Tris (1-chloro-2-propyl) phosphate exposure to zebrafish causes neurodevelopmental toxicity and abnormal locomotor behavior

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Tris (1-chloro-2-propyl) phosphate exposure to zebrafish causes neurodevelopmental toxicity and abnormal locomotor behavior Min Xia, Xuedong Wang ⁎, Jiaqi Xu, Qiuhui Qian, Ming Gao, Huili Wang ⁎ National and Local Joint Engineering Laboratory of Municipal Sewage Resource Utilization Technology, School of Environmental Science and Engineering, Suzhou University of Science and Technology, Suzhou 215009, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Acute and chronic TCPP exposure induced zebrafish abnormal behavior. • Acute TCPP exposure triggered a series of neurodevelopmental toxicity to larvae. • Neurotoxicity resulted from apoptosis, oxidative stress and changes in related genes. • BDT, CPP, T-maze and SI tests confirm neurobehavioral abnormality in chronic exposure. • Abnormal behavior contributes to changes in biomarkers and histopathological injury.

a r t i c l e

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Article history: Received 10 September 2020 Received in revised form 27 October 2020 Accepted 31 October 2020 Available online xxxx Editor: Daqiang Yin Keywords: Tris (1-chloro-2-propyl) phosphate Neurotoxicity Abnormal locomotor behavior Gene expression change Zebrafish

a b s t r a c t The organophosphate flame retardant, tris (1-chloro-2-propyl) phosphate (TCPP), is ubiquitous in environmental matrices; however, there is a paucity of information concerning its systemic toxicity. Herein, we investigated the effects of TCPP exposure on zebrafish neurodevelopment and swimming behavior to elucidate the underlying molecular mechanisms of neurotoxicity. Under TCPP gradient concentration exposure, the hatching rates were declined by up to 33.3% in 72 hpf, and the malformation rates increased from 15% to 50%. Meanwhile, TCPP led to abnormal behaviors including decreased locomotive activity in the dark and slow/insensitive responses to sound and light stimulation of larvae. TCPP caused excessive apoptosis and ROS accumulation in early embryonic development, with hair cell defects and structural deformity of neuromast. Abnormal expression of neurodevelopment (pax6a, nova1, sox11b, syn2a, foxo3a and robo2) and apoptosis-related genes (baxa, bcl2a and casp8) revealed molecular mechanisms regarding abnormal behavioral and phenotypic symptoms. Chronic TCPP exposure led to anxiety-like behavior and excessive panic, lower capacity for discrimination and risk avoidance, and conditioned place preference in adults. Social interaction tests demonstrated that long-term TCPP stress resulted in unsociable, eccentric, lonely and silent behaviors in adults. Zebrafish memory and cognitive function were severely reduced as concluded from T-maze tests. Potential mechanisms triggering behavioral abnormality were attributed to histopathological injury of diencephalon, abnormal changes in nerve-related genes at transcription and expression levels, and inhibited activity of AChE by TCPP stress. These findings provide an important reference for risk assessment and early warning to TCPP exposure, and offer insights for prevention/ mitigation of pollutant-induced nervous system diseases. © 2020 Elsevier B.V. All rights reserved.

⁎ Corresponding authors. E-mail addresses: [email protected] (X. Wang), [email protected] (H. Wang).

https://doi.org/10.1016/j.scitotenv.2020.143694 0048-9697/© 2020 Elsevier B.V. All rights reserved.

Please cite this article as: M. Xia, X. Wang, J. Xu, et al., Tris (1-chloro-2-propyl) phosphate exposure to zebrafish causes neurodevelopmental toxicity and abno..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2020.143694

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1. Introduction

morphological deformity and abnormal locomotor behavior were disclosed through integration of physiological and biochemical metrics, histopathological observations and changes in transcription and expression levels of neurodevelopment-related genes. Results of this research offer new insights on TCPP-induced neurotoxicity in aquatic animals, and provide theoretical guidance for early intervention of druginduced abnormal neurodevelopment in mammals and as an early warning indicator for environmental exposure.

Due to adverse health effects of polybrominated diphenyl ethers, many developed countries have banned the use of these compounds as flame retardants. Consequently, alternative chemicals such as organophosphate flame retardants (OPFRs) have become popular substitutes. OPFRs are widely used in multiple fields, including electronic products, furniture and floor polish (van der Veen and de Boer, 2012). Because OPFRs are primarily used as an additive product rather than being chemically bonded to materials, they are prone to release to the environment via volatilization, leaching and/or abrasion (van der Veen and de Boer, 2012). Several OPFRs are ubiquitously detected in environmental matrices including indoor air, dust, water, sediment and soil (Ali et al., 2017; Guo et al., 2017). Among them, tris (1-chloro-2-propyl) phosphate (TCPP) is one of the most frequently detected halogenated OPFRs in both water and sediments. TCPP is of particular concern due to its high volume of use, frequent detection in the environment and potentially high toxicity (Xu et al., 2017). Around Bohai Bay (China), TCPP was detected in the range of 4.6–921 ng/L and reached concentrations up to 1392 ng/L in the East China Sea (Zhong et al., 2017). Additionally, TCPP is frequently detected in marine fish and mussels, indicating the potential for bioaccumulation/biomagnification within aquatic food webs (Wu et al., 2018). Consequently, the long-term presence of TCPP in aquatic environments poses great risks to aquatic organisms and human health. The potential toxicity and adverse health risks associated with TCPP have captured much attention in recent years (Wu et al., 2018). However, the paucity of TCPP toxicity studies hinder our assessment of health risks to aquatic organisms and humans. From the perspective of proteomics and metabolomics, Ji et al. (2020) revealed the molecular responses in rockfish upon TCPP exposure. A total of 143 proteins were found to change significantly, some of which were related to nerve system development; however, the authors did not conduct further experimental verification on the relationship between these proteins and neurodevelopment. Wu et al. (2018) reported that TCPP inhibited the innate immunity in Mytilus galloporvincialis through expression changes in the related genes; however, there is a lack of systematic evidence on phenotypic malformations and physiological indicators. Although abnormal swim activity of zebrafish was observed after TCPP exposure for 5 days, no mechanistic exploration was concerned (Dishaw et al., 2014). The reports on TCPP from 2014 to 2020 mainly focused on its degradation and environmental behavior (Dishaw et al., 2014; Ji et al., 2020). Since TCPP is structurally similar to organophosphate pesticides (OPPs—an established neurotoxicant), it is critical to gain additional neurotoxicity information to properly assess the environmental/human impacts of TCPP. Although the neurotoxicity of TCPP has been demonstrated in Ji et al.'s (2020) report, the underlying molecular mechanisms remain ambiguous. For example, a series of scientific problems need to address: What abnormal behaviors and which target organ abnormalities can be caused by TCPP-induced neurotoxicity? What are the key biomarkers and physiological and biochemical indicators? And what are the differential neurotoxicity effects on embryonic-adult zebrafish under acute and chronic TCPP exposure? Zebrafish (Danio rerio) are a valuable model organism in the field of neuroscience as their blood-brain barrier develops gradually at 3 days post fertilization (dpf), leading to high sensitivity to chemical toxins (Zhang et al., 2016). Moreover, zebrafish circadian rhythm, cognitive functions, autonomic responses and other behaviors can be adopted as evaluation metrics to study nerve injuries (Zhao et al., 2018). In addition to their similar brain structure, zebrafish have similar regulatory processes to those underlying human behaviors, which make them a valuable model for neuropsychiatric disease studies (Zhao et al., 2018). Building upon previous research, we systematically explored the effects of TCPP exposure on zebrafish neurodevelopment and neurobehavior at different development stages, as well as at different system levels. The underlying mechanisms regarding TCPP induced

2. Materials and methods 2.1. Chemicals and reagents Tris (1-chloro-2-propyl) phosphate (TCPP; CAS No. 13674-84-5; >97% purity), dissolved in dimethyl sulfoxide, was purchased from Tokyo Chemical Industry (Tokyo, Japan). TRIzol reagent and reverse transcription kits were obtained from Takara (Dalian, China). All reagents used in this study were of analytical grade unless otherwise stated and used as received. 2.2. Zebrafish maintenance and exposure protocols Wild-type zebrafish (AB strain) and transgene zebrafish line Tg (flk1: mCherry) were acquired from the China Zebrafish Resource Center (Wuhan, China). Zebrafish maintenance followed methods previously reported by our group (Li et al., 2018). Briefly, adult zebrafish were raised at 28 ± 1 °C with a 14 h/10 h (light/dark) rhythm cycle, and fed live brine shrimp three times each day. Three TCPP concentrations (5, 15 and 25 mg/L), equivalent to 1/10 to 1/2 the LC50 value (49.7 mg/L), were selected for continuous exposure to zebrafish from embryos (6 hpf) to adults (90 dpf, days post fertilization). Control group was treated with 0.005% dimethyl sulfoxide, referring to the highest 25 mg/L TCPP exposure treatment. After TCPP exposure, we randomly collected the 24-, 48- and 120hpf embryos/larvae for morphological observation using an optical microscope with camera (DM2700 M, Leica, Heidelberg, Germany). The 120-hpf larvae and sexually mature adults were anesthetized with 0.03% tricaine (buffered MS-222, Sigma, St. Louis, USA). Recorded indices included 72-hpf hatching, 120-hpf malformation, 120-hpf mortality and 48-hpf heartbeat rates. We provide a schematic representation of the overall experimental design in Fig. S1. 2.3. Acridine orange (AO) staining and reactive oxygen species (ROS) quantification After TCPP exposure from 6 to 72 hpf, 20 larvae from each treatment were collected for AO staining in the dark for 30 min (Qi et al., 2016). Following staining, specimens were washed with phosphate buffered saline (PBS) solution three times. Apoptotic cells, which appeared as distinct bright spots, were identified using a fluorescence microscope. ROS in control and treatment groups was detected at 96 hpf using ROS assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). After TCPP exposure, larvae were incubated with 30 μM of DCFH-DA (a fluorescent probe) for 30 min in the dark, washed with PBS solution three times, and anesthetized with 0.03% MS-222. We acquired a lateral image of each larva using a fluorescence microscope, and quantified fluorescence intensity using Image-Pro Plus software. 2.4. qRT-PCR and whole-mount in-situ hybridization (WISH) for identification of neurodevelopment-related genes After TCPP exposure from 6 to 120 hpf, zebrafish larvae were collected and rinsed using PBS solution. Then, we isolated total RNA from 50 larvae homogenized for each replicate of each treatment (0, 5, 15 and 25 mg/L) using TRIzol reagent. Primers used for qRT-PCR are summarized in Table S1 with elfa as the endogenous reference. All PCR 2

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analyses utilized three biological replicates and each biological replicate included three technical replicates. The cDNA probe sequences for two genes (pax6a and nova1) were labeled with digoxigenin (DIG) for WISH (Supplementary Fig. 2). The 72- and 120-hpf embryos in each treatment were treated with 0.5% N-phenylthiourea (Aladdin, Shanghai, China) and collected in 1.5 mL RNase-free EP tubes, each tube containing 15 embryos.

T-maze experiments were used to study zebrafish learning/memory capacity. TCPP-exposed and non-exposed treatments were subjected to an exploratory assay in which zebrafish were put in the starting arm location of a T-maze: food bait was used as a reward in the right side (“r” zone), and tapping as a sound stimulation in the left side (“w” zone). Before the test, zebrafish were trained for 7 days (15 min each time; 4 times per day). Then, adult zebrafish were individually placed in the “s” area, and the barrier (red line of “s” zone) was quickly removed. After a 5-min acclimatization period, locomotor activity was videorecorded for 10 min; videos were analyzed firstly by direct observation, and then with Noldus Ethovision XT software (Noldus IT, Wageningen, Netherlands).

2.5. Lateral-line neuromast staining and formation observation of hair cell bundles After TCPP exposure from 6 to 120 hpf, 20 larvae from each treatment were collected for IM FM™ 1–43 Dye (GeneBio, Shanghai, China) staining in the dark for 1 min. Following staining, live larvae were rinsed in PBS solution for three 30 s rinse cycles and then anesthetized in MS-222. Specimens were immediately imaged using an optical microscope (Nikon, Tokyo, Japan). Additionally, the 120-hpf larvae were fixed, dehydrated and dried for observation following Liu et al. (2019). We used scanning electron microscopy (SEM; Hitachi S-2400, Tokyo, Japan) to observe the hair bundles of L1 neuromasts in the posterolateral lines.

2.7. Histopathological observation, and immunofluorescence (IF) and AChE activity tests for adult brain After TCPP exposure from 6 hpf to 90 dpf, adult zebrafish were anesthetized on ice, and the brain was carefully dissected. Dissected tissues were washed in PBS solution, fixed in 4% paraformaldehyde overnight, paraffin-embedded and section-dried according to Zang et al. (2019). The resultant brain tissue was used for histopathological observation after haematoxylin and eosin (HE) staining (Solarbio, Beijing, China). Brain tissue was cut into 10-μm sections, mounted on glass slides and stored at −20 °C prior to observation. IF staining was carried out based on Wang et al.'s (2020) report. Histological sections were imaged and recorded with a fluorescence microscope/camera (Nikon, Tokyo, Japan). AChE activity was detected using kits manufactured by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.6. Locomotor behavior of embryonic/larval/adult zebrafish upon exposure to TCPP Embryonic and larval locomotor tests were performed in 96-well plates at 27.5 ± 1 °C during the light phase between 9 a.m. and 4 p.m. The 24-hpf embryonic spontaneous movements per minute were calculated as tail coil alternations over a 5 min period as recorded using a microscope video camera (SZX16, Olympus, Japan). After TCPP exposure to larvae from 6 to 96 hpf, a tapping stimulation test was conducted. Following a 5-min adaptation period, video was collected for 5 min with tapping intensities set at eight different levels. Further, autonomous movement and light-to-dark tests were conducted at 120 hpf. For autonomous movement of larvae, video was collected for 5 min after a 5-min dark adaptation. Two repeats of the light-to-dark cycle were used prior to the start the light-to-dark test, which consisted of 10 min of 100% light and 10 min of complete darkness. All locomotor activities in the light-dark cycle were analyzed and swim velocities were averaged into 1-min time bins. The video was analyzed using ethoVision XT software (Noldus IT, Wageningen, Netherlands) to track the movement of individual zebrafish in the 96-well plates. Four different behavioral tests were conducted using adult zebrafish. The bottom dwelling test (BDT) assessed zebrafish anxiety and abnormal locomotion (Cachat et al., 2010; Stewart et al., 2011). Zebrafish from all groups were individually placed in a trapezoidal tank (15 × 28 × 23 × 7 cm; height × top-length × bottom-length × width). The water tank was divided into two equal virtual/horizontal portions with a line marking on the outside walls. Conditioned place preference (CPP) was assessed by recording zebrafish behavior in the non-preference side to measure the zebrafish guts. The testing tank dimension was 25-cm length × 15-cm width × 20cm depth. Distinct visual cues divided the experimental tank into two halves: one half was colored light-brown and the other half colored white, with two black spots placed at the bottom of the tank. The water depth was kept at 2.5 cm from the bottom to relieve pressure. Zebrafish prefer to swim in cohorts because they are social aquatic organisms. The social interaction test (SI) tank (52-cm length × 26-cm width × 30-cm depth) was divided into two compartments, left zone (social zone) and right zone (non-social zone), and zebrafish could freely swim from one zone to the other through the hole of middle plugboard. The social area was connected to a transparent glass water tank with 8 adults (4 males/4 females). Zebrafish in the social area could see each other through the transparent glass cylinders. In contrast, the interior of the non-social zone was opaque (Supplementary Fig. 3).

2.8. Statistical analysis Each TCPP-exposure treatment and control group included three biological and technological replicates as summarized in Table S2. One-way analysis of variance examined the effect of TCPP exposure, followed by Dunnett's tests to independently compare the TCPP treatments with the control group. Video was analyzed using EthoVision XT software (Noldus IT, Wageningen, Netherlands) to track individual movement of zebrafish. All data were reported as mean ± standard deviation (SD), and significance levels recorded at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). 3. Results 3.1. TCPP-induced embryonic development toxicity The control group malformation rate was <5.3% in 48-hpf larvae, indicating that embryo quality satisfied requirements for subsequent TCPP-exposure experiments. After gradient concentration TCPP exposure from 6 to 120 hpf, mortality percentages for 120-hpf larvae were significantly increased (p < 0.01 or p < 0.001) in a dose-dependent manner (Fig. 1A). The calculated 120-hpf LC50 value for TCPP was 49.7 mg/L (R2 = 0.996). Likewise, 72-hpf hatching rates were significantly decreased (p < 0.01 or p < 0.001) by 4.7–37.7% with increasing TCPP concentrations (Fig. 1B). With increasing TCPP concentrations, total malformation rates showed a significant increasing trend from 12.3 to 42.4% (p < 0.01 or p < 0.001) (Fig. 1C). Heart beat was normal (~120 beats/min) and showed regular rhythm in the control group; while TCPP exposure prominently inhibited heart rhythm (~90–100 beats/min) and showed arrhythmia in different individuals (Fig. 1D). Most TCPP-exposed zebrafish possessed two or more malformation symptoms, such as yolk sac edema, bent spine, closure of swim sac and pericardial edema (Fig. 1E). After TCPP exposure from 6 to 24 hpf, embryos showed pronounced developmental retardation anormogenesis of head and eye, as well as yolk cyst and bent spine from 48 to 120 hpf. In the 5-mg/L treatment, yolk sac edema, delayed absorption and sacculus formation were similar to that of the control group. 3

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Fig. 1. TCPP induces developmental toxicity to embryonic zebrafish. Note: (1) A, zebrafish mortality rate at 120 hpf after exposure to TCPP; (2) B, zebrafish hatching rate at 72 hpf after exposure to TCPP; (3) C, statistical analysis of malformation rate at 72-hpf zebrafish larvae exposed to TCPP; (4) D, zebrafish heart rate at 48 hpf after exposure to TCPP; (5) E, zebrafish malformation from 24 to 120 hpf after exposure to TCPP; (6) arrows in Fig. 1E: YS, yolk cyst; BS, bent spine; SS, swim sac; PC, pericardial cyst; VB, venous sinus bleeding; Ey, eye; (7) “*“, “**” and “***” indicate significance levels of p < 0.05, p < 0.01 and p < 0.001, respectively.

However, in the 15 and 25-mg/L treatments, yolk sac edema, pericardial cyst and developmental defects in the swim sac were commonly present, while a few individuals showed venous sinus bleeding (Fig. 1E). These observations provide compelling evidence that TCPP exposure led to several adverse effects on cardiovascular development.

development of movement neurons and implementation of nerve conduction function. Abnormal locomotive behavior can be characterized in terms of opening/closure of swim sac, absorption of yolk, heart arrhythmia and abnormal development of neurons (McKeown et al., 2009). Average swimming velocity in the 5-mg/L treatment was about 0.75 mm/s, which was significantly lower (p < 0.05) than that of the control group (~1.26 mm/s) (Fig. 2C). Similarly, the 5-min total distances traveled in the 15 and 25-mg/L treatments were significantly decreased (p < 0.01 or p < 0.001) by 21.2% and 36.4%, respectively, compared to the control group (Fig. 2C). Larvae in all three treatment groups showed abnormal movement paths, such as tremor, rotation in situ or circular motion (Supplementary Fig. 4). To explore the effects of TCPP exposure on sound and light sensitivity, we analyzed the larval average speed traced under tapping stimulation and the distance of motion under dark-light alternation. Compared to the control group, larval average speed in the 5-mg/L treatment showed a slight decrease in high or low speed response to tapping stimulation. However, larval average speed was decreased by 24.2% and 30.1% in the 15 and 25-mg/L treatments, exhibiting a horizontal line for movement speed (Fig. 2B). These observations imply that TCPP exposure inhibited larval sensitivity to acoustic stimulation, i.e., a retardation phenomenon. Under light-dark alternation, larval zebrafish in the

3.2. Effect of TCPP exposure on embryonic/larval behavior Zebrafish embryos are often used to investigate the regulation of the neural networks coordinating locomotive behavior. Zebrafish exhibit a strong spontaneous twitch early in their embryonic development before hatching and have relatively simple and accessible nervous systems compared to mammalian systems (McKeown et al., 2009). Embryonic spontaneous movement is the initial movement of zebrafish as similar to fetal movements in mammals. With further development of the motor nervous system, movement behavior is gradually controlled by the central nervous system (CNS) (Jin et al., 2009). Spontaneous movements averaged 8.0 (tail swings per minute) for 24-hpf embryos in the control group. In contrast, movement frequency decreased by ~27–36% (p < 0.01 or p < 0.001) in the 15 and 25-mg/L treatments, but the 5mg/L treatment showed no difference from the control group (Fig. 2A). The inhibition of embryonic spontaneous movement in the 15 and 25-mg/L treatments demonstrates that TCPP impaired normal 4

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Fig. 2. Effect of TCPP exposure on larval zebrafish swimming behavior. Note: (1) A, zebrafish embryonic spontaneous movements per minute after exposure to TCPP from 6 to 24 hpf; (2) B, swimming speed of zebrafish larvae, exposed to TCPP from 6 to 96 hpf, when subjected to a tapping stimulation at 96 hpf; (3) C, distance traveled and swimming speed of zebrafish larvae at 120 hpf after exposure to TCPP; (4) D, swimming speed of zebrafish larvae at 120 hpf after exposure to TCPP when subjected to a 40-min dark-to-light photoperiod at 120 hpf; (5) E, effects of TCPP on path angles of larval zebrafish at 120 hpf (dark period); (6) F, effects of TCPP on path angles of larval zebrafish at 120 hpf (light period); (7) “*”, “**” and “***” indicate significance levels of p < 0.05, p < 0.01 and p < 0.001, respectively.

control group showed normal movement rhythm, namely low movement under light and high movement under dark. Upon TCPP exposure, larvae continued to display a light-dark rhythm response, but movement distance was significantly decreased in a concentrationdependent manner (Fig. 2D). As for zebrafish turning habit and rotation angle, the number of clockwise movements under light or dark periods were decreased by 3.8–47.7% with increasing TCPP concentrations. Additionally, nearly 50% of rotational movements occurred under dark conditions in the 25-mg/L treatment (Fig. 2E–F). To characterize rotation angle, six classes were established from −180° to +180° (Table S3). Under both light and dark conditions, zebrafish larvae seldom exhibited a responsive turn of >90°, while always displaying a preference to turn to the right (+) rather than to the left (−). Overall, these abnormal behaviors and motor habits indicate that TCPP exposure triggered neurodevelopmental toxicity to larval zebrafish.

mitochondrial energy supply (Coffin et al., 2013). Under TCPP exposure, expression of nova1 was monotonically down-regulated (p < 0.01 or p < 0.001) with increasing TCPP concentrations in a dose-dependent manner (Fig. 3A). Both pax6a and foxo3a are involved in development of the nervous system; pax6a as a brain marker gene is widely expressed in various parts of the brain and foxo3a is essential for maintaining neural development (Peng et al., 2010). TCPP significantly down-regulated expression of pax6a and foxo3a in the 15-mg/L treatment (p < 0.01 or p < 0.001), but up-regulated expression of foxo3a in the 25-mg/L treatment (p < 0.01). Pax6a gene expression plays an important role in the cortex division of central nervous system and the differentiation of neural stem cells (NSCs) into different neurons, and pax6a may also be related to spinal cord dorsal ventral polarization mediated by spinal cord induction signal (Halluin et al., 2016; Samadi et al., 2015). Under the low concentration of TCPP exposure, the expression of pax6a decreased, which could potentially be explained by the disruption of gene expression during critical time points in early neurodevelopment, and pathological tissue damage also confirmed these findings. Therefore, abnormalities in biological functions are closely related to dysregulation of gene expression. Sox11b plays a critical role in development of the vertebrate eye and retina, and syn2a and robo2 are related neurodevelopment marker genes. Notably, transcription levels for all

3.3. TCPP-induced expression of neurodevelopment-related genes and excessive apoptosis Neurobehavioral and movement capacity is dominated by the expression of multiple genes, especially related to neural regulation and 5

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Fig. 3. Differential expression of two neurodevelopment-related genes (pax6a and nova1) by qRT-PCR and WISH. Note: (1) A, differential expression of neurodevelopment-related genes by qRT-PCR; (2) “*”, “**” and “***” indicate significance levels of p < 0.05, p < 0.01 and p < 0.001, respectively; the significance codes apply to comparisons among the different experimental groups; (3) B, WISH of pax6a expression in 72-hpf zebrafish larvae and nova1 expression in 120-hpf zebrafish larvae; (4) abbreviations in Fig. 3B: B, brain; Sc, spinal cord; H, heart; Ey, eye.

three genes were all inhibited following TCPP exposure (p < 0.05 or p < 0.01) (Fig. 3A), with the exception of robo2 in the 15 mg/L treatment. To elucidate the distribution and expression changes of neurodevelopment-related genes in the CNS, two differentially expressed genes, pax6a and nova1, were selected for WISH to directly observe the expression position and changes within the whole larvae. The quantitative expression results for the two genes are shown in Fig. S5A. In the control group, pax6a was mainly expressed in the eye, with lower expression in the forebrain. Upon TCPP exposure, pax6a expression levels were significantly decreased in the eye and forebrain of 72hpf larvae (p < 0.05 or p < 0.01) (Fig. 3B). Meanwhile, nova1 was mainly expressed in the brain and almost invisible in the spine and eye of 120hpf larvae, but displayed a significant decrease (p < 0.05 or p < 0.01) in TCPP treatments (Fig. 3B). WISH results for pax6a and nova1 were generally consistent with those of qRT-PCR. Therefore, from the perspective of gene expression location and level, TCPP-induced abnormal expression of neurodevelopment-related genes promoting behavioral abnormalities. Oxidative damage and mitochondrial dysfunction contribute to severe cell apoptosis. Hence, we examined the expression changes of three apoptosis-related genes (baxa, bcl2a and caspase-8). Both baxa and caspase-8 were over-expressed (p < 0.05, p < 0.01 or p < 0.001),

while the apoptotic suppressor gene bcl2a was significantly inhibited by TCPP exposure (p < 0.01 or p < 0.001) (Fig. 4A). Based on these data, we posit that the abnormal neurobehavior caused by TCPP exposure is likely associated with excessive apoptosis of nerve cells. 3.4. Excessive apoptosis and ROS accumulation under TCPP exposure Neurotoxicity is closely associated with CNS cell apoptosis (Liu et al., 2018). Based on AO staining, TCPP showed a dose-dependent induction in fluorescence intensities of apoptotic cells in 72-hpf zebrafish brain, pericardium and visceral mass (p < 0.05 or p < 0.01) (Fig. 4C and F). Consistent with our expectation, the occurrence of excessive apoptosis phenomenon coincided with changes in the expression of apoptotic genes. Oxidative stress is an important biological index for evaluating biological toxicology. Accumulation and abnormal metabolism of ROS cause direct oxidative stress to organisms. Oxidative stress mediates apoptosis through mitochondria, death receptor and endoplasmic reticulum stress, and may also induce apoptosis by activating mitogenactivated protein kinase pathway, nuclear transcription factor κB and Caspases (Cui et al., 2020). Oxidative stress activates the apoptotic signaling cascade pathways to induce apoptosis in a variety of cells, preferentially activating inflammatory cytokines (Li et al., 2018). ROS is the 6

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Fig. 4. Effects of TCPP exposure on zebrafish AchE activity, apoptosis, cranial vasculature and ROS level. Note: (1) A, effects of TCPP exposure on expression of three apoptotic genes (baxa, bc12a and casp8); (2) B, ROS generation identified as green fluorescence on black background after TCPP exposure; (3) C, lateral view of control and treatment groups after AO staining; (4) D, changes in networks of cranial vasculature; (5) E, quantitative analysis of ROS generation after TCPP exposure; (6) F, relative fluorescence intensity units for AO staining; (7) G, effect of TCPP on AchE activity in larvae; (8) “*”, “**” and “***” indicate significance levels of p < 0.05, p < 0.01 and p < 0.001, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

second messenger in cell apoptosis, and cells receive pro-apoptotic signals causing the accumulation of ROS (Zhang et al., 2016). Larvae in the control group displayed faint fluorescence, whereas TCPP exposure greatly enhanced the fluorescence intensity indicating an accumulation of ROS (p < 0.05 or p < 0.001) (Fig. 4B). ROS in the 15 and 25-mg/L treatments was significantly increased by 1.2 and 4.8-fold, respectively, in comparison to the control group (Fig. 4E). These findings confirm that TCPP exposure caused oxidative stress in larvae due to the scavenging barrier of ROS. Blood vessels and the neural network occur together, and the cranial vasculature is essential for survival and development of the CNS (Khor et al., 2016). The fluorescence image of 120-hpf Tg (flk1: mCherry) zebrafish showed prominent brain vascular ablation (Fig. 4D). Intersegmental vessels (ISVs) displayed a typical “S” shape in the control group, whereas they became nearly linear in TCPP treatments. Additionally, the ISVs were comparatively slim and vague in TCPP treatments (Fig. 4D). The branching pattern of arterial blood vessels plays an important role in supplying nutrients and transporting metabolic products for all tissues. Blood vessels and nerve fibers are distributed throughout the body in a systematic pattern. The crooked body shape following TCPP exposure might result from abnormalities in arterial blood vessel development and further contribute to abnormal swim patterns. Consequently, the abnormalities in vascular development, expression of nerve-related genes and locomotive behavior mutually contributed to TCPP-triggered neurodevelopmental toxicity.

These neurodevelopmental toxicity phenomena motivated further exploration of whether or not TCPP induced changes in neurotransmitters and related biomarkers. AChE activity was slightly suppressed at the low TCPP concentration (5 mg/L), but significantly inhibited in the 25 mg/L treatment (p < 0.05) (Fig. 4G). AChE activity is characterized as a biomarker of neurotoxicity in aquatic organisms by playing a role in ACh degradation, a key cholinergic neurotransmitter (Brinza et al., 2020). Thus, our findings indicate that TCPP contributed to developmental toxicity and altered locomotive behavior by disrupting the cholinergic nerve system. 3.5. TCPP impact on neuromasts and hair cells The zebrafish lateral line system is an important sensory organ derived from the skin and contributes to sensory functions of water flow, pressure and temperature, as well as hearing via converting external acoustic signals into neural electrical stimulations in the brain. Therefore, it plays an important role in training, predation and direction discrimination by providing responses to various stimuli (Liu et al., 2019). Neuromast is the basic unit of the lateral-line system and is widely distributed in the head, trunk and tail (Fig. 5A) with a fixed pattern of development (Hamilton et al., 2014). Thus, it is an excellent model to study nervous system development, regeneration and apoptosis of hair cells. Through FM™1-43 dye staining and ultrastructural observation of hair cells by SEM, the neuromast L1–L6 in segment and 7

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T1–T3 in tail of the control group displayed clear fluorescence and orderly arrangement along the lateral line. In sharp contrast, the trunk and terminal neuromasts of the 15 and 25-mg/L treatments exhibited an irregular distribution and imperfect development, such as deficiency of L5–L6 and T2–T3 (Fig. 5B). Similar abnormal phenomena also occurred in the head. SEM observations documented morphological changes in hair bundles of L1–L6 neuromast (white arrow of Fig. 5C). In the 15 and 25-mg/L treatments, hair bundle morphology became sparse and short, with additional hair cell defects and structural deformity of neuromast (Fig. 5C). These abnormal neurodevelopmental incidences lead to aberrant neurobehavior and responses to sound/light stimuli in larval zebrafish.

treatment relative to the control group (p < 0.01 or p < 0.001) (Fig. 6B). The number of transitions between the upper and lower tank showed no difference at 5 mg/L; however, the number of transfers and the activity time in the upper region were significantly reduced in 15 and 25-mg/L treatments (p < 0.001) (Fig. 6A and C). Consequently, adult zebrafish exposed to TCPP decreased their motility and increased their preference for quiet and isolation in the bottom portion of the tank (Fig. 6A).

3.6. Abnormal behavior of adult zebrafish caused by TCPP exposure

A series of CPP tests examined chronic TCPP exposure on panic reaction, risk avoidance, behavioral preference, visual sensitivity and color discrimination. Adult zebrafish prefer relatively dim or soft colored areas rather than bright colored areas, and they have strong risk avoidance (Wang et al., 2016). Relative to the control group, distance traveled and velocity were decreased at the highest TCPP concentration (25 mg/L) (p < 0.05 or p < 0.001), but showed no significant difference at 5 mg/L (Fig. 6A and D). Notably, the number of transitions to the white side monotonically increased with increasing TCPP concentrations (Fig. 6E) and reached a significantly higher level (p < 0.01) in the 15 and 25-mg/L treatments. Time spent in the brown side gradually decreased concomitant with increasing time spent in the white area as TCPP-exposure concentration increased (Fig. 6F). In general, higher TCPP concentrations induced a higher frequency to swim over the black dangerous area. These results provide strong evidence that TCPP

(ii) Conditioned place preference (CPP): TCPP reduces discrimination and risk avoidance.

Four behavioral tests investigated the effects of TCPP exposure on adult swim behavior with four endpoints recorded for each test: velocity, distance traveled, numbers of transition to the left side and time spent in the left side (Table S4). (i) Bottom dwelling test (BDT): TCPP-treated zebrafish prefer lower tank compartment Zebrafish in the control group actively shuttled between the upper and lower layers of the tank with a high movement rate and no discernable preference for upper vs. lower layers. Upon TCPP exposure, mean swim velocity and distance traveled decreased with increasing TCPP concentrations, reaching an ~40–50% reduction in the 25 mg/L

Fig. 5. Lateral-line neuromast staining in 120-hpf zebrafish by FM 1-43. Note: (A) lateral-line system pattern in normally developed zebrafish; (B) neuromast staining after TCPP exposure (×63); (C) SEM images of L1 neuromast hair bundles with different concentrations of cisplatin; (D) L1–L6 represents the trunk neuromast on one side of larval zebrafish; T1–T3 represents the terminal neuromast on one side of larval zebrafish; green arrows point to each trunk neuromast and yellow arrows point to the terminal neuromast formed in the tail. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 8

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Fig. 6. Effects of TCPP exposure on 90-dpf adult behavior and AchE activity in zebrafish brain. Note: (1) A, heatmap of 90-dpf adult zebrafish behavior; (2) B, mean velocity and distance traveled; C, number of transitions to upper compartment and time in the upper tank; (3) D, mean velocity and distance traveled; E, number of transitions to white side of the CPP test; F, time in each side in the CPP test; (4) G, mean velocity and distance traveled in the social interaction test; H, number of transitions to the social area and time in the social area for the social interaction test; (5) I, mean velocity and distance traveled; J, number of transitions to the right side and time in the right side; (6) K, effect of TCPP on AchE activity in the brain; (7) B–C, novel tank test; D–F, CPP test; G–H, social interaction test; I–J, T-maze test; (8) A, ‘S’ arrow, start; ‘R’ arrow, bait zone (right side); ‘W’ arrow, stimulating zone (left side); (9) blue to green = lower frequency, yellow and red = higher frequency (For interpretation of colored in this legend, the reader is referred to the web version of this article); (10) “*”, “**” and “***” indicate significance levels of p < 0.05, p < 0.01 and p < 0.001, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

swam to the right area (Fig. 6A; ‘r’ represents the decoy area) and remained there for capturing bait. This demonstrates that zebrafish had a strong memory capacity and quickly distinguished the bait arm. With increasing TCPP-exposure concentrations, the mean velocity and distance traveled were significantly decreased (Fig. 6I; p < 0.01 or p < 0.001), as was the number swimming to and staying in the bait arm (p < 0.01 or p < 0.001) (Fig. 6J). Notably in the 25 mg/L treatment, zebrafish had a greater tendency to swim to the risk area than to the bait arm (Fig. 6A). Based on these results, we posit that TCPP exposure affects zebrafish memory, judgment, cognitive functions and survival competitiveness.

exposure altered zebrafish swim behavioral habits/capacity, such as preferred place, color discrimination and risk evasion. (iii) Social interaction behavior: TCPP induced depression, seclusion and non-sociability Zebrafish are social and cooperative species (Wang et al., 2016). To assess the risk of autism caused by TCPP exposure, a series of social interaction metrics evaluated social capacity, clustering and psychology. In the control group, average speed and distance traveled were similar to those observed in the BDL and CPP tests (Fig. 6G), indicating that zebrafish movement was active and vigorous. Total swim time in the social area was significantly higher than that in the background (non-social) area. With increasing TCPP concentrations, swim velocity was decreased by 12.8–43.6%, and both of the number of visits to social areas and time spent in social areas significantly decreased (p < 0.05 or p < 0.001); the response was especially severe in the 25-mg/L treatment (p < 0.001; Fig. 6H). The control group zebrafish preferred social and clustering settings rather than autism style. At 5 mg/L, the time spent in social and non-social settings was both relatively higher, demonstrating a restless state, while the zebrafish in the 25 mg/L treatment preferred a quiet and isolated life consisting of an unsociable, eccentric, lonely and silent routine (Fig. 6A).

3.7. Histopathological injury of zebrafish brain caused by chronic TCPP exposure Zebrafish diencephalon is composed of optic tectum, tegmental and semicircular occipital bones, among which the optic tectum region contains stratum marginale (SM) layer, stratum centrale (SC) and periglomerular gray zone (PGz) (Ito et al., 2010). The nervous system contains two main types of cells: neurons and glial cells. HE staining of adult brain indicated ventriculomegaly (“b”), decreased number of neurons (“a”), glial cell proliferation and formation of glial scars (“c”) (Fig. 7A). Histopathological observation showed that TCPP exposure led to a decreased number of neurons in the periglomerular gray zone (PGz), and induced chromatin margination phenomenon (Fig. 7A). Meanwhile, we observed formation of many vacuoles along with loose and hollow fiber structures in the 15 and 25-mg/L treatments, confirming the formation of glial scars and inflammation. Furthermore, the number of neurons in the SC ant TSv areas was slightly reduced (Fig. 7A). Histopathological injury of diencephalon caused by TCPP exposure might be a mechanism contributing to abnormal behavior and poor memory in adults.

(iv) T-maze experiment: TCPP exposure decreased memory and survival competitiveness T-maze experiments evaluated zebrafish memory, recognition and response sensitivity to an external tapping (frightening stimuli) and food bait (attracting stimuli). After a 10-min training using food bait and tapping stimuli, the mean velocity and distance traveled by zebrafish in the control group were normal and lively. They quickly 9

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Fig. 7. Histopathological observations on adult zebrafish brain and expression changes and translocation of PAX6 and Nova1 by IF analysis in adult brain under TCPP exposure. Note: (1) A, HE dyeing of adult zebrafish brain; (2) “a arrow” shows the decreased number of neurons in TCPP-exposure treatments; “b arrow” shows ventriculomegaly; “c arrow” shows glial cell proliferation and the formation of glial scar; (3) B, the first, third, fourth and fifth rows are the confocal images of brain sections in the three TCPP treatments and control groups; the second row is the magnified images of the red box in the first row (×400); (4) SM, stratum marginale; SC, stratum centrale; PGz, periglomerular gray zone and (5) IF denotes abbreviation of immunofluorescence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

utilized a series of behavioral parameters to assess abnormal expression changes of locomotor- and neurodevelopment-related genes at transcription, translation and protein levels. Reduction in spontaneous movement indicates that the larval CNS may be injured or developmental progress delayed by TCPP exposure. Notably, TCPP exposure prominently inhibited larval swim activity and response to light-dark stimulation. The vertebrate OFF-retinal and ON-retinal ganglion cells were affected by TCPP exposure. Transcription factor pax6a has a prominent effect on eye development (Takamiya et al., 2020). Altering light-dark cycles at a short interval produces a consistent pattern of locomotive activity in zebrafish (MacPhail et al., 2009). Both WISH and qPCR results indicated that TCPP exposure altered the transcription level of pax6a. In addition to slow locomotive activity, TCPP exposure elicited slow turning behavior, especially for rotation times associated with clockwise orientation during dark periods (Zhang et al., 2017). Moreover, TCPP triggered increased ROS levels in zebrafish brain, which possibly contributed to a stress-elicited reduction in locomotive activity. Consequently, zebrafish, as a model organism for neurodevelopment, displayed early-stage neurological injury and may serve as an appropriate model for human risk assessment. Neurotransmitters play a crucial role in CNS development, and their imbalance causes many neurological disorders. Several studies indicate that environmental contaminants have adverse effects on neurotransmitters in zebrafish (Li et al., 2018). AChE is an efficient enzyme in acetylcholine metabolism to choline, and is often used as a biomarker for exposure to neurotoxicant organophosphate pesticides (Sarasamma et al., 2018). Herein, we found TCPP exposure significantly decreased

3.8. Effect of TCPP on expression and location of PAX6 and Nova1 To elucidate the underlying molecular mechanism(s) regarding TCPP-induced brain injury, we determined protein expression changes in PAX6 and Nova1 by IF in zebrafish midbrain tissues. Compared to the control group, TCPP exposure caused significant down-regulation of Nova1 and PAX6 (p < 0.05, p < 0.01 or p < 0.001) concomitant with decreasing fluorescence intensity in the SC brain region (Fig. 7B and Fig. S5B). In contrast, expression of the two proteins showed no significant difference in the SM and PGz areas among the 5, 15 and 25mg/L treatments. Protein localization is important to studies of protein expression and modification in functional proteomics. In normal conditions, nova1 and pax6a proteins were all located in nucleus, which were displayed in the columns of DAPI staining and Merge (Fig. 7B) and quantified in Fig. S4B. Moreover, zebrafish brain functional disorder was further evidenced by inhibited AChE activity (p < 0.01 or p < 0.001) upon TCPP exposure (Fig. 6K). This was especially pronounced in the 25 mg/L treatment where AChE activity decreased by ~68.7% compared to the control group. Thus, long-term TCPP exposure triggered pronounced functional disorders in zebrafish brain. 4. Discussion Zebrafish behavioral assessment is a powerful, efficient and simple tool for neuroscience research. Behavioral response is often used as a sensitive indicator of toxicological impact within neurons (Azizullah et al., 2011). To evaluate TCPP's neurotoxicity to zebrafish larvae and elucidate possible molecular mechanisms for abnormal behavior, we 10

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zebrafish AChE activity, thereby altering neuromuscular activity and behavioral response. Overall, our results demonstrated that TCPP exposure induced zebrafish developmental neurotoxicity via inhibition of cholinesterase. Hair cells in lateral-line neuromast are similar to human inner ear hair cells in structure and function, and thus they are used to evaluate the ototoxicity of drugs (Liu et al., 2018). Based on neuromast staining, the number of neuromasts in the pLL decreased upon TCPP exposure, suggesting that TCPP exposure led to ototoxicity in zebrafish. Ear function in zebrafish was evident in behavioral analysis; zebrafish took longer to respond to sound stimulation at high-level TCPP exposure. For 120-hpf zebrafish in TCPP treatments, a decrease in the number of neuromasts in the pLL demonstrated that TCPP severely affected development of hair cells. The qRT-PCR results showed that TCPP exposure affected the expression of marker genes and apoptosis-related genes in larvae. These expression changes may present as brain function disorders as further evidenced by WISH and AO staining. Expression of key genes and proteins related to neurodevelopment was confirmed as an important factor contributing to developmental neurotoxicity (Chen et al., 2012). Mechanisms related to TCPP exposure on neurodevelopment were assessed by examining changes in expression of key genes and proteins, such as nova1, which plays an important role in neuronal differentiation and synaptogenesis (Jelen et al., 2007). We found TCPP exposure led to abnormal expression of two proteins: pax6a and nova1. Downregulation of these genes and proteins indicates a linkage between TCPP exposure and developmental neurotoxicity that affects cytoskeleton formation, axon growth, neurotransmitter release and neuron differentiation. The testing of a series of behavioral functions can reflect the effects of pollutants on the nervous system. Although effects of TCPP exposure on adult zebrafish behavior have not been reported, previous studies have demonstrated that ethanol exposure to adult zebrafish altered their behaviors, including decreased anxiety, shoaling and response to predator; these changes could be associated with impairment or death of neuronal cells, and/or impairment of the ocular system (Cole et al., 2012). In this investigation, TCPP exposure affected zebrafish memory capacity, which was reflected in the T-maze experiment. Decreased memory suggests dysfunction of nerve cells, which may contribute to Alzheimer's disease; it is a central nervous system degenerative disease mainly appearing as progressive cognitive and memory impairment (Dong et al., 2020). The behavior of Alzheimer's disease is aloof, eccentric, lonely and silent routine, which is consistent with the results of our social behavior test. Meanwhile, this also reflects the change of living habits in BDT and CPP experiments. Previous studies reported on a variety of behavioral tests and models, among which the light-to-dark stimulation, CPP, T-maze and BDT tests were the most popular models for zebrafish behavioral studies. Investigations on zebrafish anxiety-like behavior and light-dark rhythm demonstrated that the expression changes of neuro-apoptotic and circadian clock genes were closely related to anxiety (Liu et al., 2018; Sarasamma et al., 2018). Our results were consistent with these investigations, which demonstrated that CPP, BDT and SI were linked with anxiety- or autism-like diseases. Although there are a large number of behavioral endpoints, an integral parameter that reflects the level of zebrafish abnormal behavior is still lacking. Therefore, creation of an integral index based on multiple factors and endpoints may be warranted. Histopathological observations demonstrated that TCPP exposure inhibited development of primary motor neurons such as pyknosis of the nucleolus and cavitation of cytoplasm in the brain and spinal cord. These findings provide insights for understanding the neurotoxic effects of chronic TCPP exposure. We posit that the tissue damage documented in histopathological observations might be an appropriate metric for evaluating behavioral abnormalities. Although TCPP is ubiquitous in environmental matrices, there is a paucity of data regarding its toxicology. This investigation systematically evaluated the toxicity effects of acute

and chronic TCPP exposure on zebrafish neurodevelopment and locomotive behavior, and infers several underlying toxicity mechanisms. Our findings provide a benchmark for environmental risk assessment to TCPP exposure, and further suggest therapies conducive to prevention and treatment of pollutant-induced nervous system diseases. 5. Conclusions We systematically investigated the toxic effects of acute and chronic TCPP exposure on zebrafish neurodevelopment and neurobehavior. Acute TCPP exposure resulted in a series of developmental toxicities, such as decreased hatching and survival rates, and increased malformation rates; and behavioral abnormalities, including reduced locomotive activity in dark stimulation and slow/insensitive responses to sound and light in larvae. Chronic TCPP exposure triggered excessive apoptosis and ROS accumulation during early larval development, and abnormalities in sensory and neurotransmission organs, such as formation and development of neuromasts and hair cells. These phenotypic malformations and abnormal neurodevelopmental manifestations mainly resulted from expression changes in nerve-related genes and oxidative stress induced by TCPP. Adult zebrafish showed a series of abnormal behaviors upon long-term TCPP exposure, such as anxiety, hyperactivity, restlessness, excessive panic, autism and unsocial, memory disorder and cognitive impairment symptoms. These neurobehavioral abnormalities were mainly attributed to the changes in the expression and distribution of neuro-related genes, further inhibition of AChE activity and neurotransmitter production. These molecular changes manifest as histopathological injury and formation of glial scars in the CNS. These findings offer beneficial insights for performing risk assessment and early warning indicators for the toxicological effects of TCPP, and further enhance our understanding of molecular mechanisms regulating zebrafish neurotoxicity in responses to pollutant stress. CRediT authorship contribution statement Min Xia: Conceptualization, Methodology, Data curation, Writing original draft. Xuedong Wang: Writing - review & editing. Jiaqi Xu: Visualization, Investigation, Supervision. Qiuhui Qian: Writing - review & editing. Ming Gao: Visualization, Investigation, Supervision. Huili Wang: Visualization, Investigation, Supervision. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was jointly supported by the National Natural Science Foundation of China (31770552 and 32071617), the Natural Science Foundation of Jiangsu Province (BK20191455) and the Graduate Research and Practice Innovation Program of Jiangsu Province (KYCX19_2029). Also, the authors thank Professor Randy A. Dahlgren (Davis, University of California) for his correction of the whole text in English language. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2020.143694. References Azizullah, A., Richter, P., Häder, D., 2011. Comparative toxicity of the pesticides carbofuran and malathion to the freshwater flagellate Euglena gracilis. Ecotoxicology 20, 1442–1454. https://doi.org/10.1007/s10646-011-0701-6. 11

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