details, so superficial

The details should be added

8- All abbreviations should be defined for the first time

9- For the statistical analysis, the normality test should be performed before the ANOVA

Results

1- All tables titles should be rephrased and rewritten

2- Table footnotes should be added and the full names of all measured parameters should be inserted in the footnote

3- Line 196, the author written (propyl paraben), the authors used ethyl paraben or propyl paraben

4- All data in the figures and tables should be carefully revised by the authors

Discussion section

This part should be revised and improved

Line 320-321 why written in italic form

Many language errors are present

Conclusion section: this section not present

Reviewer #2: 1. The abstract has not been written in a clear and concise manner. For example the paragraph starting with ethyl paraben induced oxidative .....; is not stating the findings clearly.

2. There is reference to genotoxicity in the abstract. However, it is not clear which marker for genotoxicity was used to arrive at this conclusion. If this was based on the COMET Assay, then indicate that "based on the comet assay, ....

3. Under methods: it is not clear how the dosages of 2000, 4000, and 6000 were chosen. Is this based on actual estimated concentrations of paraben in contaminated waterways? How much paraben is fish exposed to in water? Do they bio-accumulate in fish tissues? What steps were taken to ensure that prior exposure to paraben and related chemicals did not occur in regard to fish used for the study? This needs to come out clearly.

4. Quite significantly, the manuscript has recurring grammar issues, and poor prose/sentence construction. For example, the sentence that reads..." all experiments were formed.... The word "formed" is quite out of place. This is repeated throughout (line 75). Experiments are "conducted or performed" etc... Do not use don't..., rather use do not etc...

5. Did the study investigate levels of glutathione or glutathione reductase? In the results, the sub-title is GSH, which is used to abbreviate glutathione and not glutathione reductase. In the discussion and figures, references have been made in regard to antioxidants including glutathione (GSH). Where does glutathione reductase come in line 140-144?

6. Hematology data has been presented in a table format. This is not correct. Dose response studies are best presented using line graphs to easily decipher the trend. This observation applies to antioxidant analysis (GSH, TBARS, ROS, SOD, CAT etc..)

7. The data on antioxidant assay is quite confusing for liver/kidney. ROS is going up, consistent with TBARS; but antioxidant enzymes that are meant to be up-regulated in response to exposure to chemical toxicants such as paraben are actually being down-regulated. This is a paradox. Note that there is a dose-dependent rise in ROS. Antioxidants are expected to rise in concert to the rise in ROS. For this study, antioxidants are going down as the dose of paraben goes up. However, many studies have demonstrated depletion of GSH levels in response to overwhelming chemical-induced oxidative stress. This may explain the decrease in GSH. Not sure about the SOD, CAT etc..,. This scenario may be possible when there is overwhelming oxidative stress, depleting all the antioxidants. Clarify this phenomenon. Notably, liver function markers are all up-regulated, signaling severe paraben-driven liver injury.

8. Data for hematology shows evidence for paraben-driven significant up-regulation of platelets. This elevation will have serious implications for blood-related disorders like DVT and patients who may be on blood thinning medications. This aspect needs to be discussed more broadly in the discussion section to highlight implications in various circumstances. Infact, RBCs and HGB will be expected to be down-regulated due to a possible paraben-driven impediment to hematopoiesis.

9. It is not clear why paraben will result in elevation of HGB, MCV, MCHC and platelets.

10. Notably, at low dosages, the effect of paraben on blood indices was low, validating the premise of this study. This, probably needs to come out well in the discussion.

11. It is very important that the grammar issues are corrected, and the manuscript is written carefully in a concise and logical manner using appropriate english.

12. There is need for a more concise title tied to the major findings of the study.

**********

6. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

Reviewer #2: Yes: Alfred Orina Isaac

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: reviewer comments to PONE-D-23-29563.docx

pone.0302691.s004.docx^{(15.4KB, docx)}

PLoS One. 2024 May 6;19(5):e0302691. doi: 10.1371/journal.pone.0302691.r002

Author response to Decision Letter 0

20 Nov 2023

01 Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Responce: Revised and corrected

02 Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution, ....

Responce: The authors received no specific funding for this work.”

03 Please remove any funding-related text from the manuscript.

Responce: Revised, edited and corrected

04 In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety.

Respone: All data set are present in submitted manuscript. Raw data from this study are available upon request.

05 We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly.

Responce: No legal or ethical restrictions on sharing data publicaly.

06 Please ensure that you refer to Figure 3 in your text as, if accepted, production will need this reference to link the reader to the figure.

Responce: corrected

07 Please include a caption for figure 7.

Responce: corrected

08 Please upload a copy of Figure 7, to which you refer in your text on page 19 in PDF submission. If the figure is no longer to be included as part of the submission please remove all reference to it within the text.

Responce: Revised, edited and corrected

09 We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 in your text; if accepted, production will need this reference to link the reader to the Table.

Responce: Revised, edited and corrected

Respond to comments of Reviewer #1

01 The manuscript needs language editing.

The introduction section should be improved.

The methodology needs more details.

The aim of work not clear

Responce: Revised and corrected

02 Abstract section

-line 18, insert a full stop after (Products)

-line 19 to 21 rephrase

Responce: Revised and corrected

03 The methodology should mention briefly in the abstract.

-Add a space between T0(0.00 µg/L).

Responce: Revised and corrected

04 the full name of all abbreviations written in the abstract should be written

- (Due to higher oxidative enzymes activity antioxidant enzymes potential to overcome their stress activity reduced) this sentence should be deleted, no explanations were written in the abstract

Responce: Revised and corrected

05 which level of ethyl paraben that produced the results written in the abstract

06 Keywords: remove the word, aquatic toxicology

Responce: Revised and corrected

07 Introduction section

-the introduction section lack the information about the toxic impact of paraben on fish tissue, health

Also the residues of such paraben in water bodies, how paraben gain access to the water bodies. The uses of paraben in agriculture practices

-the significance of the studied fish (rohu, Labeo rohita) should be added in the introduction section

Why the authors used this fish as toxicological model

-the aim of work not clear because the problem not clarified well in the introduction section

Responce: Revised and corrected

08 Material and method section

1-line 91, replace made with assigned

2-number of fish and weight should be mentioned

3-what is the LC50 value for ethyl parapen, why authors evaluated it for 48 hours not for 96 hours

4- What is the relation between the lC50 and the different doses of parapen used in the experiment?

5-line 109, add the dose of clove oil and reference

6-the number of samples should be written for each group in all methodology (blood samples, histological samples, ….etc)

7-the procedures of histological examinations is lack many details, so superficial

The details should be added

8- All abbreviations should be defined for the first time

9- For the statistical analysis, the normality test should be performed before the ANOVA

Responce: Revised and corrected

09 Results

1- All tables titles should be rephrased and rewritten

2- Table footnotes should be added and the full names of all measured parameters should be inserted in the footnote

3- Line 196, the author written (propyl paraben), the authors used ethyl paraben or propyl paraben

4- All data in the figures and tables should be carefully revised by the authors

Discussion section

This part should be revised and improved

Line 320-321 why written in italic form

Many language errors are present

Responce: Revised and corrected

10 Conclusion section: this section not present

Responce: Revised and corrected

Respond to comments of Reviewer #2

01 The abstract has not been written in a clear and concise manner. For example the paragraph starting with ethyl paraben induced oxidative .....; is not stating the findings clearly.

Responce: Revised and corrected

02 There is reference to genotoxicity in the abstract. However, it is not clear which marker for genotoxicity was used to arrive at this conclusion. If this was based on the COMET Assay, then indicate that "based on the comet assay,

Responce:Revised and corrected

03 Under methods: it is not clear how the dosages of 2000, 4000, and 6000 were chosen. Is this based on actual estimated concentrations of paraben in contaminated waterways? How much paraben is fish exposed to in water? Do they bio-accumulate in fish tissues? What steps were taken to ensure that prior exposure to paraben and related chemicals did not occur in regard to fish used for the study? This needs to come out clearly.

Responce: As per previous work, LC50 was determined. So, according to this, LC50 doses were divided into ½, 1/3, and ¼. These doses were actually based on laboratory work, and their estimation in natural or contaminated water is under process. We did not work on the estimation of paraben in fish. Fish were taken from fresh water fish form, where treated water was used.

04 Quite significantly, the manuscript has recurring grammar issues, and poor prose/sentence construction. For example, the sentence that reads..." all experiments were formed.... The word "formed" is quite out of place. This is repeated throughout (line 75). Experiments are "conducted or performed" etc... Do not use don't..., rather use do not etc...

Responce:Revised and corrected

05 Did the study investigate levels of glutathione or glutathione reductase? In the results, the sub-title is GSH, which is used to abbreviate glutathione and not glutathione reductase. In the discussion and figures, references have been made in regard to antioxidants including glutathione (GSH). Where does glutathione reductase come in line 140-144?

06 Hematology data has been presented in a table format. This is not correct. Dose response studies are best presented using line graphs to easily decipher the trend. This observation applies to antioxidant analysis (GSH, TBARS, ROS, SOD, CAT etc..)

Responce: Revised and corrected

07 The data on antioxidant assay is quite confusing for liver/kidney. ROS is going up, consistent with TBARS; but antioxidant enzymes that are meant to be up-regulated in response to exposure to chemical toxicants such as paraben are actually being down-regulated. This is a paradox. Note that there is a dose-dependent rise in ROS. Antioxidants are expected to rise in concert to the rise in ROS. For this study, antioxidants are going down as the dose of paraben goes up. However, many studies have demonstrated depletion of GSH levels in response to overwhelming chemical-induced oxidative stress. This may explain the decrease in GSH. Not sure about the SOD, CAT etc..,. This scenario may be possible when there is overwhelming oxidative stress, depleting all the antioxidants. Clarify this phenomenon. Notably, liver function markers are all up-regulated, signaling severe paraben-driven liver injury.

Responce: Explained in line 345-379

08 Data for hematology shows evidence for paraben-driven significant up-regulation of platelets. This elevation will have serious implications for blood-related disorders like DVT and patients who may be on blood thinning medications. This aspect needs to be discussed more broadly in the discussion section to highlight implications in various circumstances. Infact, RBCs and HGB will be expected to be down-regulated due to a possible paraben-driven impediment to hematopoiesis.

Responce: Explained in line 285-290

09 It is not clear why paraben will result in elevation of HGB, MCV, MCHC and platelets.

Responce: Revised and corrected

10 Notably, at low dosages, the effect of paraben on blood indices was low, validating the premise of this study. This, probably needs to come out well in the discussion.

Responce: Revised and corrected

11 It is very important that the grammar issues are corrected, and the manuscript is written carefully in a concise and logical manner using appropriate english

Responce: Revised and corrected

12 There is need for a more concise title tied to the major findings of the study.

Responce: Revised and corrected

Attachment

Submitted filename: Response to Reviewers.docx

pone.0302691.s005.docx^{(21.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0302691.r003

Decision Letter 1

Amel Mohamed El Asely

8 Dec 2023

PONE-D-23-29563R1A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

Please submit your revised manuscript by Jan 22 2024 11:59PM. 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.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: the authors made all comments and the revised manuscript now accepted for publication

no further correction were needed

Reviewer #2: I gave detailed review proposals in my first analysis of this work. The authors have made reasonable effort to correct the figure for dose response studies as i guided. However, the grammar issues have not been addressed across the board starting with the abstract, through to the discussion section. A lot of work needs to be done here.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

Reviewer #2: No

**********

PLoS One. 2024 May 6;19(5):e0302691. doi: 10.1371/journal.pone.0302691.r004

Author response to Decision Letter 1

23 Jan 2024

errors have been corrected

Attachment

Submitted filename: Response to Reviewers.docx

pone.0302691.s006.docx^{(14KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0302691.r005

Decision Letter 2

Amel Mohamed El Asely

19 Feb 2024

PONE-D-23-29563R2A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

Please submit your revised manuscript by Apr 04 2024 11:59PM. 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.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2024 May 6;19(5):e0302691. doi: 10.1371/journal.pone.0302691.r006

Author response to Decision Letter 2

23 Feb 2024

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results.

answer: Not applicable

Answer: We have reviewed all references. No cited paper has been retracted.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,

Answer: Corrected and submitted in supporting information

Attachment

Submitted filename: Responce to reviewer.docx

pone.0302691.s007.docx^{(12.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0302691.r007

Decision Letter 3

Amel Mohamed El Asely

2 Apr 2024

PONE-D-23-29563R3A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. I have observed that your response to the reviewers' comments was not addressed carefully, and the quality of English in your manuscript still needs improvement. Therefore, we recommend that you adhere to the instructions by providing detailed responses to the reviewers in a separate file and enhancing English in your manuscript. The attached file contains the comments from the reviewers. Please carefully consider every point and respond to them individually, addressing each one separately.

Please submit your revised manuscript by May 17 2024 11:59PM. 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.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2024 May 6;19(5):e0302691. doi: 10.1371/journal.pone.0302691.r008

Author response to Decision Letter 3

4 Apr 2024

Respond to comment Response

01 I have observed that your response to the reviewers' comments was not addressed carefully, and the quality of English in your manuscript still needs improvement.

Ans: Revised and improved English.

02 Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.

Ans: We cross checked all references and we haven’t found any retracted reference. Please let us know any retracted reference.

03 While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.

Ans: Figures have been uploaded and resized according to plos requirements and uploaded as figure and raw data in supporting information title “figure. Rar”.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0302691.s008.docx^{(14.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0302691.r009

Decision Letter 4

Amel Mohamed El Asely

10 Apr 2024

A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and Genotoxicity

PONE-D-23-29563R4

Dear Dr. Shahzad,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Associated Data

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

Supplementary Materials

S1 Data. Genotoxicity.

(XLSX)

pone.0302691.s001.xlsx^{(8.8KB, xlsx)}

S2 Data. Hematology and biochemical.

(XLSX)

pone.0302691.s002.xlsx^{(10.5KB, xlsx)}

S3 Data. Oxidative stress.

(XLSX)

pone.0302691.s003.xlsx^{(10.7KB, xlsx)}

Attachment

Submitted filename: reviewer comments to PONE-D-23-29563.docx

pone.0302691.s004.docx^{(15.4KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0302691.s005.docx^{(21.3KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0302691.s006.docx^{(14KB, docx)}

Attachment

Submitted filename: Responce to reviewer.docx

pone.0302691.s007.docx^{(12.2KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0302691.s008.docx^{(14.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; hemato-biochemical assays, histology, oxidant and antioxidant activity and genotoxicity

Hasnain Akmal

Shabbir Ahmad

Muddasir Hassan Abbasi

Farhat Jabeen

Khurram Shahzad

Roles

Abstract

Introduction

Materials and methods

Laboratory setting and animal handling

Chemical

Experimental design and treatment

Haematological and biochemical analysis

Histology

Biochemical assay

ROS assay

TBARS

Catalase

Superoxide dismutase

Glutathione

Peroxidase

Comet assay

Statistical analysis

Results

Hematology

Fig 1. Showing variations in blood parameters of L.rohita treated with different concentrations of ethylparaben.

Biochemical parameters

Fig 2.

Fig 3.

Oxidative stress and antioxidant response

Table 1. Oxidative stress in the liver, gills and kidney of L.rohita exposed to different concentrations of ethylparaben.

Fig 4. Showing decrease in level of antioxidant enzymes in different tissues (liver, gills and kidney) of Labeo rohita after exposure to higher concentrations of ethyl paraben.

Histology

Fig 5. Shows the gills histopathology of L. rohita after exposure to ethyl paraben for 21 days (40X).

Fig 7. Shows the liver histopathology of L. rohita after exposure to ethyl paraben for 21 days (40X).

Fig 6. Shows the kidney histopathology of L. rohita after exposure to ethyl paraben for 21 days (40X).

Genotoxicity

Fig 8.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Amel Mohamed El Asely

Roles

Author response to Decision Letter 0

Decision Letter 1

Amel Mohamed El Asely

Roles

Author response to Decision Letter 1

Decision Letter 2

Amel Mohamed El Asely

Roles

Author response to Decision Letter 2

Decision Letter 3

Amel Mohamed El Asely

Roles

Author response to Decision Letter 3

Decision Letter 4

Amel Mohamed El Asely

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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