Effects of Sublethal Concentrations of the Herbicide, Glyphosate, on Embryonic Development of the Indian Major Carp, Labeo rohita

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Introduction
Glyphosate [N-(phosphonomethyl) glycine], CAS Number: 1071-83-6, is a chemical used for weed control.Te half-life period of the molecule ranges from 2 to 215 days in soil and 2 to 91 days in water [1].Weeds compete with cultivated crops for nutrients and sunshine.Tey adversely afect the growth of plants [2].Glyphosate has inhibitory efects on EPSPS (5enolpyruvylshikimate 3-phosphate synthase).EPSP inhibition leads to the depletion of the aromatic amino acids tryptophan, tyrosine, and phenylalanine that are needed for protein synthesis.Tis organophosphate group of pesticide was frst synthesized and commercialized in 1950 by a pharmaceutical company of Switzerland, but its herbicidal properties were unknown.E. Franz of Monsanto Company studied the herbicidal properties of glyphosate under the trade name Roundup [3].Since then, it has been used in agriculture, horticulture, and forestry [4].Its use became widespread after the discovery of glyphosate-resistant crops in 1980, and it has been increased by 100 fold; till date, it is the most abundantly used herbicide worldwide [5].Tis polyprotic molecule has phosphonate, carboxyl, and amino groups as three polar functional groups [6].Glyphosate has been used as a herbicide for many years [5,7].But during the last few years, it has been banned in some countries worldwide, since it afects soil, water, and soil microbiota.In India, it has been banned in Kerala and Punjab.In other states, it is permitted for restricted use only.In Odisha, glyphosate is commonly used for weed management in rice, maize, cotton, and vegetable cultivations [8][9][10].Glyphosate (GLY) has been classifed as a Group 2B and Group 2A carcinogens for humans [11,12], respectively, and frequently found in aquatic ecosystems.To date, GLY has been detected in natural waters at concentrations generally between 3 and 700 μg/L in many countries [13].
Aquaculture is the controlled process of breeding, raising, and harvesting fnfsh, shellfsh, and aquatic plants for human consumption.Fish gives hope for fghting malnutrition since it is a food that is rich in protein, calcium, omega-3 fatty acids, and vitamins like A and B 12 [14].Currently, 50% of global fsh consumption is fulflled by aquaculture [15].Besides, it creates scope for employment and contributes to economic growth.Fish and aquaculture support the livelihood of 12% of the global population.It is one of the fastest-growing sectors worldwide.
According to the State of World Fisheries and Aquaculture study, the growth of aquaculture, particularly in Asia, has raised the total production of fsheries and aquaculture to 214 million tons in 2020 comprising 178 million tons of aquatic animals and 36 million tons of algae [16].Globally, India is the second largest producer of fsh after China.Around 95% of total aquaculture production is contributed by freshwater aquaculture.Te application of advanced technologies has resulted in an increase in aquaculture production in India by 7%, with an annual production of 5.77 million tons.Tis upward shifting of the growth curve has been achieved by induced breeding of fshes like the three Indian major carps (Labeo catla, Labeo rohita, and Cirrhinus mrigala) and composite culture of major carps as well as exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, and Cyprinus carpio) [8].Out of these, L. rohita is a highly consumed farmed fsh.Te presence of pollutants in water bodies afects the growth and development of fsh and ultimately adversely afects the aquaculture output in terms of quality and quantity.Te development of fsh is dependent upon the factors present in the surrounding environment.So, any alteration in surroundings can induce developmental changes and these changes may persist to adulthood, even if exposure to pollutants is ceased.Tis study aims to determine the developmental toxicity of one of the widely used herbicides, glyphosate, on the commercially important fsh, L. rohita.

Collection of Eggs.
Fertilized eggs of L. rohita were collected from ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, Carp Hatchery Unit.A pair of 3-year-old male and female fsh was bred.Te female fsh weighed 2.2 kg with 38.5 cm length.Te male fsh was of 1.8 kg weight with 33 cm length.

Screening of Fertilized Eggs.
Te fertilized eggs were screened in the laboratory depending on their transparent appearance which was visible by naked eyes.Te unfertilized dead embryos were opaque in appearance.Te embryos were observed under microscope and those showing cleavage were selected for the toxicity test.
To prepare 1, 10, 20, 30, 40, and 50 mgL −1 concentrations of glyphosate, 0.25, 2.5, 5.0, 7.5, 10, and 12.5 µl volumes were added in 80 ml of water, respectively.Ten fertilized eggs were released to each concentration 4 h after fertilization.Te number of dead embryos was counted and noted down at an interval of 24 h, and the rate of mortality was calculated.Ten, the probit value was noted down from Finney's table .Te logarithm values of respective concentrations were calculated.A graph was plotted taking logarithm values of concentrations along the X-axis and probit values of mortality in respective concentrations along the Y-axis.Ten, probit of 5 was located on Y-axis.Ten, by moving down the X-axis, we could fnd the logarithm of LC 50.By calculating the anti-logarithm, we could determine the LC 50 .

Exposure of Embryos to
Glyphosate.Te 4 hpf (hours post fertilization) embryos were exposed to 1/3 rd , 1/5 th , and 1/10 th of the median lethal concentration of glyphosate.Te three treatments were designated as treatment-I, II, and III depending upon the concentration of glyphosate.Te dosages were 2.08, 4.17, and 6.96 mgL −1 for treatment-I, treatment-II, and treatment-III, respectively.Te concentrations were loaded 30 min before the release of embryos.Fifty embryos were released to each Petri plate containing 500 ml of water.Te experiment was conducted in a set of 3 replicates.Te embryos were observed under a microscope and photographed.Te observations include duration of hatching, heart, and optic cup formation, eye development, formation of the branchial artery, edema, and kink formation.Te percentage of deformed embryos was calculated for 24, 48, 72, and 96 h as a function of time.Te percentage of deformed embryos was calculated as the ratio of deformed embryos/ larvae to the number of alive embryos/larvae at 24 h.For 48, 72, and 96 h, it was calculated as a cumulative percentage.

Results
Te median lethal concentrations for 72 and 96 h were 26.3 and 20.89 mgL −1 (Figure 1), respectively.Te diferentiation of the head, yolk sac, and tail was observed in all treatments and control at 13 hpf.Te twitching movement was also observed after the 13 th hour of development.In control and treatment-I, hatching occurred between the 16 th and 27 th hpf, whereas in treatment-II and treatment-III, it occurred 2 Journal of Applied Ichthyology between the 16 th and 22 nd hpf.Te optic cup formation was delayed in all three treatments.In control, it occurred at 24 hpf, but in all three treatments, it was found at 27 hpf.At the 16 th hour of development, the red blood cells (RBCs) were formed in all treatments and control except two embryos in treatment-II.Te branchial arteries were formed at 37 hpf in control and all treatments.Other deformities observed were yolk sac edema, pericardial edema, abdominal curvature, and kink formation in the tail.At 24 h, 2 deformed embryos were observed in treatment-I with yolk sac edema, pericardial edema, and elongated yolk sac.In the rest of the treatments and control, all embryos were normal (Figure2).At 48 h, more abnormalities were observed at higher concentrations as compared to lower concentrations (Figure 3).At 2.08 mgL −1 , 4 deformed embryos were found, of which 3 contained yolk sac edema and 2 had pericardial edema.In treatment-II, 7 deformed embryos were observed, of which 4 had deformed yolk sacs and pericardium, and 6 embryos contained deformed spinal cords.Eight deformed embryos were observed in treatment-III, out of which 4 embryos had yolk sac and pericardial edema and 2 were with kink formation in the tail.At 72 h, 6 embryos were detected with yolk sac edema, 4 with pericardial, 1 with abdominal curvature, and 2 with tail kink (Figure 4).In treatment-II, 6 embryos were having yolk sac edema, 4 with pericardial edema, and 3 with kink formation in the tail.In the highest concentration of glyphosate, yolk sac edema, pericardial edema, and abdominal curvature were noticed in 6, 5, and 1 embryos, respectively.At 96 h, more changes were observed in the high concentration as compared to the lower concentrations (Figure 5).Te tail kink formation was observed with 1 embryo in both treatment-I and treatment-II.In treatment-III, abdominal curvature was observed in one embryo.Te embryos treated with glyphosate showed less pigmentation.Besides, the air bladder was not properly fattened in the embryos of treatment-III.Te percentage of deformed embryos in various concentrations of glyphosate at diferent time intervals is mentioned in Table 1.
Te heart rate was calculated in embryos of all the treatments and control at an interval of 24 h (Figure 6).It was found that in all four observations, the heart rate of control embryos was higher than treatments and it decreased in a concentrationdependent manner.

Discussion
In this experiment, we studied the sublethal efects of glyphosate on the development of embryos of L. rohita.Te 96 hpf is a crucial period for embryos.In zebrafsh, more developmental abnormalities were observed when the embryos were exposed to glyphosate for 96 h after 4 h of fertilization as compared to the exposure for 96 h at 3 days post fertilization [18].Hence, in this experiment, we exposed the 4 hpf embryos to diferent concentrations of glyphosate for a period of 96 h.It could be observed that the deformities in embryos increased with an increase in glyphosate concentration and duration of exposure.Te same observations were reported in zebrafsh exposed to 1, 5, 10, and 100 mg/L concentrations of glyphosate for 24, 48, 72, and 96 h [19].Before hatching, no deformities were observed in any treatment or control due to the presence of chorion membrane.Te chorion is a secondary membrane secreted by the developing oocyte.It is a cellular layer present immediately above the plasma membrane of an ovum [3,20].Tis membrane protects the embryo from pollutants present in the external environment.According to the study by Villalobos et al. [21], the chorion membrane protects the medaka embryos from the toxic efects of Tiobencarb.Tis information supports our fndings.
Te heart rate was found to be decreased in all the treatments compared to the control.It slowed down with an increased concentration of glyphosate.In the case of medaka embryos, the heart rate increased at frst and then it decreased.Te increased heart rate was described as an adaptation to stress environment, and the decreased heart rate might have resulted due to destruction of cells in the heart wall [22].Our fndings difered from it.Te heart rate was decreased in glyphosate-treated fsh in a dose-dependent manner.Te same results were observed in the case of zebrafsh exposed to 50 μg/ ml of glyphosate.Tey described it as a result of cell death in the heart and the concentrations taken were higher, so the embryos could not acclimate to the stress condition [23].
Te frst deformities were observed in 2.08 mgL −1 concentration at 23 rd h.We could observe 2 deformed embryos in the 23 rd h of development at 2.08 mgL −1 only, but no deformities were observed at higher concentrations.Te embryos are more susceptible to lower concentrations of glyphosate during early stages within 24 h.During later stages of development, they were more susceptible to higher concentrations (Table 1).Such changes were observed in zebrafsh exposed to methyl mercury.More number of deformed embryos were observed at 20 μg/l compared to 30 μg/l after 16 hpf [24].Te spinal curvature and kink formation in the tail were observed in all the treatments, but not in the control.Hence, it is clear that these changes resulted due to exposure to glyphosate.Tese could be due to decreased collagen synthesis.In the case of medaka embryos, several developmental deformities were reported, such as bent tail and abdominal enlargement after exposure to glyphosate.Te concentrations used in this experiment (100, 200, 300, 400, and 500 mgL −1 ) were much higher than the concentrations used in our experiment [22].Tis indicates that the embryos of L. rohita are more sensitive to glyphosate as compared to medaka.
Spinal curvature was reported in zebrafsh exposed to 0.620 (0.436-0.963), 0.475 (0.302-0.801), and 0.341(0.177-0.617)mg•L −1 concentration of organophosphate insecticide, Sumithion [1].Glyphosate was identifed as a potent teratogen to the Japanese medaka fsh (Oryzias latipes) embryos and can induce developmental abnormalities at a concentration of 0.5 mg/L [25].Te abnormalities include spinal curvature, enlarged yolk sac, and greying of the yolk sac.Te yolk sac edema was noticed in the African catfsh (Clarias gariepinus) exposed to a group of metallic chemical elements, such as chromium, cadmium, copper, and agrochemicals like sodium pentachlorphenol (NaPCP) and malathion [26].Te yolk sac is the site for early blood fow.Remodeling of blood vessels occurs at this site.Glyphosate (TCDD) [27].In this experiment, the frst yolk sac edema was observed at 24 h at a 2.08 mgL −1 concentration of glyphosate.Te well-developed swim bladder was observed at 96 hpf.Tis organ serves as a bouncy device.In teleost, it   consists of two chambers, anterior and posterior.Te posterior chamber is vascularised and gas release occurs in this chamber [28].In the case of carp, the one-chambered swim bladder develops at 96 hpf and the two-chambered swim bladder develops at 192 hpf [29].In our experiment, the swim bladder was improperly fattened only at a 6.96 mgL −1 concentration of glyphosate.A similar observation was made in zebrafsh exposed to glyphosatebased herbicides.Te diferent concentrations were 11.7, 35, and 58.3 mgL −1 .Te abnormality was observed in a dose-dependent manner [10].A reduced pigmentation was observed in glyphosate-treated embryos.Such a reduction in pigmentation has been observed in algae treated with various pesticides.However, no such study has been conducted on animals.Te efects of glyphosate on chromatophore cells can be explored further.

Conclusion
Tis research presents an enormous efect of glyphosate in L. rohita embryos.Te teratogenic efects of glyphosate led to deformed embryos, decreased heart rate, and high mortality in a dose-dependent manner.Tis chemical would also adversely afect the natural population of the carps and pond culture system which receives runof from agricultural land.
To sustain the production of food fsh from aquaculture, an amelioration mechanism (feed supplementation) can be developed further for combating the impact of the herbicide.

Figure 1 :
Figure 1: Te graph showing the 96 h LC 50 value of glyphosate for embryos of Labeo rohita.

Figure 2 :Figure 3 :
Figure 2: Development of embryos of Labeo rohita at 24 hpf.(a) Normal hatchling in control.(b) Kink formation in the tail and pericardial edema in T-I.(c) Elongated yolk sac and incomplete diferentiation of tail in T-I.(d) Normal hatchling in T-II.(e) Normal hatchling in T-III.

Figure 4 :
Figure 4: Developing embryo of Labeo rohita at 72 hpf: (a) normal developing embryo in control, (b) tail curvature in T-I embryo, (c) yolk sac and pericardial edema with an embryo of T-I, (d) yolk sac and pericardial edema with tail kink formation in T-II embryo, (e) yolk sac edema and pericardial edema in an embryo of T-II, (f ) pericardial edema in T-III embryo, and (g) abdominal curvature in T-III embryo.

Figure 5 :
Figure 5: Developing embryo of Labeo rohita at 96 hpf: (a, b) normal developing embryo in control, (c, d) an embryo of T-I showing reduced pigmentation, (e) embryo with spinal deformity in T-II, (f ) reduced pigmentation in T-II embryo, (g) developing embryo in T-III with abdominal curvature and improperly fattened swim bladder, and (h) reduced pigmentation in T-III embryo.

Table 1 :
Te percentage of deformed embryos of Labeo rohita at various concentrations of glyphosate at diferent time intervals.