Neurobehavioral, physiological and inflammatory impairments in response to bifenthrin intoxication in Oreochromis niloticus fish: Role of dietary supplementation with Petroselinum crispum essential oil

Journal Pre-proof Neurobehavioral, physiological and inflammatory impairments in response to bifenthrin intoxication in Oreochromis niloticus fish: Role of dietary supplementation with Petroselinum crispum essential oil M.R. Farag, H.K. Mahmoud, Sabry A.A. El-Sayed, Sarah Y.A. Ahmed, M. Alagawany, Shimaa M. Abou-Zeid

PII:

S0166-445X(20)30464-1

DOI:

https://doi.org/10.1016/j.aquatox.2020.105715

Reference:

AQTOX 105715

To appear in:

Aquatic Toxicology

Received Date:

21 October 2020

Revised Date:

30 November 2020

Accepted Date:

2 December 2020

Please cite this article as: Farag MR, Mahmoud HK, El-Sayed SAA, Ahmed SYA, Alagawany M, Abou-Zeid SM, Neurobehavioral, physiological and inflammatory impairments in response to bifenthrin intoxication in Oreochromis niloticus fish: Role of dietary supplementation with Petroselinum crispum essential oil, Aquatic Toxicology (2020), doi: https://doi.org/10.1016/j.aquatox.2020.105715

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Neurobehavioral, physiological and inflammatory impairments in response to bifenthrin intoxication in Oreochromis niloticus fish: Role of dietary supplementation with Petroselinum crispum essential oil

M.R. Farag1*, H.K. Mahmoud2, Sabry A.A. El-Sayed3, Sarah Y.A. Ahmed4 , M. Alagawany5 *, Shimaa

Forensic Medicine and Toxicology Department, Veterinary Medicine Faculty, Zagazig University,

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M. Abou-Zeid6

Zagazig 44519, Egypt; 2Animal Production Department of, Faculty of Agriculture, Zagazig

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University, Zagazig, Egypt; 4Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt; 5Microbiology Department , Veterinary Medicine

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Faculty, Zagazig University; 5Poultry Department, Faculty of Agriculture, Zagazig University,

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Zagazig 44511, Egypt; 6Forensic Medicine and Toxicology Department, Faculty of Veterinary Medicine, University of Sadat City, 32897, Egypt

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Running title: Bifenthrin toxicity and parsley supplementation in fish *Corresponding authors: [email protected] (M. Farag), [email protected]

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(M. Alagawany)

HIGHLIGHTS  

Bifenthrin (BF) insecticide is one of the most frequently recognized SPs in surface waters and sediments of many water resources Bifenthrin has been reported to be highly toxic, as it exhibited hepatotoxic, immunotoxic and tumorigenic impacts

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Using of natural substances is widely employed to mitigate these undesirable impacts of pesticides Co-supplementation of parsley oil with bifenthrin alleviated the BF-induced neurotoxic insult

Abstract

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This study was conceptualized in order to assess the 96-h LC50 of bifenthrin (BF) in O. niloticus and also to measure the biochemical, behavioral, and molecular responses of the fish suchronically

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exposed to a sub-lethal concentration of the insecticide. The role of Petroselinum crispum essential oil (PEO) supplementation in mitigating the resulted neurotoxic insult was also investigated. The

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acute toxicity study revealed that the 96-h LC50 of BF is 6.81 µg/L, and varying degrees of

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behavioral changes were recorded in a dose-dependent manner. The subchronic study revealed reduction of dissolved oxygen and increased ammonia in aquaria of BF-exposed fish. Clinical signs

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revealed high degree of discomfort and aggressiveness together with reductions in survival rate and body weight gain. The levels of monoamines in brain, and GABA and amino acids in serum were

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reduced, together with decreased activities of Na+/K+-ATPase and acetylcholine esterases (AchE). The activities of antioxidant enzymes were also diminshed in the brain while oxdative damage and DNA breaks were elevated. Myeloperoxidase (MPO) activity in serum increased with overexpression of the pro-inflammatory cytokines in the brain tissue. BF also upregulated the

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expression of brain-stress related genes HSP70, Caspase-3 and P53. Supplemention of PEO to BF markedly abrogated the toxic impacts of the insecticide, specially at the high level . These findings demonstrate neuroprotective, antioxidant, genoprotective, anti-inflammatory and antiapoptic effects of PEO in BF-intoxicated fish. Based on these mechanistic insights of PEO, we recommend its use as an invaluable supplement in the fish feed.

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Key words: Oreochromis niloticus; Bifenthrin; Petroselinum crispum; Neurobehavioral toxicity. 1. Introduction The human and animal toxicity by organophosphorus insecticides makes them a societal health and environmental concern; hence the environmental protection agency (EPA) constrained most residential uses of them in 2001. Bifenthrin (BF) insecticide is one of the most frequently

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recognized synthetic pyrethroids (SPs) in surface waters and sediments of many water resources, where fish and aquatic invertebrates are continuously exposed to different toxicants at potentially

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hazardous levels (Zhang et al., 2020). The estimated level of BF in the water and sediment are over

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100 ng/L, and 135 ng/g dry weight, in southern rivers of China (Zheng et al., 2016), and 0.03 - 0.08 mg/L and 0.11 - 0.18 mg/kg, respectively, in the Challawa river in Nigeria (Akan et al., 2014). In

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addition, the runoff of residential areas was reported to be 0.12–6.12 μg/L (Beggel et al., 2011). The measured concentrations of BF in the water of Sacramento-San Joaquin Delta have reached up to

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5.3 μg/L; such concentration is higher than the levels (≤1.5 μg/L) found to induce olfactory and neuroendocrine alterations in juvenile Chinook (Giroux et al., 2019). Unlike other SPs, BF elicits

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higher toxicity, as it relatively persists in the soil, easily binds to the organic matter and flows into the aquatic environments prompting ecological hazards and adverse effects on the aquatic organisms (Manzoor and Pervez, 2017).

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BF is used to control termites, cockroaches, mosquitos, and flies by interfering with the ATPase enzymes and voltage-gated ion channels leading to convulsions, tremors, hyperexcitability, and ultimately death .Moreover, neurotoxic effects in mammals' brain and apoptotic and inflammatory events in murine brain were recorded as a result of BF exposures (Syed et al., 2018; Gargouri et al., 2018). However, the neurotoxicity of BF in fish has been less studied.

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Bifenthrin has been reported to be highly toxic, as it exhibited hepatotoxic, immunotoxic and tumorigenic impacts through oxidative stress mechanisms in mammalian species and it exerted an endocrine-disrupting effect in murine and several fish species (Xiang et al., 2019). Recent findings stated that BF at sublethal concentrations could induce developmental vascular malformation and immunotoxicity during zebrafish embryogenesis (Park et al., 2020), and

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alter the behavioral responses in fathead minnow (Pimephales promelas) larva, chinook salmon (Oncorhynchus tshawytscha) and adult Corbicula fluminea (Beggel et al., 2011; Giroux et al., 2019;

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Zhang et al., 2020). Xiang et al. (2019) found that the parental exposure of zebrafish to cis-BF

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induced neurotoxic impacts and growth retardation in unexposed offspring. Additionally, BF caused changes in detoxification process, antioxidant status, metabolism, and histopathological

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characteristics in Xenopus laevis and Corbicula fluminea (Zhang et al., 2019, 2020). In the Mediterranean countries, aromatic plants including thyme, parsley, lavender, or

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peppermint are widely used for multiple purposes, like ornamental cropping, flavoring agents in food, and fragrance in cosmetics and perfumes (Alagawany et al., 2020a,b,c,d,e; Alagawany et al.,

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2021). Petroselinum crispum, commonly known as parsley, is belonging to Umbelliferae family. It originated in the Mediterranean region and is currently cultivated worldwide. Oil extracted from leaves has a flavor similar to that of the fresh herb. There is a blend of active compounds that have

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been extracted from parsley including phenolics, flavonoids especially apiin, apigenin and 6"Acetylapiin, and essential oil components mainly myristicin , apiol and

coumarins which

responsible for antioxidant effects of the oil (Farzaei, et al., 2013). Wong and Kitts (2006) found that parsley essential oil (PEO) exhibited significant free radical scavenging activity in vitro. Parsley was found to exhibit many pharmacological activities

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including cytoprotective, cardioprotective, hepatoprotective, nephroprotective, neuroprotective, anti-diabetic, spasmolytic, diuretic , antibacterial and antifungal properties (Abdellatief et al., 2017). This study was appointed to assess the median lethal concentration (96h-LC50) of BF in O. niloticus. It also aimed to investigate the possible role of oxidative stress in BF-induced neurotoxic effects and to state the influence of the dietary supplementation of PEO to O. niloticus fish

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concurrently exposed to sublethal concentration of BF. This was done by evaluating the growth performance, physiological, behavioral, and neurological endpoints and the mRNA expression of

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stress and inflammatory cytokines-encoding genes.

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2. Materials and methods 2.1. Tested compound and plant

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Bifenthrin (BF) PESTANAL®, analytical standard, [C23H22ClF3O2; 3-[(1Z)-2-Chloro-3,3,3-

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trifluoro-1-propenyl]-2,2-dimethylcyclopropanecarboxylic acid (2-methylbiphenyl-3-yl)methyl ester], CAS Number 99267-18-2], is purchased from Sigma-Aldrich International GmbH (St. Louis,

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MO, USA). The fresh Petroselinum crispum plant was purchased from a local vegetables market in Zagazig city, Egypt.

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2.2. Extraction of PEO

Fresh parsley leaves were cut into small pieces and subjected to hydrodistillation according to the method of Farouk et al. (2017) using a Clevenger-type apparatus (El-Gomhouria Company, Zagazig, Egypt) for 3h. After extraction, the oil was dried by anhydrous sodium sulfate and kept in air-tight glass vials wrapped in aluminum foil and stored until analysis at –20°C. 2.3.Gas chromatography–mass spectrometry (GC–MS) analysis of PEO bioactive constituents 5

The PEO components were characterized using a GC–MS analysis (Trace GC Ultra Chromatography system model; Thermo Scientific, USA) equipped with an ISQ-mass spectrometer with a 60 m × 0.25 mm × 0.25-μm-thick TG-5MS capillary column (Thermo Scientific, USA) following the method of (Badee et al., 2020). 2.4.

Experimental Fish and tested diets formulation

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440 O. niloticus fish (10-12 g BW) were allocated to the two experiments (Acute toxicity and

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antidotal studies). They were obtained in a good health condition from El-Abbassa Fish Hatchery, Al-Sharkia, Egypt. They were subjected to 14 days acclimatization period (in glass aquaria, filled

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with dechlorinated tap water). They fed on a formulated basal diet at a rate of 5% of the fish biomass, 3 times daily, without PEO or BF. The parameters of water quality as recommended by

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the American Public Health Association (APHA, 1998) were followed. The same rearing conditions

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including pH, temperature, dissolved oxygen, and ammonia with a 10 h light: 14 h dark controlled photoperiod, were adjusted in all aquaria.

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The extracted PEO was supplemented to the ingredients of the basal diet (Table 1), at the rate of 1 mL or 2 mL/kg , and then all dietary ingredients were mechanically mixed, pelletized and subjected to air for 24 h at 25 °C to dry thoroughly, and kept at 4 °C until be used . Acute toxicity study (Median lethal concentration; 96-h LC50)

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2.5.

Eighty O. niloticus fingerlings were randomly assigned to eight groups with ten fish per

each. A control group was kept in dechlorinated water and the other seven groups were subjected to different levels of BF (0.5, 1, 2, 4, 6, 8 and 10 µg/L). Throughout the investigation period (96 h), the contaminated water of the glass aquaria was not changed and the fish did not receive feed. The resultant mortalities were recorded at 24, 48, 72, and 96 h, and the dead fish were rapidly discarded.

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Live fish in each group were monitored daily (at the same time, 8:00 a.m.) for recording of any behavioral alterations. The 96-h LC50 value of BF was calculated by Finney's probit method with 95% confidence limits (Finney, 1971). 2.6.Antidotal study: Role of PEO in attenuating the neurotoxicity of BF 360 O. niloticus fish were distributed into six equal groups (n=60 fish/group); each group with 3

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replicates (20 fish/replicate). Each replicate was kept in 100 × 50 × 40 cm-sized glass aquaria with 160 L of dechlorinated tap water capacity. The 1st group (control) was kept on clean water glass

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aquaria and fed on a basal diet only. The 2nd and 3rd groups were fed on basal diet supplemented

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with PEO at level of 1 mL or 2 mL/kg diet (PEO1 and PEO2), respectively. The 4th group of fish (BF) was fed on a basal diet and exposed to 1/10th LC50 of BF (0.68 μg/L). The 5th and 6th groups

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(PEO1/BF and PEO2/BF) were fed on a basal diet supplemented with PEO at levels of 1 mL and 2 mL/kg diet, respectively, and exposed to 1/10th LC50 of BF. Feed was introduced to fish three times

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daily (7:00 a.m., 11:00 a.m., and 4:00 p.m.) throughout the experiment (60 days) and feed intake was adjusted every 2 weeks as per the fish weight gain. All the experimental procedures were

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approved by the Institutional Animal Care and Use Committee of Zagazig University (ZU-IAURC). 2.6.1. Water quality parameter

Water quality was consistently measured twice weekly throughout the experiment using portable

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digital instruments for estimating the water temperature, dissolved oxygen (DO), and pH levels (Martini Instruments Model 201/digital), whereas total ammonia concentration was assessed calorimetrically.

2.6.2. Survivability and growth of O. niloticus fish

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The daily mortality was recorded to estimate the survivability percentage as follow: Survivability (%) = (final fish number/initial fish number) × 100. At the end of the experiment, final body weight (FBW) of the fish was recorded for the calculation of weight gain (WG) in each experimental group [Weight gain (g/fish) = (Wf)–(Wi)]. The SGR was calculated as follow: (SGR) (%/day) = 100 × [(ln Wf–ln Wi)/T]. Where ‘Wf' is a final weight

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(g), ‘Wi’ is an initial weight (g), and ‘T' is the experimental duration (days).

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2.6.3. Behavioral pattern in O. niloticus fish

Comfort and aggressive behaviors were monitored in each aquarium in all experimental groups in

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the last week of the experiment (one observation/day). All observations were conducted in constant

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time (7:00- 9:00 a.m.) and appropriately blinded by one observer.

Comfort behaviors included, chafing or scratching, resting, surfacing, schooling and

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eliminative behavior. Moreover, the aggressive behavior was monitored including chasing, fin tugging (biting) and mouth pushing as described by El-Hawarry et al. (2018)

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2.6.4.Blood and brain tissue sampling

On the last experimental day, blood were collected from the caudal veins by sterile syringes without anticoagulant, and then centrifuged at 1075 ×g for 20 min to obtain serum. Sera were utilized to

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measure the level of the inflammatory biomarker MPO and amino acid neurotransmitters. Spinal cord section was applied to sacrifice fish, and then the brain specimens were dissected. Brain samples were kept at −20°C, till the determination of oxidant/antioxidant biomarkers, and another set of samples were quickly frozen in liquid nitrogen and then stored at −80 °C until the total RNA extraction. 2.6.5.Antioxidant status, oxidative injury assays and MPO 8

Brain samples were homogenized to estimate antioxidants including the activity of CAT and SOD and the GSH, MDA, protein carbonyl (PC) and MPO levels via commercial kits, following the manufacturer's protocols. The levels of oxidative DNA damages in the brain tissues were determined by comet assay following the method affirmed by Singh et al. (1988).

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2.6.6. Brain monoamine neurotransmitters, GABA, and serum amino acids

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Monoamines (dopamine; DA, serotonin; 5-HT and norepinephrine; NE) and γ-aminobutyric acid (GABA) were evaluated in brain homogenate by reverse-phase high-performance liquid

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chromatography equipped with electrochemical detector (HPLC-ECD) using a C-18 column. All

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other characters and conditions applied were the same as mentioned in Khalil and Hussein (2015). The serum amino acid neurotransmitters (aspartate , glycine and glutamine) levels were

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estimated via the fluorescence detection of o-phthaladehyde/2- mercaptoethanol (OPA/MCE) derivatives of the analytes then a gradient elution reversed phase HPLC (Pre-column derivatization

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and reversed phase HPLC) were used for and their separation (Khalil and Hussein,2015). 2.6.7. AChE and Na+/K+-ATPase enzyme activities in the brain tissues The AChE activity was evaluated spectrophotometrically at 412 nm according to Ellman et al.

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(1961). The activity of Na+/K+-ATPase was measured according to Svobaca and Mossinger (1981). 2.6.8. Transcriptional analysis of stress and inflammatory cytokines -encoding genes in the brain tissue

Frozen brain specimens were subjected to total RNA extraction using TRIzol reagent (easyREDTM, iNtRON Biotechnology, Korea). Quantitect® Reverse Transcription kit (Qiagen, Germany) was used for the synthesis of the first-strand cDNA from the extracted RNA. The RNA 9

extraction and cDNA synthesis were performed following the kits prtotocol. The forward and reverse sequences of primers of the studied genes are as follows: the primers for tumor suppressor protein (P53) were F: 5- GCATGTGGCTGATGTTGTTC-3 and R : 5GCAGGATGGTGGTCATCTCT-3 , for Caspase-3 were F: 5- GGCTCTTCGTCTGCTTCTGT-3 ad R: 5- GGGAAATCGAGGCGGTATCT-3 , for heat shock protein-70 (HSP-70) were F: 5-

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CTCCACCCGAATCCCCAAAA-3 and R: 5- TCGATACCCAGGGACAGAGG-3 , for interleukin 1β (IL-1β) were F: 5- CAAGGATGACGACAAGCCAACC-3 and R: 5-

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AGCGGACAGACATGAGAGTGC-3, for interferon - γ (IFN-γ) were F: 5-

AAGAATCGCAGCTCTGCACCAT-3 and R: 5- GTGTCGTATTGCTGTGGCTTCC-3 and for

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tumor necrosis factor - α (TNF-α) were F: 5- GGAAGCAGCTCCACTCTGATGA-3 and R: 5-

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CACAGCGTGTCTCCTTCGTTCA-3 in addition to the housekeeping gene β-actin with the primers F: 5- CAGCAAGCAGGAGTACGATGAG-3 and R: 5-

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TGTGTGGTGTGTGGTTGTTTTG-3 . The Rotor-Gene Q instrument with a QuantiTect® SYBR® Green PCR kit (Qiagen, Germany) was used for performing the qPCR analysis under the

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following thermocycler condition: 10 min at 95 °C , followed by 40 cycles of 95 °C for 15s and 60 °C for 03s and 72 °C for 03s. Melt-curve analysis was performed to verify the specificity of PCR. The relative mRNA expression pattern for each gene was calculated using the comparative 2-ΔΔCt

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method approved by Livak and Schmittgen ( 2001). 2.7. Statistical analysis

Data were statistically analyzed by one-way ANOVA, using SPSS. Tukey's multiple comparisons post hoc test was applied to compare means among groups, where the statistical significance was approved at p < 0.05. The analyzed data were expressed as means ± SE. 3. Results 10

3.1. Gas chromatography-Mass spectrometry (GC-MS) analysis of PEO components A total of 24 active constituents were detected in the PEO by GC-MS analysis with different retention times, molecular weights, and area % (Table 2, Fig. 1). The main identified compounds were limonene (18.82%), oleic Acid (14.52%), á-cyclocitral (11.75%), and globulol (11.24%) followed by 2á,8á-(Dimethoxy)-3,5,7-trioxatetracyclo[7.2.1.0(4,11).0(6,10)] dodecane (8.51%), á-

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selinene (7.78%), à-guaiene (7.34%), and apiol (5.45%).

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3.2. Estimated 96-h LC50 value and behavioral response in BF-exposed fish

The control group showed neither mortalities nor abnormal behaviors during the 96h study

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duration. The BF exposed groups showed mortalities in a dose-dependent manner. The estimated 96-h LC50 of BF was determined to be 6.81 µg/L with lower and upper confidence limits of 5.32

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and 8.48 µg/L, respectively( Fig. 2). Varying degrees of behavioral changes were recorded either in

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live or dead fish before death, in a dose dependent manner. Air-gulping and respiratory distress were observed in fish at a minimum concentration of 2 µg/L, while other disorders including

(Table 3).

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uncoordinated swimming, sluggish movement, and hyperventilation, were recorded at ≥4 µg/L

3.3. Role of PEO in attenuating the neurotoxic impacts of BF (antidotal study)

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3.3.1. Water quality parameters

No changes were recorded in temperature and pH in all aquaria. BF significantly decreased the levels of DO while increased the ammonia levels compared to control. In the PEO1/BF and PEO2/BF groups, the diminished DO level was modulated to control value. Moreover, the elevated level of ammonia was also decreased in both groups, but still higher than control (Table 4). 3.3.2. Behavioral impairments 11

Fish exposed to BF (1/10th LC50; 0.68 µg/L) for 60 days, showed a high degree of discomfort represented by decreases in the monitored chafing, resting, surfacing, elimination and schooling behaviors. Moreover, such fish exhibited a high degree of aggressiveness act, reflected by increasing chasing, fin tugging and mouth push, relative to control. PEO supplementation to BF groups modulated the comfort behavior indices, and reduced the level of the aggressiveness compared with the BF group particularly at 2 mL/kg diet which restored the elimination, schooling,

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3.3.3. Survival percentage and growth performance

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mouth push, fin tugging, and total aggressive behaviors to normal (Table 5).

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BF significantly reduced the survival rate, while PEO, at two dose levels with BF increased the survivability percentage among fish to normal levels. In the PEO1 and PEO2- supplemented groups,

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significant increases were recorded in FBW and WG, while specific growth rate (SGR) did not significantly increase compared to control. Sublethal exposure to BF significantly reduced WG and

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FBW, while a non-significant decrease of SGR was noted, compared to control. A significant improvement of the previously mentioned indices was observed in PEO2/BF, compared with BF,

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but not in PEO1/BF group (Table 6). 3.3.4. Antioxidant status variables

The SOD and CAT activities were significantly lowered in BF group, compared to control.

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While, the co-administration of both PEO levels with BF significantly improved their activities, and restored CAT activity to control value. While the GSH level did not significantly differ among all studied groups (Table 6). 3.3.5. Oxidative stress and DNA damage variables

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The brain MDA and PC contents showed non-significant differences in the two PEO-supplemented groups relative to control. On contrary, these contents were significantly higher in BF group, compared to the control. MDA content was significantly diminished in both PEO1/BF and PEO2/BF groups to the control level. Moreover, the PC level was improved to the control value in PEO2/BF, but still significantly higher in PEO1/BF group (Table 6).

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A significant increase in the comet variables was found in BF group compared to control. On the other hand, both PEO1/BF and PEO2/BF showed a significant improvement in all indices of

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DNA damage compared to BF group, particularly PEO2/BF. However, most indices were still

3.3.6. Inflammatory response marker (MPO)

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higher than control, except tail DNA % that did not significantly differ than control (Fig. 3).

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MPO activity showed no significant difference in the two PEO-supplemented groups, compared

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with control. Its activity showed a significant enhancement in BF-exposed group, while was significantly improved in both PEO1/BF and PEO2/BF groups. The control value was attained in

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PEO2/BF group, but not in PEO1/BF group (Table 6). 3.3.7. Monoamine neurotransmitters, GABA and amino acids levels A significant decline was observed in the monoamine indices in BF group, compared to control. A significant modulation was recorded in PEO1/BF and PEO2/BF groups. PEO1/BF revealed a

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significant improvement in the 5-HT and NE concentrations to be restored to control, but did notsignificantly affect the DA value. PEO2/BF restored these indices to the control value. The GABA concentration was decreased in the BF group than control. GABA level was significantly improved in PEO1/BF and PEO2/BF but still under control value (Fig. 4).

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The levels of amino acid neurotransmitters significantly declined in BF group compared to control. PEO significantly modulated the levels of these neurotransmitters. The glutamine was restored to the control level but not for aspartate. On the other hand, the level of glycine was significantly modulated in PEO2/BF group, but not restored to control. 3.3.8. AChE and Na+/K+-ATPase enzymes activity

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The Na+/K+-ATPase and AChE activities were significantly decreased in the BF-exposed group. Upon the PEO co-supplementation with BF, an obvious restoration was found. The improved

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activities were found to be within normal value in PEO2/BF group, but still significantly differ in

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PEO1/BF group (Fig. 5).

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3.3.9. Effects on stress-related genes and pro-inflammatory cytokines BF elicited significant upregulations of P53, Caspase-3 and HSP70 mRNA expressions compared to

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control. Supplementation of BF-intoxicated fish with PEO improved the upregulated pattern of such genes particularly at 2 mL/kg, whereas P53 and HSP70 expression was modulated to control values but

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not for Caspase-3.

The same trend was observed for pro-inflammatory cytokines, where a significant upregulation was found in response to BF, compared with control. On contrast, in PEO1/BF and PEO2/BF groups , significant modulations of the expressions of TNF-α and IFN-γ was observed and control

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values were attained, but not for IL1β (Fig. 6). 4. Discussion

Fish are aquatic organisms enduring prime hazard from the constant exposure to a wide variety of xenobiotics released into their environment. The 96-h LC50 is estimated for the accurate evaluation of acute toxicity of various contaminants. Herein, the probit analysis showed the 96-h 14

LC50 of BF in O. niloticus fish to be 6.81 µg/L. Several acute toxicity datasets in different fish species have showed the 96-h LC50 value to be 2.08, 1.47, and 0.26 μg/L in common carp, rainbow trout, and fathead minnow larva, respectively (Liu et al., 2005; Werner and Moran, 2008; Velisek et al., 2009). The variation could be attributed to the species difference, water quality, kinetic parameters, age, size and experimental conditions.

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Our data demonstrated improved survivability among BF-intoxicated fish co-supplemented with PEO to reach control values. Similarly, El-Barbary and Mehrim (2009) reported that parsley reduced

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the mortalities in O. niloticus exposed to aflatoxins. Mooraki et al. (2014) reported that Cyprinus carpio

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fed on 0.5% parsley for 60 days showed increased survival rate. This could be attributed to the various protective influences of PEO including antioxidant, anti-inflammatory, antiapoptic and neuroprotective

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effects.

The present study showed behavioral, physiological and transcriptomic alterations in O. niloticus

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subchronically exposed to BF with poor growth performance. This could be returned to the toxicity stress which could induce changes in metabolism of carbohydrate and protein resulting in low available

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energy required for growth and increasing the mortalities. Additionally, in this study, ammonia tended to accumulate in the aquaria with BF and decreased the fish survival. Similarly, growth inhibition has been reported upon exposure to other pyrethroids (Werner and

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Moran, 2008; Khalil et al., 2020). While, pure BF or its formulation Talstar did not affect the growth rate of larval fathead minnow (Beggel et al., 2010). This variation from our results may be due to the difference in experiment duration (7 days) and the stage of fish development. Our data demonstrated that PEO2 improved the FBW and WG in BF-intoxicated O. niloticus. Similarly, Mooraki et al. (2014) reported that parsley could enhance growth and feed conversion of Cyprinus carpio. Parsley juice administered to pregnant mice ameliorated the 15

cadmium toxicity on neonatal BW by the 3rd week of age (Allam et al., 2016). This growth stimulant effect of PEO may be attributed to improvement of antioxidant status and enhancement of detoxication and elimination of BF from intoxicated fish. In addition, parsley was reported to have spasmolytic, gastroprotective, antibacterial and antifungal activities (Farzaei et al., 2013) which may improve growth through modulation of gastrointestinal microbiota and improvement of food

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digestion and utilization. The behavioral alterations by BF were displayed in a dose dependent manner and this

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agreed with Velisek et al. (2009) and Jin et al. (2009) . These clinical signs were reported with other

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pyrethroids (Khalil et al., 2020).

The respiratory distress observed in BF-exposed fish may be due to disturbance of

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respiratory and osmoregulation processes resulting from the structural alterations in gills and excessive mucus secretion. It may be also due to the effects of BF on the DO content in the test

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aquaria, where the exposed fish try to compensate the diminished oxygen via hyperventilation. The altered swimming activity could result from BF inhibitory effect on AChE in all brain regions

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(Syed et al., 2018). Accumulation of ACh at the synaptic cleft changes the activity and locomotor behavior of fish and disrupts the nervous-muscular synchronization (Pamanji et al., 2016). This may decline the fish swimming performance and affect their ability to forage, migrate, avoid predation,

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and successfully reproduce. The present findings suggest a close relation between these behaviors and the recorded declines in monoamine neurotransmitters, GABA and amino acids and the activity of Na+/K+-ATPase in the brain and serum of intoxicated fish as they elicit important roles in these processes (Ullah et al., 2018). Herein, O. niloticus exhibited changes in their behaviors in all aquaria in response to BF. These impairments were reflected as a decrease of comfort behaviors and an elevation of the 16

aggressive behaviors. PEO co-supplemented with BF improved the comfort behavior indicators, and reduced the level of aggressiveness in a dose-dependent manner may be due to the recovery of brain biochemical variables by PEO. In addition, the reduced aggression may result from the effect of volatile constituents in the oil on serotonin reuptake, and thereby slowing the time that 5-HT is accessible for neurotransmission . The neuroprotective effect of parsley was previously reported in newborn mice from cadmium- intoxicated dams (Allam et al., 2016). Salahshoor et al. (2020)

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showed that parsley extract improved the reduced number of neurons and dendritic thorns in

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prefrontal cortex of morphine-intoxicated rats, this may be attributed to the control of

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neurodegeneration and apoptosis by the plant extract.

Furthermore, our data revealed that BF induced oxidative injury in the brain of O. niloticus. The

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inhibited SOD and CAT activities in the exposed fish reflected a deficiency in the defense against ROS, which may lead to superoxide radical formation and H2O2 accumulation resulting in peroxidation of the

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polyunsaturated fatty acids of cellular membranes, protein oxidation and initiation of DNA damage. This is supported by the increased MDA, enhanced formation of PC, and elevated DNA damage

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indices in the BF-exposed fish suggesting that BF could disrupt the antioxidants expression and reduce the antioxidant capacities. Pyrethroids have been documented to cause oxidative damages in various organs of aquatic organisms (Khalil et al., 2020).

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The enhancement of antioxidant enzymes activity is substantial as buffer in the detoxication and interception of ROS and other free radicals. Herein PEO markedly offset the BF-provoked oxidative injury particularly at high level. This reflects the protective potential of PEO against oxidative-mediated damage which may be due to the high antioxidant activity of its components, mainly flavonoids, phenolics and carotenoids (Abu-Serie et al., 2019). Similarly, parsley juice or extract elicited antioxidant effect in brain of newborn mice exposed in utero to cadmium (Allam et 17

al., 2016) and in D-galactose-intoxicated mice (Vora et al., 2009). The antioxidant effect of PEO or aqueous extract was demonstrated in liver and heart of cisplatin-intoxicated rats (Abdellatief et al., 2017) and CCl4 -treated mammalian kidney (Vero) cells (Abu-Serie et al., 2019). The BF-genotoxicity was markedly observed in the brain of BF-exposed fish, evidenced by a noteworthy increase in the comet endpoints. The genotoxic potential of other pyrethroids in fish was

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previously documented (Ullah et al., 2018). PEO ameliorated the DNA oxidative damage in BFexposed fish, probably due to the antioxidant and free radical scavenging potential of the oil. Hassan

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and Abdel-Wahhab (2006) reported that parsley seed oil diminished the chromosomal aberrations in

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testis of zearalenone-treated mice. Tang et al. (2015) observed that the parsley extract protected mouse fibroblasts against H2O2-induced DNA damage.

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The oxidative stress is defined as the existence of metabolic and radical constituents or socalled reactive (chlorine, oxygen or nitrogen) species. The HSP-70 expression was up-regulated in BF-

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exposed fish as a mechanism of adaptation against environmental stressor. This upregulation indicates the improvements in the intracellular buffer systems, which protect against oxidative damage (Elnesr et

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al., 2019; Elwan et al., 2019).

The transcriptomic data revealed upregulation of P53 and Caspase-3 expressions in BF-exposed fish as a response to cellular stress. BF induced overexpression of Caspase-3 in rat brain (Gargouri et

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al., 2019), up-regulated the expression of Bax protein and down-regulated BCL-2 in human hepatocarcinoma cells (Liu and Li, 2015) and upregulated p53 and caspase-3, with downregulated BCL-2 in RAW 264.7 cells (Wang et al., 2017). Supplementation of PEO to BF-intoxicated fish ameliorated the upregulated pattern of HSP70, Caspase-3 and P53 genes, especially at 2ml/kg. The antiapoptotic activity of parsley was shown in hepatocytes and cardiomyocytes of cisplatin-intoxicated rats (Abdellatief et al., 2017), testis of 2,3,7,8 18

Tetrachlorodibenzo-p-dioxin (TCDD, dioxin)-treated rats (Edrees et al., 2015) and in heart of diabetic rats (Soliman et al., 2015). Herein, BF induced oxidative injury in the brain, which facilitated or triggered secondary inflammatory events. This was indicated by the recorded up-regulation of IL-1β, IFN-γ and TNF-α expression. Neuroinflammation were previously demonstrated in murine brain after BF treatment

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(Gargouri et al., 2018). The inflammatory cytokines are produced in the brain from microglia and astrocytes

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together with other inflammation mediators such as NO and prostaglandin. Furthermore, our

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findings showed that BF significantly induced serum MPO activity, whereas it released into the extracellular spaces during the inflammatory processes when neutrophils are activated. It acts as a

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catalyst in the hypochlorous acid synthesis, which is considered as a toxic agent to the different

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cellular components and consequently could intensify the oxidative damage (Arnhold et al., 2001). Upon concurrent PEO supplementation with BF-exposure, significant modulations in the

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expression of IFN-γ, IL-1β and TNF-α gene and in the serum MPO were recorded. The antiinflammatory potential of PEO was reported by Abdellatief et al. (2017) and Abu-Serie et al. (2019). The PEO anti-inflammatory effect originates from polyphenols, flavonoids and carotenoids, as shown in our GC-MS analysis. These components were previously shown to suppress

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inflammatory responses (Hussain et al., 2016). 5. Conclusion

The current investigation demonstrated that BF, at sublethal concentration, induced neurobehavioral deficits in O. niloticus accompanied with alterations in the brain levels of monoamines and serum amino acid neurotransmitters, with suppression of AChE and Na+/K+-ATPase activities. Brain

19

oxidative stress with consequent inflammatory response and modulation of the expression of stress - encoding genes are involved in eliciting these neurotoxic effects. Notably, co-supplementation of PEO with BF alleviated the BF-induced neurotoxic insult, especially at the highest dose applied (2 mL/kg diet). This was evidenced by improving the neurobehavioral variables, survival and growth rates, oxidative stress biomarkers, antioxidant status, neurochemistry, expressions of inflammation and stress genes. Our data collectively recommend supplementation of fish diet with PEO to

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enhance survival and BWG and to minimize any environmental toxic impacts on the fish.

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Additional studies should be aimed at understanding the biochemical pathways underlying the

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individual components in the PEO oil. Author Statement

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All authors (M.R. Farag, H.K. Mahmoud, Sabry A.A. El-Sayed, Sarah Y.A. Ahmed, M. Alagawany and

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Shimaa M. Abou-Zeid) have read and agreed to the published version of the manuscript.

Declaration of Competing Interest None

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https://doi.org/10.1016/j.chemosphere.2015.09.050.

29

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Figures

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Figure 1. GC-MS analysis of the active components of PEO.

30

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Figure 2. Probit analysis of 96-h LC50 calculation of bifenthrin in O. niloticus.

31

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Figure 3. Effect of supplementation of PEO (1 and 2 mL/kg diet) on the Comet variables in the

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brain of O. niloticus exposed to BF (96 LC50; 0.68 µg/L) for 60 days. Values are mean ± SE, bars are not sharing a common superscript letter (a,b,c,d) differ significantly at p < 0.05.

32

of ro -p re lP ur na Jo Figure 4. Effect of supplementation of PEO (1 and 2 mL/kg diet) on the brain monoamine neurotransmitters, GABA and serum amino acids levels of O. niloticus exposed to BF (96 LC50; 0.68 µg/L) for 60 days. Values are mean ± SE, bars are not sharing a common superscript letter (a,b,c,d) differ significantly at p < 0.05.

33

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Figure 5. Effect of supplementation of PEO (1 and 2 mL/kg diet) on the brain AChE and Na+/K+-ATPase

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enzymes activity of O. niloticus exposed to BF (96 LC50; 0.68 µg/L) for 60 days. Values are mean ± SE, bars

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are not sharing a common superscript letter (a,b,c,d) differ significantly at p < 0.05.

34

35

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re

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Figure 6. Effect of supplementation of PEO (1 and 2 mL/kg diet) on the expression pattern of stress-related genes and pro-inflammatory cytokines in brain of O. niloticus exposed to BF (96 LC50; 0.68 µg/L) for 60 days. Values are mean ± SE, bars are not sharing a common superscript letter (a,b,c,d) differ significantly at p < 0.05.

36

Table 1. Formulation and calculated composition analysis of the basal diet fed to experimental O. niloticus fish. Items

(g/kg)

210

Soybean meal 48% CP

200

Fish meal

150

Corn gluten 60% CP

130

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ro

Yellow corn

110

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Rice bran Wheat middlings

Premix-Vit**

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Corn oil

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Premix-Min*

Total

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Ingredient (%)

150 10 10 30 1000

Calculated composition (g/kg)

320.5

Lipid

45.50

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Crude protein

Crude fiber

42.45

Ash

73.01

Nitrogen free extract***

518.54

*Composition of mineral premix kg−1: manganese, 53 g; zinc, 40 g; iron, 20 g; copper, 2.7 g; iodine, 0.34 g; selenium, 70 mg; cobalt, 70 mg and calcium carbonate as carrier up to 1 kg.

37

**Composition of vitamin premix kg−1: vitamin A, 8,000,000 IU; vitamin D3, 2,000,000 IU; vitamin E, 7,000 mg; vitamin K3, 1,500 mg; vitamin B1, 700 mg; vitamin B2, 3,500 mg; vitamin B6, 1,000 mg; vitamin B12, 7 mg; biotin, 50 mg; folic acid, 700 mg; nicotinic, 20,000 mg; pantothenic acid, 7,000 mg.

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***Nitrogen free extract = 100 − (crude protein + Crude lipids + ash + crude fiber).

38

Table 2. Bioactive chemical constituents assigned in PEO by GC-MS analysis No. Bioactive chemical constituents

Chemical

Mass Weight

Formula

(MW)

Retention Time (RT) (min)

Area %

D-Limonene

C10H16

136

8.61

18.82

2

Oleic Acid

C18H34O2

282

32.66

14.52

3

á-Cyclocitral

C10H16O

152

13.18

11.75

4

Globulol

C15H26O

222

C11H16O5

228

204

18.57

7.78

204

18.73

7.34

222

22.76

5.45

256

29.05

4.59

C10H16

136

7.38

1.56

C12H8O4

216

30.75

1.09

C11H12O3

192

19.32

1.05

2á,8á-(Dimethoxy)-3,5,7trioxatetracyclo[7.2.1.0(4,11).0(6,13)] dodecane

19.52

11.24

29.78

8.51

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5

of

1

á-Selinene

C15H24

7

à-Guaiene

C15H24

8

Apiol

C12H14O4

9

Hexadecanoic acid (

C16H32O2

10

α-Pinene

11

Bergapten

12

Myristcin

13

2,4-Decadienal, (E,Z)-

C10H16O

152

14.80

1.05

14

Trans-carane

C10H16O

152

11.26

0.86

15

3,5-Decadiene, 2,2-dimethyl-,

C12H22

166

14.33

0.76

16

Dasycarpidan-1-methanol, acetate

C20H26N2O2

326

32.86

0.62

C15H24

204

18.21

0.59

C15H24

204

18.06

0.52

C11H24O

172

15.63

0.47

re

lP

ur na

Jo 17 18 19 20

-p

6

Caryophyllene

á-Chamigrene

1-Decanol, 2-methyl1,2-Epoxycyclooct-3-ene, 5,5dimethyl-8-methylene-

C11H16O

39

164

23.86

0.32

21

Lucenin 2

C27H30O16

610

42.01,

0.32

42.10

0.24

á-elemene

C15H24

204

16.18

0.27

20

à-acorenol

C15H26O

222

23.02

0.18

24

5á,7áH,10à-Eudesm-11-en-1à-ol

C15H26O

222

22.44

0.11

Jo

ur na

lP

re

-p

ro

of

22

40

Table 3. Behavioral response changes in O. niloticus exposed to different concentrations of BF for 96 h (Acute toxicity study).

avior

Experimental groups (BF; µg/L) BF 0.5

BF 1.0

BF 2.0

BF 4.0

BF 6.0

BF 8.0

BF 10.0

gulping

-

-

-

+

+

++

+++

++++

piratory distress

-

-

-

+

+

++

+++

++++

ggish movement

-

-

-

-

+

+

++

++++

oordinated swimming

-

-

-

-

+

+

++

++++

erventilation

-

-

-

-

+

++

+++

++++

-p

ro

of

Control

Jo

ur na

lP

re

The score of behavioral responses were recorded as follows: None (-), mild (+), moderate (++) strong (+++), very strong (++++)

41

Table 4. Water quality measurements in the aquaria in response to BF exposure (96h LC50; 0.68 µg/L) and supplementation of PEO (1 and 2 mL/kg diet) for 60 days. Measurements

P value*

Experimental groups PEO1

PEO2

BF

PEO1 /BF

PEO2/BF

Temperature (ºC)

27.73±0.26

26.95±0.37

27.06±0.19

27.04±0.53

27.15±0.11

27.12±0.41

0.544

pH

7.68±0.39

7.38±0.27

7.39±0.26

7.23±0.04

7.23±0.06

7.27±0.33

0.861

Dissolved oxygen (mg/L)

6.45±0.42 abc

7.03±0.04ab

7.30±0.13a

5.19±0.08d

5.71±0.32 cd

6.19±0.12bc

0.000

Total ammonia (mg/L)

0.13±0.01c

0.11±0.01c

0.09±0.01c

0.51±0.04a

0.31±0.01b

0.24±0.02b

0.000

ro

of

Control

Jo

ur na

lP

re

-p

Values are mean ± SE for three replicate/group, values are not sharing a common superscript letter (a,b,c,d) differ significantly at p < 0.05. *P- overall treatment.

42

Table 5. Effect of supplementation of PEO (1 and 2 mL/kg diet) on behavioral response in O. niloticus exposed to BF (96h LC50; 0.68 µg/L) for 60 days.

havior

Experimental groups

Pv

Control

PEO1

PEO2

BF

PEO1 /BF

PEO2/BF

afing (%)

33.42±0.85 a

33.13±2.45 a

33.21±1.87 a

6.99±0.75 b

12.25± 0.35b

11.91±0.39b

0.0

ting (%)

15.97±0.42 a

15.71±0.54 a

15.72±0.30 a

3.14±0.45 d

7.03±0.78 c

9.02±0.33 b

0.0

facing (%)

17.33±0.36 a

17.29± 0.27a

17.17±0.91a

8.10±0.05 c

11.61±0.68b

12.17±1.52 b

0.0

ooling (%)

8.71±0.36 a

8.69±0.42 a

8.29±0.25 a

5.31±0.24b

7.76±0.49 a

8.44±0.17 a

0.0

mination (%)

10.06±0.05 a

9.99±0.66 a

10.41±1.07 a

3.75±0.09 b

4.68±0.55 b

8.13±0.80 a

0.0

al comfort act )%(

82.73±1.18 a

82.55±0.57 a

82.82±0.78 a

26.19±0.62 d

43.26±0.43 c

50.54±0.27 b

0.0

4.34± 0.09c

3.12±0.01 d

2.46± 0.14e

6.20± 0.02a

5.62± 0.28b

5.36± 0.17b

0.0

tugging (%)

1.49± 0.16bc

0.93± 0.18cd

0.63± 0.02d

2.25±0.12 a

2.28± 0.33a

1.95± 0.11ab

0.0

uth push (%)

9.24±0.57 ab

8.37±0.23 ab

5.84±1.97 b

11.08± 0.21a

10.66± 0.28a

10.21±0.26 a

0.0

al aggressive act )%(

16.39±0.08 c

11.88±0.30 d

10.44± 9.46e

19.05± 0.29a

17.43±0.18 b

16.78±0.20 bc

0.0

ur na

asing (%)

ro

-p

re

lP

gressive behavior

of

mfort behavior

Values are mean ± SE, values are not sharing a common superscript letter (a,b,c,d) differ significantly at p <

Jo

0.05. *P- overall treatment.

43

Table 6. Effect of supplementation of PEO (1 and 2 mL/kg diet) on survivability, growth performance, brain oxidant/antioxidant status and serum inflammatory response of O. niloticus exposed to BF (96h LC50; 0.68 µg/L) for 60 days. Experimental groups

Pv

Control

PEO1

PEO2

BF

PEO1 /BF

PEO2/BF

96.91±0.17ab

98.06±0.11 a

98.15 ±0.06a

78.79±3.43 c

87.64±3.32b

89.65±3.65 ab

0.0

al body weight (Wi) (gm)

10.50±0.18

10.52±0.15

10.51±0.19

10.47±0.11

10.50±0.22

1.0

l body weight (Wf) (gm)

40.22±1.05 b

43.10±0.53 a

45.22±1.51 a

36.06±1.05 c

36.06±0.97 c

38.21±0.55 bc

0.0

y weight gain (gm)

29.56±0.57 b

32.58±0.16 a

34.70±0.73 a

25.58±0.50 c

25.59±0.39 c

27.70±0.12 bc

0.0

ific growth rate (SGR) (/day)

1.94±0.10 ab

2.53±0.23 a

2.55±0.23 a

1.50±0.10 b

1.69±0.02 b

1.90±0.05 ab

0.0

D ( U/g tissue)

8.87±0.30a

8.90±0.28 a

re

avior

8.89±0.32a

4.03±0.72c

5.38±0.07b

5.61±0.25b

0.0

T ( U/g tissue)

6.38±0.03a

6.20±0.01 a

6.26±0.12a

3.89±0.33b

6.21±0.12a

6.38±0.03a

0.0

H (nmol/g tissue)

18.16±0.04

18.11±0.07

18.11±0.03

17.95±0.31

18.15±0.03

18.15±0.03

0.9

17.94±0.49b

16.67±0.33b

16.39±0.35b

31.60±2.16a

16.07±1.04b

17.91±0.13b

0.0

6.11±0.03c

6.10±0.03c

6.17±0.18c

16.30±1.55a

12.70±0.79b

7.57±0.34c

0.0

1.60±0.18c

1.81±0.08c

7.36±0.47a

4.20±0.14b

1.40±0.21c

0.0

ival %

A (nmol/g tissue)

Jo

ein carbonyl (nmol/g tissue)

ur na

dative injury variables

10.46±0.17

ro

-p

lP

oxidant status variables

of

wth performance

mmatory injury variable

O (U/L)

1.80±0.08c

Values are mean ± SE, values are not sharing a common superscript letter (a, b, c, d) differ significantly at p < 0.05. *P- overall treatment.

44

Superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), malondialdehyde (MDA),

Jo

ur na

lP

re

-p

ro

of

myeloperoxidase (MPO)

45