Effects of chlorpyrifos on the crustacean Litopenaeus vannamei.

Shrimps can be used as indicators of the quality of aquatic systems exposed to a variety of pollutants. Chlorpyrifos is one of the most common pesticides found in environmental samples. In order to evaluate the effects of chlorpyrifos, adult organisms of Litopenaeus vannamei were exposed to two sublethal concentrations of the pesticide (0.7 and 1.3 μg/L) for four days. The LC50 (96-hours) value was determined and Lipid oxidation levels (LPO) and the activities of catalase (CAT), glutathion peroxidase (GPx), glutathion-S-transferase (GST) were assessed on the muscle, hepatopancreas and gills from the exposed organisms. In addition, inhibition of acetylcholinesterase (AChE) was determined in the brain. LC50 (96-hours) was 2.10 μg/L of chlorpyrifos. Catalase activity and LPO were elevated in the three tissues, whereas a decrease of AChE activities in the brain and an increase of GST activity in the hepatopancreas were observed.

Introduction

Marine organisms can be used as indicators of the quality of aquatic systems exposed to a variety of environmental pollutants. Invertebrates are exposed to xenobiotics resulting from agricultural fields via the surface runoff of water or indirectly through the trophic chain of the ecosystem [1].

Pesticides play an important role in sustaining agricultural production by protecting crops from pests and vector-borne diseases [2-6]. Organophosphosphorus (OP) compounds are toxic, and their toxicity mechanism is related to the inhibition of acetylcholinesterase (AChE), resulting in accumulation of acetylcholine in the cholinergic receptors of peripheral and central nervous systems [7].

Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl) phosphorothionate] (CAS Number 2921-88-2) (), is an OP insecticide widely used in Colombia for agricultural and domestic pest control, but this also results in non-targeted organisms being exposed to either lethal or sublethal concentrations of this contaminant [8-12].

Crustaceans exposed to OP insecticides have shown an inhibition of AChE, as well as oxidative stress, oxidative metabolism alterations, osmoregulation and immunological responses [11]. Even though AChE inhibition is a widely employed biomarker of OP contamination, other oxidative stress-related parameters have been used as indicators of exposure to xenobiotics in aquatic invertebrates as well [12-13].

Once chlorpyrifos reaches target organisms, an oxidative desulfuration of the P = S moiety to P = O occurs, a reaction which is catalyzed by cytochrome P-450 (CYP 1A1) (Phase I), resulting in the toxic intermediate chlorpyrifos-oxon, which can inhibit AChE activity. In additon, chlorpyrifos can undergo dearylation while catalyzed by CYP1A1, resulting in 3,5,6-trichloro-2-pyridinol (TCP), or conjugation by glutathion-S-transferases (GST), sulfotransferases and glucuronil-transferases to form the corresponding glutathion, glucuronide and sulfate conjugates (detoxification) [9,10]. The pathways of chlorpyrifos in shrimp are shown in .

Chlorpyrifos biotransformation pathway in invertebrates.

Chlorpyrifos-Oxon; TCP, 3,5,6-trichloro-2-pyridinol; AchE, acetylcholinesterase; CYP 1A1, cytochrome P-450; NADPH, nicotinamide adenine dinucleotide phosphate.

The antioxidant defense system includes enzymes such as catalase (CAT), glutathion peroxidase (GPx) and glutathione S-transferase (GST) [14]. In addition, increased lipid peroxidation (LPO) is one of the major contributors to the loss of cell function in oxidative stress situations. CAT is an essential enzyme to promote the degradation of H2O2, a precursor of the hydroxyl radical that induces DNA damage, protein degradation and lipid peroxidation [14]; GSTs are major phase II-related enzymes, which conjugate electrophilic compounds with reduced glutathion (GSH). For these reasons, OPs are less toxic and more soluble in water, and, therefore, can be quickly excreted from cells after further metabolism [15]. LPO determination has also been successfully employed in aquatic animals to indicate oxidative stress induced by organic contaminants, including OP pesticides [16]. Moyano et al., (2017) reported that chlorpyrifos induces, after acute and long-term exposure, apoptosis and necrosis, partially mediated through AChE overexpression, by using septal SN56 basal forebrain cholinergic neurons [17].

Invertebrates do not have a spinal cord or a spine; instead, most of them have an exoskeleton that spans the entire body. They do not have lungs since they breathe through their skin [18]. The liver participates mainly in the metabolism and regulation of nutrients in aquatic animals and plays an important role in oxidative stress and lipid peroxidation [19]. Crustaceans do not have a liver; they have a hepatopancreas, with functions similar to those of the liver in fish. Therefore, the investigation of possible biochemical changes in response to xenobiotics in crustaceans are done through the hepatopancreas [20]. In the Fig 3 the anatomy of shrimp (L. vannmaei) can be seen. However, an understanding of the influential mechanism underlying the pesticides in the hepatopancreas is still limited and needs further investigation, since most of the research efforts are aimed at biological models of vertebrate organisms [3,4,10], and very few at that of invertebrates, such as L. vannamei, which, precisely because of its economic importance and participation in the food supply of humans, must be ensured in terms of food safety.

Fig 3

Anatomy of Litopenaeus vannamei.

(Fully owned source; the final illustration was made by using the GIMP Software v2.8.14 (GNU Program for Image Manipulation available at https://www.gimp.org/downloads/).

It has been observed that shrimps are more sensitive to pesticides than fish and mollusks, and have been proposed as indicators of estuarine health due to their worldwide distribution [21]. Thus, L vannamei can be used as a test organism for in situ assessment of water contamination [8]. Moreover, crustaceans represent an advantageous tool to monitor environmental contamination as their communities are numerous and can be easily grown under laboratory conditions [18]. Therefore, the aim of this study was: (a) to determine LC50 of chlorpyrifos on the Litopenaeus vannamei shrimp and (b) assess the effects of sublethal concentrations of chlorpyrifos in this species by using a variety of potential biomarkers including, CAT, GST, GPx, AChE activities, and LPO levels.

Materials and methods

Animal collection and maintenance

Shrimps (Litopenaeus vannamei) of both genders with an average weight of 48 ± 3 g were collected at CENIACUA (Aquaculture Research Center) located about 20 kilometers (12.4 miles) from Cartagena (Colombia). The animals were transported to the Agrochemical laboratory at University of Cartagena (Colombia) and acclimatized for seven days in 32-liter plastic (polypropylene) tanks containing aerated seawater (30% salinity, pH 8.0, dissolved oxygen between 7.8–9.5 mg/L and 12h/12h dark/light cycles) at 26± 0.6 oC (). The shrimps were fed only during acclimation with pellets, formulated shrimp diet (Solla, Colombia), and the water was renewed every two days.

For this study, the gender of L. vannamei was not considerated. Shrimps were not currently undergoing moults. Experimental procedures were performed in accordance with the principles of the National Animal Protection Status (Colombian law 84 of 1989) established by the University of Cartagena’s Central Committee of Ethics in Scientific Research (Resolution 01597 of 2014).

Toxicity tests

A preliminary analysis was carried out to find the range of chlorpyrifos concentrations. Finally, five concentrations (13.0, 5.4, 2.1, 1.4 and 0.7 μg/L) were used in each toxicity test with three replicates per concentration and one control. The test solutions and control water were renewed daily.

Experiments were carried out in plastic polypropylene tanks (35 L capacity), filled with 30 ppt aerated saline water, pH: 8.0, dissolved oxygen between 7.8–9.5 mg/L and 12h/12h dark/light cycles and 26± 0.6 oC. Eight adult shrimps were used in each replicate. A chlorpyrifos stock solution was prepared by diluting 1 mL Lorsban® 4E in 1 L of distilled water. The specimens were not fed the day before or during toxicity tests. Dead shrimps were counted after 24, 48, 72 and 96 h. The LC50 after 96 h was calculated using the probit method by following OECD Guideline 203 [22].

Sampling and chromatographic analysis

500 mL of each aqueous sample was transferred to separatory funnels. Samples were extracted with 100 mL of dichloromethane (DCM) (three times) and dried using sodium sulphate. The extracts were combined in a 500 mL round bottom flask. Then, 5 mL of isooctane were added in a rotary evaporator. The concentrates were quantitatively transferred to 15 mL centrifuge tubes with two 2 mL of hexane and concentrated by nitrogen evaporation up to 1 mL. Finally, 1 μL was injected into the gas chromatograph coupled to a mass detector (GC-MS) [23].

Chromatography separation was performed using an HP-5 MS capillary column (30 m × 0.25 mm × 0.5μm) (J & W Scientific, USA), and a 7890A gas GC-MS chromatograph system (Agilent Co., Palo Alto, CA, USA) equipped with an Agilent Technologies 5975N GC-MS series mass selective detector (MSD). The injection temperature (Agilent 4513A) was 250°C. The carrier gas was Helium (99.99%). The flow rate was 1 mL/min. The column temperature was held at 120°C for 1 min and then raised to 300°C at a rate of 15°C/min. Transfer interface and ion source temperatures were 230 and 300°C, respectively. Ions were generated by a 70-eV electron at full scan mode (m/z 40–600).

Sublethal toxicity assay

The sublethal toxicity of chlorpyrifos was evaluated using biomarkers of oxidative stress (CAT, GPX, and GST activities), lipid peroxidation and AChE inhibition in four tissues (brain, hepatopancreas, gills, and muscle) of L. vannamei. Two sublethal concentrations of chlorpyrifos were used (0.7 and 1.4 μg L−1) for 96 hours [24]. No mortality was observed under the experimental conditions. Three replicates were done using five shrimps each.

Tissue preparation for antioxidant enzymes assays

After 96 h exposure, shrimps of each experimental tank were collected, and brain, hepatopancreas, gills, and muscles were dissected, cleaned and immediately washed with ice-cold saline. Samples were weighed and homogenized in ice-cold Potassium Phosphate Buffer (50mM, pH 7.5, EDTA 60mM) [25] containing protease inhibitor (Sigma P2714) by using a glass homogenizer. Then, the homogenate was centrifuged at 3600 rpm for 15 min at 4°C [KUBOTA (3700), Japan] and the supernatant corresponding to the post-mitochondrial fraction was used to evaluate enzymatic (CAT, GR, GST) and lipid peroxidation levels (LPO).

AChE activity

Brains were homogenized at 1:10 w/v with ice-cold Potassium Phosphate Buffer (0.1 M, pH 7.5, EDTA 1 mM, and 0.5% Triton + protease inhibitor). Homogenates were centrifuged at 3600 rpm for 15 min at 4°C, and AChE activity was measured by using a colorimetric method [26,27]. The reaction was carried out by adding 10 μL of 75 mM substrate of ATCH (acetylthiocholine iodide) into a 990 μL reaction mixture containing 50 μL of 0.01 M dithiobisnitrobenzoate (DTNB) in 0.1 M phosphate buffer (pH 8.0). The optical density increase rate of the reaction medium was measured using a spectrophotometer at 412 nm for 240 s at room temperature. Specific activity is expressed as nmoles of product formed per min−1 mg−1 protein. One AChE unit was the amount of enzyme that hydrolyzed 1 nmol of acetylcholine/min/mg of protein.

Protein determination

Protein determination was carried out according to the Bradford method [28] with Coomassie Brilliant Blue G-250 by using bovine serum albumin as a standard. The absorbance of samples was measured at 595 nm.

Catalase (CAT) activity

Catalase activity was determined by following the spectrophotometric decomposition of H2O2 at 240 nm, in a reaction mixture containing a 50 mM potassium phosphate buffer (pH 7) and 10 mM H2O2 [29]. Results were expressed as pmol CAT per mg of protein. One CAT unit was the amount of enzyme required to catalyze 1 pmol of H2O2/min.

Glutathion-S-transferase (GST) activity

Glutathion-S-transferase (GST) activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, according to the methodology described by Sharbidre [30]. The final reaction mixture contained 1 mM CDNB, and 1 mM reduced glutathion (GSH). The results were expressed as units of GST per mg of protein. One GST unit represented the amount of enzyme required to conjugate GSH with 1 μmol of 1-chloro-2,4-dinitrobenzene/min.

Glutathion peroxidase (GPX) activity

The glutathion peroxidase (GPX) activity was measured using the method described by Li et al. (2016) [31]. The reaction mixture consisted of a 50 mM phosphate buffer (pH 7), 1 mM EDTA, 1 mM sodium azide, NaN3, 2 U mL−1 glutathion reductase (GR), 2 mM reduced glutathion, GSH, 0.2 mM NADPH. After 10 min incubation at 37°C, the overall reaction was initiated by adding 0.5 mM hydrogen peroxide, H2O2. Oxidation of NADPH was recorded spectrophotometrically at 340 nm for 5 min.

Lipid oxidation

The production of malondialdehyde (MDA) was assessed by the thiobarbituric acid reactive substances assay TBARS [14, 32]. MDA reacts with thiobarbituric acid (TBA), and the product is read spectrophotometrically at 535 nm. Homogenate was added 1:1 (v:v) to 5% trichloroacetic acid (TCA), and incubated on ice for 15 min. The solution was then mixed at a 2:1 ratio with 0.67% TBA, and centrifuged at 2200 × g at 4°C for 10 min. The whole supernatant was boiled for 10 min and refreshed at room temperature before the absorbance was recorded. A calibration curve with increasing MDA concentrations allowed the calculation of LPO expressed as nmol MDA equiv. g−1 tissue.

Statistical analysis

Data are presented as mean ± Standard deviations (SD). All values were examined for normality (Shapiro-Wilk test) as well as the homogeneity of variances (Levene’s test). 96-h LC50 (Median lethal concentrations) with 95 percent confidence limits were calculated by using the SPSS software package, version 25 (SPSS Inc. Chicago, IL, USA). One-Way Analysis of variances (ANOVA) was used to identify differences between control and exposed groups, followed by Tukey’s post hoc test (). The level of significance was set at p<0.05.

Results and discussion

Chlorpyrifos concentrations in water samples were measured by gas chromatography (GC) coupled to a mass spectrometer detector (MS), before and after their application. The quantification limit was 0.1 μg/L and the detection limit was 0.01 μg/L. Linear calibration curves with concentrations ranging from 0.1 to 5 μg/L were employed, with determination coefficients over 0.98. Recoveries of chlorpyrifos from water were in the range of 106–119%.

shows the fragmentogram of chlorpyrifos obtained by GC-MS. The m/z 198 was obtained due to the elimination of–C4H10O2PS molecule from a parent ion molecule m/z 350, leading to the formation of structure at m/z 198 corresponding to 3,5,6-trichloro-2-pyridinol (TCP); this is a principal metabolite from chlorpyrifos [33].

Determination of LC50

Percentages of mortality of L. vannamei exposed to chlorpyrifos are shown in the (). The 96-h acute LC50 value determined for L. vannamei was 2.10 μg/L of chlorpyrifos with 95% confidence limits from 2.04 μg/L to 2.16 μg/L. This result allowed for the selection of the pesticide concentrations towards the biomarker analysis. Control mortality was zero. The results are shown in Table 1.

Table 1

Acute 96-h toxicity of chlorpyrifos in adult shrimp (Litopenaeus vannamei).

Concentration (μg/L)	Percentage of mortality ± SE	LC50 (μg/L)	95% confidence level		Regression equation	Chi square
			LCL	UCL		df
Control	0 ± 0
23	100
23	100
5.4	83.3 ± 2.3
2.1	50 ± 1.4	2.1	2.04	2.16	Y = 2.14x + 2.97	6.10
1.4	33.3 ± 1.3
0.6	16.7± 1.1

Values of mortality percentages are presented as the overall mean of three replicates ± SE (Standard error). p < 0.05

Percentage of mortality of L. vannamei exposed to chlorpyrifos.

Values represent the mean of three determinations+/-SD. (n = 8).

L. vannamei was found to be very sensitive to chlorpyrifos toxicity in comparison with other crustacean species, including Palaemonetes argentinus [34], Paratya australiensis [35] and Macrobrachium rosenbergii [36]. Therefore, it can be concluded that L. vannamei is an organism that is sensitive to the exposure to chlorpyrifos, which could serve as a bioindicator of contamination with this pesticide. Thus, the study of the effects of sublethal concentrations of chlorpyrifos on some biomarkers was carried out.

Acetylcholinesterase inhibition

After four days of sublethal exposure to 0.7 and 1.3 μg/L of chlorpyrifos (), activity of AChE in brain tissue of L. vannamei decreased by 30.8 and 46.2%, respectively (Tukey’s test, p<0.05) in a dose-dependent manner compared to the effects observed in the control group ( and ). There are investigations in invertebrates that associate the presence of OP compounds in the aquatic environment with AChE activity in different tissues [37-40].

Effect of chlorpyrifos on Acetylcholinesterase (AchE) activity in L. vannamei during sublethal exposure on the brain (0.7 and 1.3 μg L−1) for 96 hours.

Data are presented as mean±SD. (p<0.05). (n = 5).

Some studies have reported that OP pesticides cause effects through the inhibition of AChE, which leads to an accumulation of the neurotransmitter acetylcholine. In the neuromuscular junction, overstimulation of postsynaptic cholinergic receptors leads to muscle fasciculation and eventual paralysis [41,42].

Sublethal effects of chlorpyrifos on enzymatic antioxidant defense

The effect of chlorpyrifos on the enzymatic antioxidant defense was evaluated on the hepatopancreas, gills, and muscle tissues of L. vannamei. The main defense system of invertebrates is the antioxidant because these organisms have a deficiency of antibodies and acquired immunity. Because of that, the production of high levels of reactive oxygen species (ROS) that generate oxidative stress is considered to be the manifestation of several metabolic pathways resulting in an imbalance of pro-oxidant and antioxidant defense mechanisms [21]. It has been demonstrated that OP compounds can activate oxidative biotransformation processes by generating free radicals and altering antioxidant levels of free radical scavenging enzymes [43, 44].

Oxidative stress as a result of pesticide concentrations has been reported previously [44-48]. Antioxidant enzyme systems have been investigated in aquatic organisms to find biochemical biomarkers that could be used in environmental monitoring systems [48-50]. In this work, alterations in the activity of antioxidant enzymes of L. vanammei (CAT, Gpx, and GST) were found after exposure to two sublethal concentrations of chlorpyrifos, thus suggesting that these changes could be adaptive responses to ROS.

CAT activity

After four days of sublethal exposure to 0.7 and 1.3 μg/L of chlorpyrifos, activities of CAT in tissues of L. vannamei (muscle, gills, and hepatopancreas) were found to be significantly different from those of the control (Tukey's test, p<0.05). In muscle tissues, CAT activity increased (9.1%, 27.27%). In the gills and hepatopancreas, CAT also showed a dose-dependent elevation (5.12%, 15.38%; and 35%, 100%, respectively) ().

Effect of chlorpyrifos on CAT, GST, and Gpx activities in heopatopancreas, gills and muscle of L vannamei.

Data are presented as mean±SD. (p<0.05). (n = 5).

CAT is an enzyme that regulates high concentrations of H2O2 [29]. In this research, a significant effect was found in the activity of CAT in the hepatopancreas of L. vannamei exposed to chlorpyrifos. In all treatments applied in this study, the action of the CAT enzyme in the hepatopancreas was higher than in the other tissues. Rőszer (2014) showed that mollusks and crustaceans integrate immune functions to a metabolic organ, that is, the mid-intestinal gland (hepatopancreas) [51]. This is considered to be a gland with high metabolic activity, in which a large production of ROS is expected due to its role as a detoxifying organ [21,51].

Researches have found a dose-dependent increase in the activities of SOD and CAT in diferents tissues of fish exposed to dichlorvos [52], methyl-chlorpyrifos [53], methyl paration [54], fenitrothion [55], and diazinon [56]. Thus, the existence of an inducible antioxidant system may reflect an adaptation of organisms. CAT is reported as one of the key enzymes that condense ROS generated during the bioactivation of xenobiotics in the hepatic tissues [56].

GST activity

After four days of exposure to the lowest chlorpyrifos concentration (0.7 μg/L), the activity of GST in muscle, gills and hepatopancreas increased (24.1, 4.75 and 22.53%, respectively), but as the dose increased (1.3 μg/L), a gradual elevation was observed (29.44, 13.47 and 40.42%, respectively). Significant differences were observed when compared to the control (Tukey’s test, p<0.05). GST activity in Hepatopancreas was higher than in other tissues ().

GST is responsible for catalyzing the conjugation of GSH with a wide range of electrophilic substances that could be produced endogenously or by means of lipid peroxidation. This enzyme also has activity against xenobiotics [57].

In this work, we found a marked rise in GST activity compared to the control in all tissues. This suggests the active involvement of this enzyme in detoxification of chlorpyrifos. the hepatopancreas of crustaceans can metabolize chlorpyrifos, which is transformed into oxidized products [53]. Different studies have shown an induction of GST activity into the hepatopancreas. For instance, an increase in GST activity was observed in Chasmagnathus granulatus exposed to methyl-parathion [54], and in Ictalurus nebulosus exposed to dichlorvos [51]. Similarly, trichlorfon produced an elevation in the activity of this enzyme in M. rosenbergii [58].

GPx activity

Shrimps were exposed to sublethal concentrations of chlorpyrifos (0.7 and 1.3 μg/L). After four days, GPx activities were evaluated in hepatopancreas, gills, and muscles (Tukey’s test, p <0. 05) (). Three tissues showed a higher GPx activity than the control (gills: 6.12 and 18.36%; hepatopancreas: 21.4 and 39.3%; and muscle: 9.5 and 25.7%, respectively). GPx activity in the hepatopancreas reached a high increase (39.2%) and was greater than that of gills (18.6%) and muscle (25.7%) ().

The GPx enzyme plays a critical role in the antioxidant defense of crustaceans. This is a multifunctional enzyme that prevents the oxidative damage and the formation of lipid hydroperoxides [57]. In this study, the activity of GPx increased in all tissues, which suggests an increase in the mechanisms to decrease the oxidative damage due to exposure to chlorpyrifos. Some studies have also found an elevation in GPx activity by exposing different marine organisms to OP pesticides [59, 60].

Levels of malondialdehyde (MDA) in L. vannamei exposed to two concentrations of chlorpyrifos (0.7 and 1.3 μg/L) showed a significant elevation of lipid peroxidation in the three tissues (muscle, gills, and hepatopancreas) when such results were compared to the control (p < 0.05). Muscle tissues showed the highest level of MDA (111.3%) ().

Effect of chlorpyrifos on LPO in heopatopancreas, gills and muscle of L vannamei.

Data are presented as mean±SD. (p<0.05). (n = 5).

The peroxidation of polyunsaturated fatty acids (PUFAs) is used as a biomarker of effect [32,49,50]. LPO is considered as the first step of cellular membrane damage by OP pesticides [60]. In this study, a significant increase in the lipid peroxidation of membrane lipids of all assessed tissues (hepatopancreas, gills, and muscles) indicates that ROS-induced damage is one of the main toxic effects of chlorpyrifos ( [60]. These results are congruent with high levels of lipid peroxidation observed in marine organisms (Mytilus edulis, Gambusia affinis, Oreochromis niloticus and Poecila reticulate) exposed to sublethal concentrations of chlorpyrifos [60-62]. The observed LPO resulting from exposure to chlorpyrifos may lead to cell death [25]. Hence, L vannamei can be used as a test organism for in situ assessment of lipid oxidation.

Conclusions

This work provides evidence of chlorpyrifos toxicity in L. vannamei that is caused by OP-induced oxidative stress, thus indicating that the biological response to sublethal concentrations of the contaminant could be a valuable tool for monitoring OP contamination in freshwater environments. The availability of this robust and economical method sustains the use of L. vannamei as a model organism in monitoring studies.

Picture of acclimation of L. vannamei in the laboratory.

(TIF)

Click here for additional data file.

File of the statistical treatment of the trials essays, using the SPSS software package, version 25.

(DOCX)

Click here for additional data file.

Lethal concentration of chlorpyrifos to reach 50% mortality of shrimp within 96 hours (96-h LC50) of the L. vannamei.

(XLSX)

Click here for additional data file.

Dates of enzimatic activities.

(XLSX)

Click here for additional data file.

9 Sep 2019

PONE-D-19-20201

Effects of chlorpyrifos on the crustacean Litopenaeus vannamei

PLOS ONE

Dear Dr. Beatriz E Jaramillo-Colorado,

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.

Specifically:

==============================

The target chemical Chlorpyrifos is a traditional pesticide and its molecular mechanism of toxicity effect is relatively clear, and the study method is also traditional. So I think the work is not innovative on the whole, and it can not be accepted to be published in the journal.

==============================

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

Yours sincerely,

Zhenguang Yan

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The work is not innovative, so it can not be accepted.

[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 to be viewed.]

- - - - -

For journal use only: PONEDEC3

6 Nov 2019

Cartagena of Indias, 6 of November /2019

Staff

PLOS ONE

Kind regards.

The authors appreciate the important comments from the academic Editor. In the adjunt file (answer for reviewer), We explain the reasons that justify the publication of this article entitled: Effects of Chlorpyrifos on the crustacean Litopenaeus vannamei.

.

Thanks so much for your valuable collaboration.

Sincerely.

Beatriz E. Jaramillo C., Chem, Ph.D

Chemistry Program, Agrochemical Research Group, University of Cartagena, Cartagena of Indias, Colombia.

The Academic Editor Wrote:

The Academic Editor Wrote:

“The target chemical Chlorpyrifos is a traditional pesticide and its molecular mechanism of toxicity effect is relatively clear, and the study method is also traditional”.

Answer:

We will write three reasons why the manuscript should be published

1) Several research papers can be found on the effect of chlorpyrifos on mammals and fish, but much remains to be studied in aquatic species, especially in invertebrates (crustaceans). For example, when reviewing the topic with keywords such as chlorpyrifos and fish in www.pubmed.com, more than 400 articles appear, while searching in the same database with the keywords: “Litopenaeus vannamei” and “organophosphorus” and “chlorpyrifos” shows one result. We did a bibliographic tracking in the following databases: Sciencedirect, ACS, Scielo and Springer on Chlorpyrifos toxicity and we have found (see table 1 in the adjunt file of answer to reviewer)

Table 1. Articles found in the databases: Sciencedirect, ACS, Scielo and Springer on Chlorpyrifos toxicity

On invertebrates only exist 0,76% of public articles.

In the Stanford Encyclopedia of Philosophy we read:

“In developing ideas about the overall value of biodiversity it has been natural to draw on existing arguments about values of individual species (for review, see World Conservation Union 1980; Norton 1988). Commodity value and other direct use values have intuitive appeal because they reflect known values. But a key problem is that species need to be preserved for reasons other than any known value as resources for human use (Sober 1986). Callicott (1986) discusses philosophical arguments regarding non-utilitarian value and concludes that there is no easy argument to be made except a moral one. Species have some “intrinsic value” — reflecting the idea that a species has a value “in and for itself” (Callicott 1986, p.140) — and there is an ethical obligation to protect biodiversity”.

2) There are differences between the physiology of vertebrate and invertebrate organisms. In our study, the target organism was an invertebrate, crustacean, the Litopenaeus vannamei. See in the manuscript pages 3-4

Invertebrates do not have a spinal cord or spine; instead, most of them have an exoskeleton that spans the entire body. They do not have lungs since they breathe through their skin [1]. The liver participates mainly in the metabolism and regulation of nutrients in aquatic animals and plays an important role in oxidative stress and lipid peroxidation [2]. The crustaceans do not have liver; they have hepatopancreas, with functions similar to those of the fish liver (see fig1). Therefore, the investigation of possible biochemical changes in response to xenobiotics in crustaceans are done through the hepatopancreas [3], (see Fig. 1). However, the understanding of the influential mechanism underlying the pesticides in the hepatopancreas is still limited and needs further investigation [4].

So in Shrimp doesn’t exist a molecular mechanism of toxicity effect relatively clear.

Is also true that the vast majority of research is directed to biological models of vertebrate organisms, and less to invertebrates, such as L. vannamei, which precisely because of its economic importance and participation in the food of humans, must be ensured their food safety.

¿How chlorpyrifos affects a crustacean as the Litopenaeus vannamei?; what biomarkers could be used to assess its toxic effect

This research contributes to the study of the toxicity of chlorpyrifos in crustaceans, using the shrimp (Litopenaeus vannamei) as a model.

• On the other hand, the cultivation of Shrimp, Litopenaeus vannamei has an important role in economic growth, employment and welfare of coastal communities, around the world. But is very sensible to xenobiotics like pesticides.

Fig 1. Anatomy of L. vannamei (http://www.parasitosypatogenos.com.ar).

1. Robalino J, Wilkins B, Bracken-Grissom HD, Chan T-Y, O’Leary MA The Origin of Large-Bodied Shrimp that Dominate Modern Global Aquaculture. PLoS One. 2016; 11(7): e0158840.

2. Lu Q, Sun Y, Ares I, Anadon A, Martinez M, Martínez-Larrañaga MR. Deltamethrin toxicity: A review of oxidative stress and metabolism. Environ. Res. 2019; 170: 260–281.

3. Silveira de Melo M, Gonçalves dos Santos TP, Jaramillo M, Nezzi L, Rauh Muller YM, Nazari EM. Histopathological and ultrastructural indices for the assessment of glyphosate-based herbicide cytotoxicity in decapod crustacean hepatopancreas. Aquat. toxicol. 2019; 210:207-214.

4. Yang Z, Zhang Y, Jiang Y, Zhu F, Zeng L, Wang Y, et al. Transcriptional responses in the hepatopancreas of Eriocheir sinensis exposed to deltamethrin. PLoS ONE. 2017; 12(9): e0184581.

3) Chlorpyrifos is one of the most utilized insecticides in Colombia for agricultural and domestic pest control, but this also results in non-target organisms exposed to either lethal or sublethal concentrations of this contaminant (García-de la Parra et al., 2006). In Colombia is permit the use the chlorpyrifos, here don’t exist none prohibition. We want to change Colombian lays about concentration limits chlorpyrifos. But politicians think that in Colombia, there is not enough research on this pesticide In Colombia, we need to disseminate research on the effects of chlorpyrifos to pressure government agencies and thus transform the laws that govern the use of this pesticide. With this work we would like to contribute to that objective. (See fig 2 and fig 3, in the adjunt file of answer to reviewer)

Fig 2. Pesticide production in Colombia. See it in the adjunt file of answer to reviewer

Fig 3. Sales of Chlorpyrifos in Colombia. See it in the adjunt file of answer to reviewer

https://www.ica.gov.co/areas/agricola/servicios/regulacion-y-control-de-plaguicidas-quimicos/estadisticas/boletinplaguicidas2015-12-12-2016.aspx

This work is addressing a problem that could be considered at the regional level, but in reality, it is a global problem. The Bay of Cartagena and its biota has been exposed to a chlorpyrifos spill, and some companies (Dow Agrosciencies, Monsanto (Syngenta) produce this compound even in its industrial zone. See fig 4. See it in the adjunt file of answer to reviewer

Fig 4. Chlorpyrifos spill in Cartagena (Colombia). See it in the adjunt file of answer to reviewer

Fig 5. Plaguicides factories in Cartagena Bay (Colombia). See it in the adjunt file of answer to reviewer

This factories to this day continue to pollute the marine environmental of Cartagena bay with Chlorpirifos (LORSBAN)

Year article

1991 Cowgill RT, Gowland CA, Ramirez V. The history of a chlorpyrifos spill:

Cartagena, Colombia. Environ. Int. 1991; 17: 61-71.

2013 https://sostenibilidad.semana.com/actualidad/articulo/cartagena-dano-ambiental-petroleo-dow-quimica/30230

2015 https://www.eltiempo.com/archivo/documento/CMS-15869078

2015 https://www.javerianacali.edu.co/sites/ujc/files/node/field-documents/field_document_file/caso_presentado_por_estudiantes_derecho_javeriana_cali_en_buenos_aires.pdf

Agrochemical Research Group, University of Cartagena, did a work where sampled marine sediments in nine sites in the Bay of Cartagena in collaboration with the University of Buffalo (New York) and the following concentrations of pesticides were found:

Fig 6. Pesticides concentration in Cartagena Bay. See it in the adjunt file of answer to reviewer

The samples were conducted in June 2015 and 2017. We can see in the figure 6 that in seven of the sampling sites the highest concentration pesticide is chlorpiryfos.

Submitted filename: answer to Plos one -Edisson 101019.docx

Click here for additional data file.

2 Jan 2020

PONE-D-19-20201R1

Effects of chlorpyrifos on the crustacean Litopenaeus vannamei

PLOS ONE

Dear Dr Jaramillo-Colorado,

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. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

We have received two referee reports ad both have pointed out the need for a major revision to address several issues raised. Kindly respond to each of referee comments.

==============================

We would appreciate receiving your revised manuscript by Feb 16 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Iddya Karunasagar and Xinghui Qiu

Academic Editors

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2) We note that Figure(s) [Anatomy of L. vannamei (http://www.parasitosypatogenos.com.ar)] in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a). You may seek permission from the original copyright holder of Figure(s) [Anatomy of L. vannamei (http://www.parasitosypatogenos.com.ar)] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b). If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

3) Thank you for your ethics statement:

Experimental procedures were performed in accordance with the principles of animal care

established by the Central Committee of Ethics in Scientific Research of the University of

Cartagena (Act No 80/2015).

a) Please amend your current ethics statement to confirm that your named ethics committee Institutional Care and Use Committee (IACUC) specifically approved this study.

b) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for animal research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research

4) Thank you for updating your data availability statement. You note that your data are available within the Supporting Information files, but no such files have been included with your submission. At this time we ask that you please upload your minimal data set as a Supporting Information file, or to a public repository such as Figshare or Dryad.

Please also ensure that when you upload your file you include separate captions for your supplementary files at the end of your manuscript.

As soon as you confirm the location of the data underlying your findings, we will be able to proceed with the review of your submission.

Additional Editor Comments (if provided):

We have received two referee reports ad both have pointed out the need for a major revision to address several issues raised. Kindly respond to each of referee comments.

[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: (No Response)

Reviewer #2: (No Response)

**********

2. 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: Partly

Reviewer #2: Yes

**********

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?

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: No

Reviewer #2: Yes

**********

5. 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: No

Reviewer #2: Yes

**********

6. 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 authors need to have the manuscript checked for English usage by someone whose first language is English. Throughout the paper there are many instances of improper English (unnecessarily capitalized words, missing words, misspelled words).

2. The manuscript as downloaded was difficult to review as there were 2 versions that were not well defined. I reviewed the one that had small type at the top that read "Revised Manuscript with Track Changes." There were no figures included with that, so I reviewed the figures associated with another version labeled "Manuscript". Then there were other figures located elsewhere in the download. This was all very confusing.

3. Figures legends were all placed oddly throughout the manuscript and do a very poor job of describing the figures.

4. Table 1 has values that do not match the values in the manuscript.

5. In the methods, it is not clear how many shrimp were placed in each replicate.

6. Reference #25 has the incorrect year.

7. I could not find if the authors made all data underlying the findings in their manuscript fully available. That does not mean it is not there, I just do not know where to find it.

8. I think the manuscript is worth publishing whenever it is satisfactorily revised.

Reviewer #2: Line 38: Last line in this paragraph seems inappropriately placed as no information on oxidative stress has been addressed at this point. Remove or re-word.

Line 102: Was shrimp sex determined or was sex not considered during selection of organisms? This is unclear.

Line 107: More detail on the brand or content of the shrimp feed may be useful.

Line 108: There are many stages of intermoult in a shrimp's life. Clarify if the shrimp were at a certain life stage or state that they were not currently undergoing moult. "The intermoult stage" is unclear.

Line 119: What type of plastic? Could this plastic affect your results at the temperature you held the shrimp at?

Line 218: Why were calibration curves so high (100-5000ug/L)? Test concentrations were only 0.7-13.0 ug/L. Clarify.

Line 229: LC50 value in this line (2.10 ug/L) differs from the LC50 value in Table 1 (0.00021 ug/L). Confirm units.

Line 268: Behaviours are mentioned but observation of behaviour is not outlined in methods.

Line 303: Was chlorpyrifos detected in muscle tissue? This is worded poorly and should be clarified. If stating a dose-dependent relationship then re-word the statement. If indeed the chlorpyrifos content of the muscle increased, include in methods how this was measured.

**********

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

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Feb 2020

Cartagena of Indias, 16 February 2020

Sir/Madam

Editor

PLOS ONE

Kind regards.

I´m sending you the answers to the reviewers, and corrections of the manuscript titled:” Effects of chlorpyrifos on the crustacean Litopenaeus vannamei”.

The authors appreciate the important comments from the reviewers. Below, we explain the corrections and changes made to the article. The changes are indicated in the manuscript with red letters.

Thanks so much for your valuable collaboration.

Sincerely.

Beatriz Eugenia Jaramillo-Colorado., Chem, Ph.D

Chemistry Program, Agrochemical Research Group, University of Cartagena, Cartagena of Indias, Colombia.

The Academic Editors Wrote:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

ANSWER

It was checked and corrected.

2. We note that Figure(s) [Anatomy of L. vannamei (http://www.parasitosypatogenos.com.ar)] in your submission contain copyrighted images. (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

ANSWER

That was changed. We made our own illustration, using the GIMP Software v2.8.14.

See Figure 3.

3. Please amend your current ethics statement to confirm that your named ethics committee Institutional Care and Use Committee (IACUC) specifically approved this study.

ANSWER

The files will be loaded in the submit

4. Thank you for updating your data availability statement. You note that your data are available within the Supporting Information files, but no such files have been included with your submission. At this time we ask that you please upload your minimal data set as a Supporting Information file, or to a public repository such as Figshare or Dryad.

Please also ensure that when you upload your file you include separate captions for your supplementary files at the end of your manuscript.

ANSWER

The Supporting information was added to the manuscript, and the files will be upload in the submission.

REVIEWERS´ COMMENTS

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

ANSWER.

The Supporting information will be upload in the submission.

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

ANSWER

The manuscript was checked and corrected by an English language professional.

Reviewer #1:

3. Figures legends were all placed oddly throughout the manuscript and do a very poor job of describing the figures.

ANSWER

This was reviewed and corrected throughout the manuscript.

4. Table 1 has values that do not match the values in the manuscript.

ANSWER

It was our mistake. Since we did not consider a unit conversión. This was corrected. See Table 1, and lines 235 to 242, page 10.

5. In the methods, it is not clear how many shrimp were placed in each replicate.

ANSWER

It was corrected. In toxicity tests, eight adult shrimps were used in each replicate. Please, to see page 5 line 124. In Sublethal toxicity assay Three replicates were done using five shrimps each. See page 6 line 151.

6. Reference #25 has the incorrect year.

ANSWER.

It was corrected. The year is 2015. See page 19, line 481

7. I could not find if the authors made all data underlying the findings in their manuscript fully available. That does not mean it is not there, I just do not know where to find it.

ANSWER.

The Supporting information was added to the manuscript, and the files will be upload in the submission. See page 15.

Reviewer #2

8. I think the manuscript is worth publishing whenever it is satisfactorily revised.

ANSWER

Thanks so much. Below, I describe the changes done.

8. Line 38: Last line in this paragraph seems inappropriately placed as no information on oxidative stress has been addressed at this point. Remove or re-word.

9. ANSWER.

It was removed.

10. Line 102: Was shrimp sex determined or was sex not considered during selection of organisms? This is unclear.

ANSWER

For this study, the gender of L. vannamei was not considerated. See page page 5 line 110.

10. Line 107: More detail on the brand or content of the shrimp feed may be useful.

ANSWER.

The feed was shrimp diet (Solla, Colombia). See Page 5, line 108

11. Line 108: There are many stages of intermoult in a shrimp's life. Clarify if the shrimp were at a certain life stage or state that they were not currently undergoing moult. "The intermoult stage" is unclear.

ANSWER

Shrimps were not currently undergoing moults. See Page 5, line 111.

11. Line 119: What type of plastic? Could this plastic affect your results at the temperature you held the shrimp at?

ANSWER.

The plastic was polypropylene. See page 5, line 106. It does not affect the experiment, since it was carried out at room temprature.

12. Line 218: Why were calibration curves so high (100-5000ug/L)? Test concentrations were only 0.7-13.0 ug/L. Clarify.

ANSWER

It was our mistake. Since we did not consider a unit conversión. This was corrected. See lines 219- 223, page 9.

13. Line 229: LC50 value in this line (2.10 ug/L) differs from the LC50 value in Table 1 (0.00021 ug/L). Confirm units.

ANSWER

It was our mistake. Since we did not consider a unit conversión. This was corrected. See Table 1, and lines 235 to 255, page 10.

14. Line 268: Behaviours are mentioned but observation of behaviour is not outlined in methods.

ANSWER.

It was removed.

15. Line 303: Was chlorpyrifos detected in muscle tissue? This is worded poorly and should be clarified. If stating a dose-dependent relationship then re-word the statement. If indeed the chlorpyrifos content of the muscle increased, include in methods how this was measured.

ANSWER

That was misspelled. Chlorpyrifos was not determined in the muscle. What was evaluated was the activity of catalase in the muscle. See page 12 line 305.

Submitted filename: Response reviewers Plos One 16-02-20.docx

Click here for additional data file.

23 Mar 2020

Effects of chlorpyrifos on the crustacean Litopenaeus vannamei

PONE-D-19-20201R2

Dear Dr. Jaramillo-Colorado,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Iddya Karunasagar

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All comments addressed satisfactorily

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: All comments have been addressed

**********

2. 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

Reviewer #2: Yes

**********

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?

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

Reviewer #2: Yes

**********

5. 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

Reviewer #2: Yes

**********

6. 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: All the previous comments seem to have been addressed. I have no further comments to make to the authors.

Reviewer #2: (No Response)

**********

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

30 Mar 2020

PONE-D-19-20201R2

Effects of chlorpyrifos on the crustacean Litopenaeus vannamei

Dear Dr. Jaramillo-Colorado:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Iddya Karunasagar

Academic Editor

PLOS ONE

Table 2

Activities of AchE enzymes in the brain of L. vannamei following four days of exposure to chlorpyrifos.

Chlorpyrifos (μg.L-1)	0	0.7	1.3
AchE (Unit.mg-1 protein)	26±2.3	18±2.7	14±2.4

Values are presented as mean ± S.D. (n = 5).