Effect of Silver nanoparticles exposure on oxidative stress in blue swimmer crab (Portunus pelagicus)

doi:10.21203/rs.3.rs-2436656/v1

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Effect of Silver nanoparticles exposure on oxidative stress in blue swimmer crab (Portunus pelagicus)

https://doi.org/10.21203/rs.3.rs-2436656/v1

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Nanotechnology is a new technology that has many potential environmental effects on living organisms, especially aquatic species. This study was carried out to determine the median lethal concentrations (LC₅₀) of silver nanoparticles and its effect on the antioxidant system in the hepatopancreas and muscle tissue of blue swimmer crab (Portunus pelagicus). The LC₅₀ value was determined according to the OECD standard method and after that, the crabs were exposed to sublethal silver nanoparticles concentrations (equivalent to 25, 50 and 75% LC₅₀ AgNPs) for 14 days. To measure the activity of catalase, glutathione superoxide dismutase, glutathione peroxidase, total antioxidant capacity and malondialdehyde, crab were sampled on days 3, 7 and 14. According to the results, the 96 h LC₅₀ value of AgNPs was 13.65 mg/L. In both hepatopancreas and muscle tissue, except glutathione and glutathione peroxidase, the level of catalase, superoxide dismutase and total antioxidant capacity of crabs exposed to 50% LC₅₀ of AgNPs were significantly decreased as compared to the control (p < 0.05). MDA level in hepatopancreas significantly increased in crab exposed to 50% LC₅₀ of AgNPs compared to all tested concentration and control group (p < 0.05), but not in the muscle. In general, crabs exposed to 50% LC₅₀ of AgNPs showed a significant decrease in the levels of catalase, superoxide dismutase and total antioxidant capacity, except in MDA level where it was significantly increased which was indicate that a dose of 50% LC₅₀ of AgNPs had a deleterious effect on the hepatopancreas antioxidant system of blue swimmer crab.

Silver nanoparticles

Toxicity

Oxidative stress

Portunus pelagicus

Nanotechnology is an emerging field that tremendously developed in the last decade deals with the manufacturing, designing, describing and applying structures, tools and systems at the nanoscale (Ansar et al. 2020). Silver nanoparticles (AgNPs) are as one of the most important kind of nanosized materials with unique properties such as small size, increased strength, chemical reactivity, and higher surface area that are well known for their worldwide application, specially due to their biological activities and in mariculture can be used for the treatment of diseases caused by pathogens (Kakakhel et al. 2019; Camacho-Jiménez et al. 2020). Nowadays extensive use of silver nanoparticles had been led to their amplified discharge into the environment. Silver nanoparticles is very toxic to aquatic fauna and cause harmful effects on the organisms that are constantly exposed to these materials (McGillicuddy et al. 2017). Exposure to metallic nanoparticles probably caused severe oxidative stress in the aquatic organisms, which was manifested as an increase in the production of reactive oxygen species and damage the antioxidant defense system (Xu et al. 2018; Yu et al. 2020). Blue swimming crab Portunus pelagicus as one of the most economically value species often is a major component of commercial fisheries among crustacean throughout the Indo-Pacific region and also considered as one of the important cultured species in China, this species is a benthic carnivore that food with benthic habits and can accumulate contaminants from the water, thus can be used as indicator organisms for assessing environmental pollution (Tadi-Beni et al., 2019; Lu et al., 2022; Wang et al., 2022)

and therefore it is necessary to establish and assess the environmental fate, transport, transformation, and toxicity of AgNPs in the test medium (Rasmussen et al. 2018). In the last decade a significant number of assays have been performed for the toxicity of nanomaterials and their effects on aquatic biota such as copepods, fishes and fish embryos, Cape River crab, Potamonautes perlatus, freshwater crab, Paratelphusa jacquemontii and Artemia, (Temleton et al. 2006; Zhu et al. 2008; Walters et al., 2016; Kadam and Raut, 2019; Rekulapally et al. 2019), although related studies are relatively scarce in crabs and there were no studies conducted to date on toxicity of NPs to P. pelagicus. As mentioned, aquatic systems exposure to NPs is inevitable and the entering of metal nanoparticles into the water is associated with high risk, therefore assessment of the interactions between the NPs and the surrounding environment are necessary, so the main objectives of this study is to investigate the toxicity of AgNPs and its effect on oxidative stress of P. pelagicus.

2.1. Crab supply and maintenance

Sub-adults of blue swimming crabs with an average initial weight of 82.14 ± 6.94 g and carapace width of 75.30 ± 6.3 mm were obtained from a local fisherman operating dealer and immediately transported to the laboratory in plastic containers with aeration provided. Prior to the start of toxicity tests, crabs were maintained in an aerated aquarium and acclimatized to laboratory conditions for five days (Wang et al. 2022). During the adaptation period, crabs were fed at a rate corresponding to 10% of body weight with squid two times a day, but were not fed for 24 h before to and during toxicity testing for minimizing the absorption of NPs in food and the production of feces (Afifi et al. 2016). Before each times of feeding, the feces and uneaten feed were removed daily through siphoning. The mean water parameters were recorded during all the experiments with a conventional photoperiod (12 h /12 h light and dark cycles) as follows (Wang et al., 2022): temperature at 20 ± 1.22 ◦C, salinity at 31 ± 0.085 ppt, pH at 7.64 ± 0.25. Silver nanoparticles which used in this study is a citrate capped colloidal AgNPs with average size 16.6 nm and concentration of 4000 mg/L are purchased from the Nano nasb Pars Company, Iran. The physiochemical properties of the nanoparticles involved in this study are estimated previously by Johari et al. (2013).

2.2. Determination of lethal concentration

After acclimation, the mortality and acute toxicity test were carried out according to the Organization for Economic Cooperation and Development (OECD 2017). To date, the lethal concentration of silver nanoparticles in blue swimmer crab has not been determined, therefore at first, during the preliminary experiments, several range finding tests were conducted to determine the range of lethal concentration. According to the data obtained from preliminary experiments, lethal range of silver nanoparticles was 1.58–105 mg/L, then for LC₅₀ determination, crabs were exposed to six different concentrations of silver nanoparticles including 3.16, 6.32, 12.64, 25.28, 50.56 and 101.12 mg/L along with control group into separated tanks which had 300 L water for 96 h in triplicate under static conditions with no replacement of the test solution. Each replicate comprised 5 crab. The mortality rate was recorded by counting the total number of dead crabs every 24 h for 96 h.

2.3. Sublethal toxicity

Based on the result, the LC₅₀ value of silver nanoparticles in this species was 13.65 mg/L, therefore, 4 tested concentrations including treatment 1: (25%LC₅₀: 3.41 mg/L AgNP), treatment 2: (50% LC₅₀: 6.82 mg/L AgNP) and treatment 3: (75% LC₅₀: 10.23 mg/L AgNP) and control treatment were chosen to determine the sublethal toxicity effects of silver nanoparticles on oxidative stress parameters of blue swimmer crab for 14 days. Each treatment contained three replicates, and each replicate encompassed 5 animals.

2.4. Oxidative stress indices

For measurement of oxidative stress indices, nine crab per treatment (3 × 3 crab/replicate) were randomly sampled at day 3, 7 and 14. The collected crabs have been carefully cryo-anesthetized in crushed ice for 5 min (Capparelli et al., 2020) and after that, the hepatopancreas and muscle tissues were excised and were stored at -80°C for further studies. The tissues of the hepatopancreas and muscle were homogenized in Tris-HCl buffer (Tris-HCl) at pH 7.4 (4°C). Homogenized samples were centrifuged at 12000 g for 20 min at 4°C and then the supernatants were used for oxidative stress parameters analysis, including catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), total antioxidant capacity (TAC) and Malondialdehyde (MDA).The catalase activity was measured based on the decomposition of hydrogen peroxide (H₂O₂) according to the method of Korolyuk et al (1988), Superoxide dismutase activity was assayed using RANSOD biochemical kit (RANDOX®, UK), Glutathione peroxidase was estimated based on the suggested method by Lawrence and Burk (1976), Total antioxidant capacity (TAC) was determined based on Benzie and Strain method (1996) and Malondialdehyde (MDA) level were also measured as lipid peroxidation (LPO) content, with commercial assay kit (Anzan Chemistry Company, Iran).

2.5. Statistical Analysis

The mortality study was done carried out 96 h and LC₅₀ was calculated using probit analysis in SPSS software (V 22.0). All the experimental data were reported as mean with standard deviation. The data were analyzed by one-way analysis (ANOVA) followed by Tukey's test. A value of P < 0.05 was considered statistically significant.

3.1. LC₅₀ determination

Results showed that with increasing concentration and exposure time, mortality rate increased. There was no mortality observed in control group during 96 h (Fig. 1). After 72 h, mortality was found in all exposed concentrations (except at concentration of 3.16 mg/L and control group). In exposed crabs, the first mortality at 24 h was observed in a dose of 50.56 mg/L AgNPs, there was no mortality found in the previous four concentrations in 24 h (Fig. 1). In concentrations of 50.56 and 101.12 mg/L AgNPs, mortality was observed in all exposure times.

The level of LC₅₀ in Portunus pelagicus at 24, 48, 72 and 96 h were determined to be 96.71, 54.25, 33.63, 13.65 mg/L, respectively (Table 1).

Table 1

Median lethal concentration (LC₅₀) of silver nanoparticles in *Portunus pelagicus*
Exposure time (h)	Median lethal concentration (mg/L)
24	96.71 ± 0.088
48	54.25 ± 0.069
72	33.63 ± 0.075
96	13.65 ± 0.060

3.2. Results of oxidative stress indices

Catalase activity in hepatopancreas and muscle of crabs exposed to 50% LC₅₀ AgNPs concentration significantly reduced compared to control and other tested concentration (p < 0.05). The lowest level of catalase in hepatopancreas and muscle was observed in a dose of 50% LC₅₀ AgNPs on days 7 and 14 respectively (Table 2).

Table 2

Changes in catalase activity (U/mg prot) of *Portunus pelagicus* after exposure to 25, 50 and 75% LC₅₀ AgNPs
Tissue	Sampling day	Control	25% LC₅₀	50% LC₅₀	75% LC₅₀
Hepatopancreas	3	48.12 ± 4.27 ^b	50.03 ± 3.98 ^b	25.16 ± 4.36 ^a	45.99 ± 2.88 ^b
	7	46.58 ± 2.93 ^b	46.79 ± 7.01 ^b	19.86 ± 3.14 ^a	48.00 ± 5.15 ^b
	14	49.70 ± 2.88 ^b	48.50 ± 4.76 ^b	19.45 ± 4.10 ^a	51.17 ± 8.12 ^b
Muscle	3	9.45 ± 0.53 ^b	9.88 ± 0.55 ^b	2.70 ± 0.15 ^a	8.14 ± 0.25 ^b
	7	9.88 ± 0.66 ^b	8.65 ± 0.45 ^b	1.95 ± 0.18 ^a	7.89 ± 0.66 ^b
	14	11.05 ± 1.03 ^b	8.96 ± 0.73 ^b	1.80 ± 0.10 ^a	7.55 ± 0.23 ^b

In all tables means not sharing the same letter (a or b) are significantly different at p < 0.05.

The level of glutathione in muscle tissue was higher than hepatopancreas. There was no significant differences in glutathione level observed in both tissues at all the concentrations of AgNPs comparing to the control group (p > 0.05). The lowest and highest level of glutathione in both tissues was observed in 50% LC₅₀ and 75% LC₅₀ AgNPs concentration on days 3 and 14 respectively (Table 3).

Table 3

Changes in glutathione activity (mg/g prot) of *Portunus pelagicus* after exposure to 25, 50 and 75% LC₅₀ AgNPs
Tissue	Sampling day	Control	25% LC₅₀	50% LC₅₀	75% LC₅₀
Hepatopancreas	3	29.27 ± 2.00 ^a	30.77 ± 3.16 ^a	28.55 ± 2.90 ^a	29.45 ± 2.00 ^a
	7	28.68 ± 2.75 ^a	29.30 ± 5.10 ^a	29.33 ± 8.12 ^a	29.84 ± 3.86 ^a
	14	28.65 ± 3.15 ^a	29.68 ± 2.69 ^a	31.00 ± 5.18 ^a	32.66 ± 3.26 ^a
Muscle	3	40.13 ± 6.44 ^a	38.60 ± 2.85 ^a	36.15 ± 2.49 ^a	39.50 ± 4.11 ^a
	7	40.90 ± 6.00 ^a	39.00 ± 6.23 ^a	36.99 ± 7.14 ^a	39.84 ± 2.16 ^a
	14	40.98 ± 1.80 ^a	40.22 ± 8.15 ^a	38.45 ± 5.35 ^a	41.00 ± 5.87 ^a

The level of glutathione peroxidase in hepatopancreas and muscle of exposed crabs did not show a significant differences compared to control group (p > 0.05), but the lowest and highest level in hepatopancreas was observed in a dose of 25% LC₅₀ and 50% LC₅₀ AgNPs on days 7 and 14 (Table 4). In muscle, the highest and lowest levels was related to days of 3 and 14 of sampling in 50% LC₅₀ AgNPs concentration, respectively (Table 4).

Table 4

Changes in glutathione peroxidase activity (U/mg prot) of *Portunus pelagicus* after exposure to 25, 50 and 75% LC₅₀ AgNPs
Tissue	Sampling day	Control	25% LC₅₀	50% LC₅₀	75% LC₅₀
Hepatopancreas	3	135.00 ± 16.24 ^a	140.13 ± 14. 25 ^a	138.22 ± 20.00 ^a	141.32 ± 19.28 ^a
	7	134.85 ± 18.20 ^a	138.13 ± 24.30 ^a	138.85 ± 16.26 ^a	143.62 ± 17.00 ^a
	14	138.45 ± 19.81 ^a	138.86 ± 10.15 ^a	144.75 ± 20.10 ^a	143.93 ± 10.74 ^a
Muscle	3	0.88 ± 0.05 ^a	0.81 ± 0.016 ^a	0.77 ± 0.03 ^a	0.84 ± 0.025 ^a
	7	0.90 ± 0.014 ^a	0.86 ± 0.014 ^a	0.83 ± 0.015 ^a	0.88 ± 0.02 ^a
	14	0.84 ± 0.01 ^a	0.80 ± 0.022 ^a	0.92 ± 0.028 ^a	0.91 ± 0.015 ^a

The superoxide dismutase activity was significantly reduced at 50% and 75% LC₅₀ of AgNPs compared to the control group in hepatopancreas and in 50% LC₅₀ of AgNPs in muscle (p < 0.05), so that the lowest level in both tissues was observed on day 14 (Table 5).

Table 5

Changes in superoxide dismutase activity (U/mg prot) of *Portunus pelagicus* after exposure to 25, 50 and 75% LC₅₀ AgNPs
Tissue	Sampling day	Control	25% LC₅₀	50% LC₅₀	75% LC₅₀
Hepatopancreas	3	73.00 ± 1.65 ^b	78.12 ± 4.35 ^b	28.75 ± 3.10 ^a	23.90 ± 5.44 ^a
	7	75.56 ± 8.00 ^b	74.47 ± 3.51 ^b	26.45 ± 0.99 ^a	30.95 ± 7.03 ^a
	14	78.12 ± 3.63 ^b	70.96 ± 4.54 ^b	22.75 ± 6.28 ^a	22.94 ± 2.89 ^a
Muscle	3	8.01 ± 0.67 ^b	7.91 ± 0.64 ^b	5.00 ± 0.45 ^a	10.03 ± 0.27 ^b
	7	8.35 ± 0.42 ^b	10.00 ± 9.61 ^b	4.86 ± 0.15 ^a	8.00 ± 0.32 ^b
	14	8.65 ± 0.64 ^b	8.00 ± 0.18 ^b	3.82 ± 0.28 ^a	7.99 ± 0.36 ^b

The level of total antioxidant capacity decreased significantly after exposure to AgNPs at a dose of 50% LC₅₀ in hepatopancreas (p < 0.05), and the lowest level was found on day 14 of sampling at this concentration (Table 6). In muscle, total antioxidant capacity level at three different concentrations of AgNPs did not show any significant differences when compared to control group (p > 0.05). The lowest level of total antioxidant capacity were obtained in 50% LC₅₀ AgNPs concentraion on day 14 of sampling (Table 6).

Table 6

Changes in total antioxidant capacity level (U/mg prot) of *Portunus pelagicus* after exposure to 25, 50 and 75% LC₅₀ AgNPs
Tissue	Sampling day	Control	25% LC₅₀	50% LC₅₀	75% LC₅₀
Hepatopancreas	3	5.50 ± 0.94 ^b	4.66 ± 0.99 ^b	2.53 ± 0.46 ^a	5.01 ± 0.25 ^b
	7	5.85 ± 0.35 ^b	4.90 ± 0.76 ^b	2.44 ± 0.83 ^a	4.00 ± 0.95 ^b
	14	6.02 ± 0.81 ^b	4.73 ± 0.80 ^b	1.95 ± 0.10 ^a	3.95 ± 0.99 ^b
Muscle	3	0.079 ± 0.010 ^a	0.080 ± 0.060 ^a	0.068 ± 0.007 ^a	0.067 ± 0.015^a
	7	0.075 ± 0.019 ^a	0.058 ± 0.013 ^a	0.073 ± 0.014 ^a	0.075 ± 0.015 ^a
	14	0.069 ± 0.012 ^a	0.079 ± 0.0040 ^a	0.056 ± 0.016 ^a	0.070 ± 0.010^a

The level of malondialdehyde increased significantly in hepatopancreas at 50% LC₅₀ of AgNPs concentraion compared to other tested concentration and control group (p < 0.05), so that its highest level were observed on day 14 of sampling (Table 7). In muscle, no significant differences were observed in MDA activity after AgNPs exposure treatments compared to control group (p > 0.05), however the highest level of malondialdehyde were obtained at a dose of 50% LC₅₀ of AgNPs concentraion on day 14 of sampling (Table 7).

Table 7

Changes in malondialdehyde activity of *Portunus pelagicus* after exposure to 25, 50 and 75% LC₅₀ AgNPs
Tissue	Sampling day	Control	25% LC₅₀	50% LC₅₀	75% LC₅₀
Hepatopancreas	3	0.32 ± 0.008 ^b	0.32 ± 0.012 ^b	1.75 ± 0.015 ^a	0.56 ± 0.005 ^b
	7	0.48 ± 0.014 ^b	0.60 ± 0.008 ^b	1.83 ± 0.016 ^a	0.80 ± 0.002 ^b
	14	0.49 ± 0.016 ^b	0.38 ± 0.0021 ^b	2.00 ± 0.087 ^a	0.93 ± 0.01 ^b
Muscle	3	0.059 ± 0.012 ^a	0.055 ± 0.070 ^a	0.096 ± 0.005 ^a	0.077 ± 0.015^a
	7	0.063 ± 0.018 ^a	0.060 ± 0.017 ^a	0.098 ± 0.018 ^a	0.085 ± 0.013 ^a
	14	0.049 ± 0.012 ^a	0.058 ± 0.0012 ^a	0.099 ± 0.021 ^a	0.091 ± 0.010^a

4.1. Determination of lethal concentration (LC₅₀) oint in acute aquatic toxicity tests.

Based on results the 96 h LC₅₀ value of AgNPs for Portunus pelagicus was 13.65 mg/L. According to the rules of the European Union (European commission 2008) AgNPs are classified as toxic to Portunus pelagicus. There are limit information regarding the acute toxicity of AgNPs for Portunus pelagicus is available. The lethal concentration of silver nanoparticles in Artemia sp, Paratya australiensis and Paratelphusa jacquemontii were determined to be 10.12, 0.77, 0.055 mg/L respectively (Rohit et al. 2018; Lekamage et al. 2018; Kadam and Raut 2019). In a study by Lam et al. (2020) the LC₅₀ values of the silver nanoparticles for Litopeaneus vannamei were calculated to be 91.2 and 35.5 mg/L during 24 and 48 h respectively. Based on the LC₅₀ values reported in different studies, different microorganisms also have different tolerance to silver toxicity (Bondarenko et al. 2013). These differences in results demonstrates that significant differences in NP toxicity to aquatic organisms exist and the test media and type of the species play a key role. The results showed that with increasing the exposure time and concentration, mortality rate was increased. Similarly in a study by Sayed and Soliman (2017) with increasing AgNPs concentration, the mortality increased remarkably in Clarias gariepinus compared with control group. After 72 h, mortality rates of crabs were greater than 50% for the all tested concentrations except in concentration of 3.16 mg/L AgNPs, which indicate that the mortality rate was directly proportional to the exposure time and concentration of AgNPs, because in addition to the concentration of AgNPs, exposure time is also directly related to its toxicity (Ivask et al. 2014).

4.2. Oxidative Stress indices

Oxidative stress is one of the cytotoxic effects of AgNPs that lead to activation of cellular antioxidant defense mechanisms that involve antioxidant enzymes, such as SOD, CAT, GPx and some other non-enzymatic antioxidants such as cellular glutathione (Xu et al. 2018). Catalase playing an important role in the cell redox equilibrium that breaks down hydrogen peroxide molecules to water and oxygen (Trasviña-Arenas et al. 2013). In the present study, exposure to 50% LC₅₀ of AgNPs caused a significant decrease in catalase activity of hepatopancreas and muscle on days 7 and 14 of sampling when compared to control group (p < 0.05). The decrease of catalase activity on both tissue that might be due to excess production of ROS, showing the direct consequence of AgNPs over oxidative stress (Choi et al. 2010). A similar response has been observed in Chapalichthys pardalis exposed to AgNPs by Valerio-García et al. (2017), who reported a diminution of catalase activity in muscle. These results are in accordance with previous studies in Oryzias latipes and Oreochromis niloticus (Wu and Zho 2013; Mansour et al. 2021). Bacchetta et al. (2017), reported that the catalase activity in Piaractus mesopotamicus after exposure to AgNPs were not statistically different from the control group (p > 0.05).

SOD as an important modulating oxidative responses, catalyze superoxide anion into oxygen and hydrogen peroxide (Valerio-García et al. 2017). SOD activities in both hepatopancreas and muscle significantly decreased in 50% LC₅₀ of AgNPs concentration on day 14 of sampling compared to control group (p < 0.05) and also decreased significantly after exposure at a dose of 75% LC₅₀ of AgNPs in hepatopancreas on day 14. In the present study, exposure to AgNPs probably produced an excessive formation of the superoxide anion, hydrogen peroxide and other oxygen radicals, which could have inhibited SOD activity followed by cellular and molecular damage caused by ROS formation in Portunus pelagicus. In contrast, Govindasamy and Rahuman (2012) reported that exposure to Ag-NPs, caused remarkable increase in SOD activities of different tissues in Oreochromis mossambicus. An et al. (2019) found that exposure to AgNPs decreased significantly SOD activity in Artemia salina. In contrast, Massarsky et al. (2013) observed that exposure to AgNPs in Danio rerio did not change catalase and SOD activities. Diminution of antioxidant enzymes level such as SOD and CAT may be related to high level of ROS such as hydrogen peroxide and molecular oxygen (Gottfredsen et al. 2013).

Glutathione peroxidases is the major source of protection against low levels of oxidative stress that catalyse the oxidation of glutathione (Kurutas 2015). Glutathione can act as a co-factor for several detoxifying enzymes that scavenge hydroxyl radical and singlet oxygen directly (Birk et al. 2013). In this study, no significant differences were observed in GSH and GPx activities of both hepatopancreas and muscle tissues between control and AgNPs exposure group (p > 0.05), however hepatopancreas GPx and GSH activities gradually increased with increased exposure time in 50% LC₅₀ and 75% LC₅₀ AgNPs concentration without reaching the statistical significance. The insignificant changes in GPx and GSH activity may be due to the fact that the organisms activate other antioxidant defenses or use existing enzymes to counteract the increased ROS after AgNPs exposure (Lekamge et al. 2019), also increase in hepatopancreas is probably due to the protective role of GPX against cells damage induced by oxyradical production and their defensive role against the effects of cellular oxidative stress (Tunçsoy et al. 2017 ). In agreement with this result Strużyński et al. (2014) reported that GSH level increased in the hepatopancreas of spinycheek crayfish, O. limosus after exposure to AgNPs. These results, however, differ from Khan et al. (2017) who reported that GPx and GSH level increased significantly in Labeo rohita after exposure to AgNPs. Massarsky et al. (2013) reported decreased glutathione content (GSH) in zebrafish embryos exposed to AgNPs. Findings of Valerio-García et al. (2017) showed significant reduction in the GPx activity of Chapalichthys pardalis liver and gills exposed to AgNPs, which inconsistent with present study.

Total antioxidant capacity indicates the activity of antioxidants against the free radicals produced in organisms, which is the sum of antioxidant enzymes (SOD, ASAFR, POD, CAT, GPx) as well as non-enzymatic antioxidants (Gu et al. 2020). In the present study, TAC levels in crabs hepatopancreas exposed to 50% LC₅₀ AgNPs concentration decreased significantly compared with other tested concentration and control group (p < 0.05), but no significant difference was observed in muscle (p > 0.05), this indicates that enzymatic and non-enzymatic antioxidants can be consumed in the hepatopancreas during exposure to AgNPs. A decreased in TAC level also can be attributed to excessive ROS production hepatopancreas than muscle in AgNPs exposed crab. Govindasamy and Rahuman (2012), reported a decrease in total antioxidant capacity in the gills and liver of tilapia exposed to silver nanoparticles, which is consistent with present study. Similarly, Long et al. (2021) reported significant reduction in TAC level of Litopenaeus vannamei hepatopancreas under the high salinity and ammonia-N exposure. According to previous studies in Danio Rerio and Gammarus fossarum no significant alterations in TAC level were observed after exposure to AgNPs (Bacchetta et al., 2016; Mehennaoui et al., 2016).

MDA is used as an indicative index of lipid peroxidation, which is considered an important endpoint measure for oxidative stress (Mensah et al. 2012). In the present study, MDA level in hepatopancreas increased significantly in concentration of 50% LC₅₀ AgNPs than other tested concentration and control group (p < 0.05) and the highest level was observed on day 14 of sampling which indicating that exposure to AgNPs at a dose of 50% LC₅₀ could influence the balance between oxidants and antioxidants in Portunus pelagicus and leading to the formation of MDA followed by induction of oxidative stress (Sayed and Soliman 2017). Previous studies also reported that after exposure to AgNPs, MDA level increased significantly in Drosophila melanogaster, Oryzias latipes, Tilapia zilli, Oreochromis niloticus (Wu and Zhou 2012; Afifi et al. 2016; Mansour et al. 2021). In muscle, although MDA values in all tested concentrations of silver nanoparticles had irregular increasing and decreasing trend, but showed no significant changes with the control group (p > 0.05). This observed pattern may be due to the formation of free radicals and the response of enzymatic and non-enzymatic antioxidants to eliminate ROS that has produced in muscle. Govindasamy and Rahuman (2012) reported that MDA contents in the liver, gill and skin of Oreochromis mossambicus exposure of 25 and 75 mg/L AgNPs were not significantly different compared with control group. Lekamge et al. (2019) found no observable effect on MDA level in freshwater shrimp, Paratya australiensis exposed to fresh and aged AgNPs except for aged C-AgNPs, which inconsistent with present study.

Based on the LC₅₀ value, silver nanoparticles are classified as toxic for Portunus pelagicus. Compared with the muscle, the hepatopancreas had higher antioxidant enzyme activities, so the hepatopancreas tissue plays an important role in the antioxidant defense of P. pelagicus. Significant reduction in catalase (CAT), superoxide dismutase (SOD) and TAC level in crabs exposed to AgNPs at concentration of 50% LC₅₀ reflected that the overall antioxidant ability crabs in hepatopancreas was impaired during AgNPs exposure as MDA level also showed significant increase at this tested concentration. Insignificant changes in GPx and GSH contents indicates limited role of glutathione-based antioxidant defense enzymes in reducing oxidative stress induced by AgNPs.

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Effect of Silver nanoparticles exposure on oxidative stress in blue swimmer crab (Portunus pelagicus)

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Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Crab supply and maintenance

2.2. Determination of lethal concentration

2.3. Sublethal toxicity

2.4. Oxidative stress indices

2.5. Statistical Analysis

3. Results

3.1. LC₅₀ determination

3.2. Results of oxidative stress indices

4. Discussion

4.1. Determination of lethal concentration (LC₅₀) oint in acute aquatic toxicity tests.

4.2. Oxidative Stress indices

5. Conclusion

References

Additional Declarations

Status:

Version 1

Effect of Silver nanoparticles exposure on oxidative stress in blue swimmer crab (Portunus pelagicus)

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Crab supply and maintenance

2.2. Determination of lethal concentration

2.3. Sublethal toxicity

2.4. Oxidative stress indices

2.5. Statistical Analysis

3. Results

3.1. LC50 determination

3.2. Results of oxidative stress indices

4. Discussion

4.1. Determination of lethal concentration (LC50) oint in acute aquatic toxicity tests.

4.2. Oxidative Stress indices

5. Conclusion

References

Additional Declarations

Status:

Version 1

3.1. LC₅₀ determination

4.1. Determination of lethal concentration (LC₅₀) oint in acute aquatic toxicity tests.