Comparison of the aquatic toxicity of diquat and its metabolites to zebrafish Danio rerio

doi:10.21203/rs.3.rs-4982746/v1

Download PDF

Article

Comparison of the aquatic toxicity of diquat and its metabolites to zebrafish Danio rerio

https://doi.org/10.21203/rs.3.rs-4982746/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Diquat (DQ) is a non-selective, fast-acting herbicide that is extensively used in aquatic systems. DQ has been registered as the substitute for paraquat due to its lower toxicity. However, the widespread presence of DQ in aquatic systems can pose an ecological burden on aquatic organisms. Additionally, DQ can degrade into its metabolites, diquat-monopyridone (DQ-M) and diquat-dipyridone (DQ-D) in the environment, whereas, the ecological risks of the metabolites remain uncertain. Herein, the aquatic ecological risks of DQ and its metabolites were compared using zebrafish as model non-target organism. Results indicated that DQ and its metabolites did not induce significant acute toxicity to zebrafish embryos at environmental relevant levels. However, exposure to DQ and DQ-D resulted in oxidative stress in zebrafish embryos. DQ treatment led to increased levels of reactive oxygen species (ROS), malonaldehyde (MDA), and glutathione (GSH) in the embryos, while DQ-D enhanced internal MDA and GSH levels. Moreover, the activities of the antioxidative enzymes, superoxide dismutase (SOD) and catalase (CAT), were significantly suppressed by DQ and DQ-D. Besides, the expression levels of antioxidative-related genes (Mn-SOD, CAT, and GPX) were disturbed accordingly after DQ and DQ-D treatments. These findings underscore the importance of a more comprehensive understanding of the ecological risks associated with pesticide substitutions and pesticide metabolites. Such knowledge is crucial for significant improvements in pesticide regulation and policy-making in the future.

Earth and environmental sciences/Environmental sciences

Earth and environmental sciences/Hydrology

diquat

pesticide metabolites

oxidative stress

ecological risk

zebrafish

Diquat (DQ) is a fast-acting, nonselective herbicide widely used for controlling vegetation in both terrestrial and aquatic environments¹. DQ has been widely used worldwide by virtue of its excellent herbicidal effect². DQ has gained popularity due to its effectiveness in weed control and its lower toxicity compared to paraquat, which has led to its replacement and widespread use as a herbicide in agriculture and household settings^{3, 4}. As one of the few herbicides registered for direct application in aquatic systems, DQ inevitably ends up in aquatic ecosystems by transfer processes, like run-off, drainage, and leaching, after repeated application near or in aquatic environments^{4, 5}. Previous study has reported that the detected concentrations of DQ were within a range of 0.002–0.038 mg/L in 114 surface water samples⁶. Environmental levels of DQ, after applications in agricultural fields or bodies of water to control weed growth, usually remain between 0.1-1.0 mg/L and often exceed recommended limits due to misuse or improper application practices⁷. Besides, DQ was also considered persistent in the water with a default dissipation time (DT₅₀) of 1000 days⁸. In some case, the DQ concentrations might maintain stable in the hydrosphere for a long time. Moreover, DQ residual in aquatic systems tended to be accumulated in weeds within a range of 0.6–2.4 mg/g (dry weight)⁹. Consequently, the presence of DQ residues in aquatic environments would be very likely to pose an ecological burden to aquatic organisms¹⁰.

Despite the weaker toxicity comparing with paraquat, DQ would still pose unexpected adverse effects to the aquatic ecological system. Sanchez et al. reported that hepatic enzymes responsible for xenobiotic metabolism in three-spined stickleback (Gasterosteus aculeatus L.) were altered after treated with DQ of 222 and 444 µg/L¹¹. DQ also induced large disturbance on the protein profiles in the liver of rainbow trout (Oncorhynchus mykiss)¹². It was reported that DQ significantly inhibited the growth of adult freshwater snail (Lymnaea stagnalis) and impaired their progeny development at the concentration of 222.2 µg/L¹³. DQ could also disturb the normal development stages of Xenopus metamorphosis and resulted in reduced lengths of the fore and hind limb¹⁴. In addition, Wang et al. had reported that DQ would disturb the behavior of zebrafish (Danio rerio) larvae¹⁵. Generally, the complex exposure of DQ induced intricate response patterns in aquatic organisms and led to more uncertainties and conflicts about its ecotoxicity.

The pesticide residues in the environment are subsequently degraded into various metabolites through various processes, including hydrolysis, photolysis, oxidation, and biodegradation¹⁶. Interestingly, some metabolites of pesticides exhibited more frequent appearance in the environment comparing with their corresponding parent compounds¹⁷. More worryingly, pesticide metabolites tend to exhibit increased environmental mobility and persistence with the occurrence of degradation, which would in return result in more potent adverse effects on the human beings and ecosystems comparing with their parent compounds^{18, 19}. Zhang et al. had reported that the hydroxylated metabolite of chlorothalonil induced more portent toxicity and endocrine disrupting effects¹⁹. Ji et al. also compared the endocrine disrupting effects of four commonly used pesticides (benalaxyl, fenoxaprop-ethyl, malathion, and pyriproxyfen) as well as their 21 metabolites, finding that approximately half of the metabolites exhibited stronger endocrine disrupting effects¹⁶. Unfortunately, the risk assessments of the pesticide metabolites are still given too little attention for the registration and use approval of pesticides, making the risks associated with pesticide metabolites an emerging issue and a scientific blind spot. For now, a few studies had reported the presence of DQ metabolites, and detection of DQ metabolites in tissues revealed that diquat-monopyridone (DQ-M) and diquat-dipyridone (DQ-D) were the main metabolites in biological materials^{20, 21}. Regrettably, few studies have concerned about the toxicity of DQ-M and DQ-D, despite the prevalent adverse effects induced by DQ. Due to the extensive use of as a contact herbicide for controlling aquatic weeds, there is a high probability of exposure of aquatic organisms to DQ and its metabolites²². Hence, it’s of great urgency to conduct the risk assessments of DQ and its metabolites to aquatic organisms.

Zebrafish has been widely used as a vertebrate model to evaluate the adverse effects of contaminants due to its unique advantages in high throughput screening, high fecundity, short reproductive cycle, rapid development, transparency during embryonic stages, orthologous genes with human beings, and so on²³. Besides, the Organization for Economic Cooperation and Development (OECD) has achieved great progress in standardizing testing guidelines for the zebrafish embryo toxicity (FET) test (OECD 203, OECD 236)²⁴. In addition to the primary development endpoints, external contaminants would induce more profound adverse damage to organisms. Exposure to xenobiotics or toxic environmental pollutants may induce an unbalance between reactive oxygen species (ROS) generation and antioxidant defenses and subsequently lead to oxidative damage in organisms²⁵. Therefore, oxidative stress evaluation has become an important endpoint in aquatic toxicology²⁶.

Herein, valuable morphometric endpoints (body length, heart rate, death rate, and malformation rate) at different stages along zebrafish embryo development as well as additional types of data about the molecular and biochemical responses (contents of oxidative damage biomarkers, activities of enzymatic antioxidants, and expression levels of related genes) to DQ and its metabolites (DQ-M and DQ-D) were collected to compare their aquatic toxicity. These findings can help to understand the potential ecological risks of DQ and its metabolites to aquatic organisms more comprehensively and contribute essential data to guide the scientific use of DQ.

2.1 Materials and reagents

Diquat (DQ, CAS#: 85-00-7, > 95.0% purity), diquat-monopyridone (DQ-M, CAS#: 54016-01-2, > 95.0% purity), and diquat-dipyridone (DQ-D, CAS#: 35022-72-1, > 95.0% purity) were purchased from Toronto Research Chemicals Co., Ltd (Canada). Diquat-D4 (DQ-D4, CAS#: 347841-65-0, > 95.0% purity) was purchased from Tianjin Alta Technology Co., Ltd (China). Detailed information regarding to DQ, DQ-M, DQ-D, and DQ-D4 are listed in Table S1. DQ, DQ-M, and DQ-D were dissolved in Milli-Q water with a stock concentration of 10^− 1 M and then diluted to working concentrations for subsequent assays.

2.2 Fish handling and exposure

Adult wide type AB zebrafish used in the present study were purchased from Institute of Hydrobiology, Chinese Academy of Sciences. Zebrafish husbandry, management, embryo collection, and all experimental protocols (developmental toxicity parameter statistics, oxidative damage biomarkers measurements, enzymatic antioxidants assessments, oxidative damage related genes transcription evaluations) were conducted strictly following the ARRIVE Essential 10 guidelines and approved by the Institutional Animal Care and Use Committee of Zhejiang Shuren University. The authors confirm that all methods were performed in accordance with the relevant guidelines and regulations. In order to distinguish the toxic effects between DQ and its metabolites, twenty zebrafish embryos (0.5-1.0 hpf) were randomly divided into 24-well plates containing 2 mL exposure solutions (3 replicates). Exposure concentrations were Hank’s embryo culture media (negative control), environmentally relevant concentrations for DQ, DQ-M, and DQ-D (10^− 7-10^− 5 M). Exposure solutions were renewed every day followed by a semi-static scenario to avoid concentration fluctuation²⁷. Embryo hatching is recorded on a daily basis starting from 48 hpf and observations are recorded every 24 h. At the end of exposure (96 hpf), developmental toxicity parameters including survival rates, deformity rates, body length, and heart rate were calculated. Following exposure, the embryos were anesthetized by MS-222 and flash-frozen in liquid nitrogen for subsequent assays.

2.3 Exposure concentration verification

To avoid the fluctuation of exposure concentrations, exposure solutions in all groups were collected for exposure concentration verification every 24 h after water renewing. Verification of the exposure concentrations of target chemicals were conducted using ultraperformance liquid chromatography (UPLC) in tandem with a micromass triple quadrupole detector (MS/MS) according to previous study^{20, 28}. Briefly, 1 mL exposure solutions collected at different time points (0 h, 24 h, 48 h, and 72 h) were spiked with 20 µL internal standard (500 ng/mL) and undergo centrifugation at 14,000 rpm for 20 min. Then, the supernatants were filtered with 0.22 µm aqueous filter membrane prior to analyze. The supernatants were subjected to an Agilent 1290 UPLC-MS/MS (Agilent, USA) for the quantification of target compounds. The MS/MS was equipped with an electrospray ionization source (ESI) operating in the positive-ion mode. The column was an UPLC® Hilic z column (2.1 mm × 100mm, 2.7 µm, Agilent, USA) and was set to 35 ℃. The mobile phase consisted of acetonitrile and water containing 20 mM ammonium formate as well as 0.1% formic acid. The gradient elution was as follows: 80% organic phase (0–1.0 min), 80%-73% organic phase (1.0–3.0 min), 73%-80% organic phase (3.0–4.0 min), 80% organic phase (4.0–5.0 min). The injection volume was 2 µL and the flow rate was 0.3 mL/min. The temperature of source gas (nitrogen) was 350 ℃ and flow rate was 8 L/min; the sheath gas temperature was 380 ℃ and flow rate was 12 L/min; the nebulizer pressure of 55 psi; capillary voltage of 1.0 kV. The samples were analyzed using full scan and product ion monitoring mode and a multiple reaction monitoring (MRM) method was used for quantification. Data acquisition was controlled using the Agilent Masshunter Software. The limit of detection (LOD) for DQ, DQ-M, and DQ-D were 0.3, 0.3, and 0.9 ng/mL, respectively. The optimized mass parameters for the determination of DQ, DQ-M, DQ-D, and DQ-D4 in positive ion mode are shown in Table S2.

2.4 Contents of oxidative damage biomarkers

Embryonic levels of ROS, malondialdehyde (MDA), and glutathione (GSH) were analyzed to reveal the oxidative stress to zebrafish according to our previous study²⁹. The total protein contents in zebrafish embryos were measured in the first place using Enhanced BCA Protein Kit (BEYOTIME, China) to normalize the actual levels of these oxidative damage biomarkers.

Briefly, two hundred zebrafish embryos were homogenized in Extracting Buffer and the supernatant was collected to record absorption values at 562 nm according to the manufacturer’s instruction. The total protein contents were calculated based on the standard curve. The ROS levels were measured using Reactive Oxygen Species Assay Kit (BOXBIO, China). One hundred zebrafish embryos were homogenized in PBS Buffer and followed by a two-step centrifugation. The sediment resuspended in DCFH-DA probe (1 µmol/L) were incubation in the dark at 37 ℃ prior to microplate reader measurement (excitation 488 nm, emission 525 nm). Internal MDA levels were measured using Malondialdehyde Assay Kit (BOXBIO, China). Briefly, two hundred zebrafish embryos were homogenized with MDA Extracting Buffer in ice bath. The supernatant was mixed with Working Solution and hatched in boiling water bath for 1 hour. Then, the mixture was cooled in ice bath and the supernatant was collected to record absorption values at 450 nm, 532 nm, and 600 nm. The GSH levels in zebrafish embryos were analyzed using Reduced Glutathione Content Assay Kit (BOXBIO, China). Generally, one hundred zebrafish embryos were homogenized Extracting Buffer and the supernatant was collected and incubated at room temperature for 2 min prior to measuring the absorption values at 412 nm.

2.5 Activities of enzymatic antioxidants

Enzyme activities of superoxide dismutase (SOD) and catalase (CAT) were analyzed to assess the neutralizing effects to harmful free radicals in zebrafish according to our previous study²⁹. The enzyme activity of SOD was measured using Superoxide Dismutase Activity Assay Kit (BOXBIO, China). Four hundred zebrafish embryos were homogenized in SOD Extract Buffer in ice bath. The supernatant was collected and incubated at room temperature for 30 min prior to measuring the absorption values at 560 nm. The enzyme activity of CAT was measured using Catalase Activity Assay Kit (BOXBIO, China). Briefly, two hundred zebrafish embryos were homogenized with CAT Extract Buffer in ice bath. The supernatant was collected and mixed with 10 µL Crude Enzyme Solution and 190 µL CAT Working Solution prior to measuring the absorption values at 240 nm at 5 s and 65 s, respectively. All results of the enzyme activities were also normalized by the total protein contents.

2.6 Transcription of oxidative damage related genes

Expression levels of oxidative damage related genes in zebrafish embryos were evaluated to reveal the underlying mechanism. Briefly, twenty zebrafish embryos of different groups were homogenized with TRIzol® Reagent (Life Technologies, USA) for total mRNA extraction. The mRNA was used for cDNA reverse transcription using PrimeScript™ RT Master Mix (Takara, Japan). The qRT-PCR procedure was conducted using Quantstudio™ Real-time PCR System (ThermoFisher, USA) with a final volume of 10 µL. The thermal cycling procedure was set as follows: 37 ℃ for 15 min and 98 ℃ for 5 min; 40 cycles of 95 ℃ for 60 s, 95 ℃ for 15 s, 60 ℃ for 60 s. The relative expression levels were normalized using 2^–∆∆Ct method with β-actin as the reference gene³⁰. The primer sequences of target genes were listed in Table S3.

2.7 Statistical analysis

The results are presented as the mean ± standard deviation (SD) from 3 independent experiments carried out in triplicates. The data were analyzed using Origin 8.0 software (OriginLab Corporation, USA). The levels of significance were set at * p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001.

3.1. Quantification of DQ, DQ-M, and DQ-D in the exposure solutions

Generally, no significant fluctuation was observed in the measured concentrations of DQ, DQ-M, or DQ-D in the water samples comparing with the nominal concentrations. As shown in Fig. 1, the initial mean concentrations of DQ, DQ-M, and DQ-D were in accord with the nominal concentrations, which were 33.73 ± 2.89, 340.05 ± 17.35, 3422.10 ± 261.21 ng/mL for DQ (equal to 9.8×10^− 8 M, 9.9×10^− 7 M, 1.0×10^− 5 M), 27.61 ± 1.25, 278.13 ± 13.36, 2783.33 ± 201.76 ng/mL for DQ-M (equal to 9.9×10^− 8 M, 1.0×10^− 6 M, 1.0×10^− 5 M), and 21.02 ± 1.87, 212.22 ± 16.45, 2129.20 ± 173.42 ng/mL for DQ-D (equal to 9.8×10^− 8 M, 9.9×10^− 7 M, 9.9×10^− 6 M). The mean concentrations of DQ in the water samples during the exposure period were 32.78–33.98 ng/mL, 329.34-336.91 ng/mL, and 3283.60-3381.68 ng/mL, respectively. For DQ-M, the mean concentrations in the water samples during the exposure period were 26.78–26.92, 268.20-275.85, and 2666.90-2751.76 ng/mL, respectively. As for DQ-D, the exposure concentrations also remained constant with the nominal concentrations, which were 20.88–21.13 ng/mL, 206.32-215.94 ng/mL, and 2071.33-2162.91 ng/mL, respectively.

3.2. Developmental toxicity to zebrafish

There may not be much of a difference in the development toxicity to zebrafish embryos induced by DQ, DQ-M, or DQ-D in the detecting concentrations. As shown in Fig. 2, no significant difference was observed in the survival rates, deformity rates, or heart rates of zebrafish embryos after 96-h exposure. However, DQ significantly suppressed the body length of zebrafish larvae at the concentrations of 10^− 5 and 10^− 6 M.

3.3. Contents of oxidative damage biomarkers

DQ, DQ-M, and DQ-D differed in generating oxidative damage biomarkers in zebrafish embryos. The ROS levels in embryos exposed to DQ were 1.07-fold higher than the control group, but DQ-M and DQ-D had no significant effects on the internal ROS levels (Fig. 3A). DQ and DQ-D posed similar effects on MDA and GSH contents. The internal MDA levels in DQ and DQ-D treated groups were 1.48- and 1.19-fold higher than the control group (Fig. 3B). As to GSH levels, DQ and DQ-D promoted GSH production by 12.96% and 16.02%, respectively (Fig. 3C). However, no significant difference was observed in the MDA and GSH levels of DQ-M treated embryos.

3.4. Activities of enzymatic antioxidants

DQ and DQ-D also altered the activities of enzymatic antioxidants except for DQ-M (Fig. 4). For SOD activities, DQ and DQ-D inhibited the enzyme activities by 11.15% and 14.86%, respectively. DQ and DQ-D treatments also significantly suppressed the enzyme activity of CAT in zebrafish embryo, which were 19.70% and 17.88% lower comparing with that of the control group, respectively.

3.5. Expression levels of antioxidative-related genes

The expression levels of antioxidative-related genes were analyzed to monitor the genetic and molecular alterations in zebrafish embryos after exposure to target chemicals. Generally, DQ and DQ-D significantly altered the transcription of antioxidative-related genes, while DQ-M not (Fig. 5). Specifically, DQ and DQ-D enhanced the transcription of GPX by 1.19- and 1.12-fold, respectively. The expression of CAT and Mn-SOD in zebrafish embryos were significantly inhibited with DQ treatment, which were 81.32% and 88.48%, respectively, of the control group. DQ-D also decreased the expression of CAT and Mn-SOD by 13.09% and 15.21%, respectively. However, none of the target chemicals significantly disturbed the transcription of GPX1a and Cu/Zn-SOD within the test concentrations.

The huge consumption of pesticides worldwide has posed serious threats to human beings and ecosystem³¹. As a nonselective, fast-acting herbicide, DQ is one of the few herbicides registered for direct application in aquatic systems, leading to its inevitable presence in aquatic ecosystems through various transfer processes after repeated application^{4, 5}. Previous studies also proved that DQ might maintain in an integrated state in the hydrosphere for extended periods and trend to be accumulated in aquatic organisms^{8, 9}. Given these, the environmental levels of DQ in aquatic system would be pretty high and would be very likely to become ecological burden to aquatic organisms¹⁰. Besides, DQ undergoes degradation via abiotic processes (including chemical and photochemical reactions) and biotic transformation processes (mediated by microorganisms, plants, or animals)³². Sufficient researches have proven that the metabolites of pesticides would pose more potent adverse effects comparing with the parent compounds^16–19. Therefore, it is imminently important for scientist to comprehensively illustrate the potential risks to aquatic organisms induced by DQ and its metabolites.

DQ has been widely used as a substitution for paraquat. Substitution, whose actual ecological risks are yet uncertain, also poses risks when replacing a well investigated pesticide. The urgency to realize the profound effects of pesticides in the environment has aggravated as researches have reflected with warnings about the adverse outcomes induced by applying certain pesticides. As an aquatic herbicide, DQ induced potential risks to non-target aquatic organisms are considerable³³. The determined 96-h LC₅₀ values of DQ on rainbow trout (Oncorhynchus mykiss) juvenile feeding fry (85 d post-hatch) was 9.8 mg/L¹². Similarly, the 96-h LC₅₀ value of DQ to common carp larvae (Cyprinus carpio L.) was 50 mg/L³⁴. Leblanc reported that the 24- and 96-h LC₅₀ values of DQ to mosquitofish were even up to 723 and 289 mg/L, respectively³⁵. As a substitution of paraquat, DQ is well-known for its significantly weaker toxicity³. The 96-h LC₅₀ of paraquat on Mesopotamichthys sharpeyi and catfish (Clarias Gariepinus) were 1.49 and 1.75 mg/L, respectively^{36, 37}. In the current study, apart from DQ suppressing body length at 10^− 5 M (equal to 3.44 mg/L), no remarkable toxicity to zebrafish embryos was observed after exposure to DQ and its metabolites at environmental relevant levels. However, many pesticides currently being used have uncertain effects (sub-lethal effects and conventional apical endpoints) on aquatic organisms than those with well-established toxicity profile. As a low-toxicity substitution of paraquat, DQ had been widely applied in aquatic system. However, the sub-lethal effects induced by DQ and its metabolite to aquatic organisms calls for a deeper and more detailed investigation.

Oxidative stress, resulting from an imbalanced generation of ROS following exposure to contaminants, has become an important issue in aquatic toxicology³⁸. ROS are the production of physiological metabolism of organisms and play a vital role in regulating physiological functions³⁹. ROS levels typically increase sharply under environmental stress and abnormity in ROS levels are closely associated with pathologic changes like inflammation and cell death^{29, 39}. Herein, ROS levels in zebrafish embryos were significantly promoted after DQ exposure, suggesting the oxidative stress induced by DQ and the possibility of significant damage to embryos. Immoderate ROS generation induced free radical attacks on membrane phospholipids can lead to the production of MDA⁴⁰. In the present study, the increased levels of MDA induced by DQ and DQ-D treatments hinted the possibility of oxidative stress. Besides, the higher MDA levels induced by DQ were in accordance with the higher internal ROS levels. The abnormal generation of ROS induced by contaminants could be subsequently eliminated by antioxidant systems in organism, including non-enzyme inhibitors (GSH) and antioxidant enzymes (e.g. SOD and CAT)⁴¹. GSH is well-known for its key role in the detoxification process by forming water-soluble substances with contaminants to be excreted from the body⁴². Therefore, GSH depletion is investigated as an indicator of oxidative stress. Herein, DQ and DQ-D significantly increased embryonic GSH levels. Pesticides such as diazinon and diuron also have been reported to increase GSH level in zebrafish embryos and larvae⁴³. As free radical scavenger, GSH serves as the first defense line for toxic chemicals and offsets the effects of oxidative stress⁴⁴. The increased GSH level implied that DQ and DQ-D caused oxidative damage to zebrafish and activated the antioxidant defense system. However, GSH levels might decrease in certain situations after exposure to contaminants. For example, long-term exposure to fluindapyr significantly down-regulated GSH levels in earthworms²⁹. The contents of GSH in zebrafish increased with low concentration microplastics exposure (0.1 and 1 mg/L) but decreased by higher concentration exposure (10 mg/L)⁴⁵. These might be attributed to the suppression on detoxification process. SOD and CAT are also vital to eliminate excess ROS⁴¹. SOD effectively sustains the oxidative balance by converting ROS to H₂O₂, while CAT subsequently catalyzes H₂O₂ into water and oxygen to protect against the oxidative damage of ROS^{26, 45}. Results showed that enzyme activities of SOD and CAT were significantly suppressed by DQ and DQ-D. The reduction in SOD and CAT activities suggested the compromised protective system of zebrafish embryos and the increasing vulnerability of zebrafish embryos to DQ and DQ-D induced oxidative damage.

Evaluating the transcription of antioxidative-related genes can help to reflect the antioxidant capacity of zebrafish. Cu/Zn-SOD and Mn-SOD encode two types of superoxide dismutase in eukaryocyte³⁸. Herein, the enzyme activity of SOD is the integrated activity of two enzymes. The downregulated expressions of Mn-SOD and CAT were in consistence with the suppressed enzyme activities of SOD and CAT. These findings suggested that SOD and CAT in zebrafish embryos might be impaired by DQ and DQ-D induced oxidative damage, and the down-regulated expression of corresponding encoding genes could account for the suppressed enzyme activities. Peroxidases (GPX, GPX1a) are a kind of cytosolic and mitochondrial isoenzymes responsible for reducing fatty acid hydroperoxides and H₂O₂ by using of glutathione⁴⁶. It was speculated that the up-regulation of GPX was probably to activate antioxidant activities to eliminate free radicals induced by DQ and DQ-D.

The profits of global pesticide application come at the cost of their universal presence in the environment. Abiotic and biotic transformations efficiently eliminate pesticides from the environment but also trigger potentially hazardous metabolites³². Previous studies have reported the presence of various pesticide metabolites in aquatic system with some metabolites exhibiting more frequent detection than their parent compounds⁴⁷. As the global consumption of pesticides can be anticipated to keep increasing, the issue of the ecological risks of pesticide metabolites remains important. For instance, it has been reported that the metabolite (4-hydroxychlorothalonil) of fungicide chlorothalonil could induce higher lethal effect to zebrafish embryo and more complex endocrine disrupting effects¹⁹. Additionally, Ji et al. reported that about half of the tested pesticide metabolites exhibited stronger endocrine-disrupting effects than their corresponding parent compounds¹⁶. In the present study, DQ and its metabolites did not induce significant acute toxicity to zebrafish embryos, but evident oxidative stress was observed after exposure to DQ and DQ-D. Oxidative stress is closely involved in the developmental toxicity during zebrafish embryogenesis⁴⁸. Therefore, the aquatic herbicide DQ and the metabolite DQ-D would inevitably pose ecological risks to aquatic organisms. Though, DQ-D induced sub-lethal effects were lower comparing with that of DQ to some degree. However, pesticide metabolites can achieve higher concentrations due to the bioaccumulation process in the terrestrial food-chain. So, even though the original effect of pesticide metabolites is typically lowered, they may still be a highly relevant issue of concern⁴⁹.

Overall, our study demonstrated that despite the low acute toxicity as paraquat substitution, DQ and its metabolite (DQ-D) still posed oxidative stress to zebrafish embryos by increasing oxidative contents and suppressing antioxidative enzymes, which would induce unavoidable ecological risks to non-target aquatic organisms. Data presented here remind us that there are still room for considerable perfection in pesticide regulation and related policy-making.

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.

Author Contribution

Lanxin Shi: Investigation, Visualization; Xinru Wang: Investigation, Methodology; Yaoyao Dai: Investigation, Simulation and Calculation; Wendong Zhou: Investigation, Visualization; Shenggan Wu: Methodology; Bo Shao: Methodology; Gorettie Nsubuga Nabanoga: Writing-review and editing; Chenyang Ji: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing-original draft, Writing-review and editing; Meirong Zhao: Funding acquisition, Resources, Supervision.

Acknowledgments

This study was supported by National Natural Science Foundation of Zhejiang Province (Q22B070573), Youth Foundation of National Natural Science Foundation of China (22106142).

Data Availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Magalhães, N., Carvalho, F. & Dinis-Oliveira, R. Human and experimental toxicology of diquat poisoning: Toxicokinetics, mechanisms of toxicity, clinical features, and treatment. Hum. Exp. Toxicol. 37, 1131–1160 (2018).
Feng, D., Fu, L., Du, X. & Yao, L. Acute diquat poisoning causes rhabdomyolysis: a case report and literature review. Am. J. Med. Sci. 364. (2022).
Choi, S., Park, Y. & Koh, H. NF-κB/p53‐activated inflammatory response involves in diquat-induced mitochondrial dysfunction and apoptosis. Environ. Toxicol. 33, 1005–1018 (2018).
Pateiro-Moure, M., Arias-Estévez, M. & Simal-Gándara, J. Competitive and non-competitive adsorption/desorption of paraquat, diquat and difenzoquat in vineyard-devoted soils. J. Hazard. Mater. 178, 194–201 (2010).
Siemering, G., Hayworth, J. & Greenfield, B. Assessment of potential aquatic herbicide impacts to California aquatic ecosystems. Arch. Environ. Contam. Toxicol. 55, 415–431 (2008).
Oh, J., Lee, J., Lee, S. & Shin, H. Ultra-trace level determination of diquat and paraquat residues in surface and drinking water using ion-pair liquid chromatography with tandem mass spectrometry: a comparison of direct injection and solid-phase extraction methods. J. Sep. Sci. 37, 2900–2910 (2014).
Ritter, A., Shaw, J., Williams, W. & Travis, K. Characterizing aquatic ecological risks from pesticides using a diquat dibromide case study. I. Probabilistic exposure estimates. Environ. Toxicol. Chem. 19, 749–759 (2000).
European Food Safety Authority. Conclusion on the peer review of the pesticide risk assessment of the active substance diquat. EFSA J. 13 (11), 4308 (2015).
Emmett, K. Final Risk Assessment for Diquat Bromide (Washington State Department of Ecology, 2002).
Xiao, Y. et al. Metabolomics analysis of the potential toxicological mechanisms of diquat dibromide herbicide in adult zebrafish (Danio rerio) liver. Fish. Physiol. Biochem. 48, 1039–1055 (2022).
Sanchez, W., Palluel, O., Lagadic, L., Aït-Aïssa, S. & Porcher, J. Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L). Mar. Environ. Res. 29–33. (2006).
McCuaig, L., Martyniuk, C. & Marlatt, V. Morphometric and proteomic responses of early-life stage rainbow trout (Oncorhynchus mykiss) to the aquatic herbicide diquat dibromide. Aquat. Toxicol. 222, 105446 (2020).
Coutellec, M., Delous, G., Cravedi, J. & Lagadic, L. Effects of the mixture of diquat and a nonylphenol polyethoxylate adjuvant on fecundity and progeny early performances of the pond snail Lymnaea stagnalis in laboratory bioassays and microcosms. Chemosphere. 73, 326–336 (2008).
Babalola, O. & van Wyk, H. Exposure Impacts of Diquat dibromide herbicide formulation on amphibian larval development. Heliyon. 7, e06700 (2021).
Wang, X., Souders, C., Zhao, Y. & Martyniuk, C. Mitochondrial bioenergetics and locomotor activity are altered in zebrafish (Danio rerio) after exposure to the bipyridylium herbicide diquat. Toxicol. Lett. 283, 13–20 (2018b).
Ji, C. et al. The potential endocrine disruption of pesticide transformation products (TPs): The blind spot of pesticide risk assessment. Environ. Int. 137, 105490 (2020).
Berton, T. et al. Development of an analytical strategy based on LC-MS/MS for the measurement of different classes of pesticides and theirs metabolites in meconium: application and characterisation of foetal exposure in France. Environ. Res. 132, 311–320 (2014).
Lu, M. et al. Endocrine disrupting potential of fipronil and its metabolite in reporter gene assays. Chemosphere. 120, 246–251 (2015).
Zhang, Q. et al. The identification of the metabolites of chlorothalonil in zebrafish (Danio rerio) and their embryo toxicity and endocrine effects at environmentally relevant levels. Environ. Pollut. 218, 8–15 (2016).
Fuke, C. et al. Analysis of paraquat, diquat and two diquat metabolites in biological materials by high-performance liquid chromatography. Leg. Med. 4, 156–163 (2002).
Nakajima, M. et al. Purification and characterization of diquat (1,1'-ethylene-2, 2'-dipyridylium)-metabolizing enzyme from paraquat-resistant rat liver cytosol. Toxicology. 154, 55–66 (2000).
Jones, G. & Vale, J. Mechanisms of toxicity, clinical features, and management of diquat poisoning: a review. Toxicol. Clin. Toxicol. 38, 123–128 (2000).
Wang, Q. et al. Identification of apoptosis and macrophage migration events in paraquat-induced oxidative stress using a zebrafish model. Life Sci. 157, 116–124 (2016).
Souders, C. L. et al. High-throughput assessment of oxidative respiration in fish embryos: Advancing adverse outcome pathways for mitochondrial dysfunction. Aquat. Toxicol. 199, 162–173 (2018).
Cai, Z. et al. Structural characterization, in vitro and in vivo antioxidant activities of a heteropolysaccharide from the fruiting bodies of Morchella esculenta. Carbohydr. Polym. 195, 29–38 (2018).
Ali, D., Alarifi, S., Kumar, S., Ahamed, M. & Siddiqui, M. Oxidative stress and genotoxic effect of zinc oxide nanoparticles in freshwater snail Lymnaea luteola L. Aquat. Toxicol. 124–125, 83–90. (2012).
Ji, C. et al. Stage dependent enantioselective metabolism of bifenthrin in embryos of zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). Environ. Sci. Technol. 55, 9087–9096 (2021).
Mao, Z. et al. Simultaneous determination of diquat and its two primary metabolites in rat plasma by ultraperformance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study. Forensic Toxicol. 40, 332–339 (2022).
Ji, C. et al. Evaluation of the toxic effects of fluindapyr, a novel SDHI fungicide, to the earthworms Eisenia fetida. Sci. Total Environ. 899, 165697 (2023a).
Ji, C. et al. AhR agonist activity confirmation of polyhalogenated carbazoles (PHCZs) using an integration of in vitro, in vivo, and in silico models. Environ. Sci. Technol. 53, 14716–14723. (2019).
Ji, C. et al. Enantioselectivity in the toxicological effects of chiral pesticides: A review. Sci. Total Environ. 857, 159656 (2023b).
Fenner, K., Canonica, S., Wackett, L. & Elsner, M. Evaluating pesticide degradation in the environment: blind spots and emerging opportunities. Science. 341, 752–758 (2013).
Moreton, M. & Marlatt, V. Toxicity of the aquatic herbicide, reward®, to the northwestern salamander. Environ. Sci. Pollut Res. 26, 31077–31085 (2019).
Chin, Y. & Sudderuddin, K. Effect of methamidophos on the growth rate and esterase activity of the common carp Cyprinus carpio L. Environ. Pollut. 18, 213–220 (1979).
Leung, T., Naqvi, S. & Leblanc, C. Toxicities of two herbicides (Basagran, Diquat) and an algicide (Cutrine-plus) to mosquitofish Gambusia affinis. Environ. Pollut. 30, 153–160 (1983).
Jaddi, Y., Safahieh, A. & Salighehzadeh, R. Determination of lethal range and the median lethal concentration (LC50 96 h) of paraquat on benny fish (Mesopotamichthys sharpeyi). (2014).
Ladipo, M., Doherty, V. & Oyebadejo, S. Acute toxicity, behavioural changes and histopathological effect of paraquat dichloride on tissues of catfish (Clarias gariepinus). Int. J. Biol. 3 (2), 67–74 (2011).
Li, H. et al. Developmental toxicity, oxidative stress and immunotoxicity induced by three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos. Chemosphere. 207, 781–790 (2018).
Pourová, J., Kottova, M., Vopršálová, M. & Pour, M. Reactive oxygen and nitrogen species in normal physiological processes. Acta Physiol. 198. (2010).
Clemente, R. et al. Combination of soil organic and inorganic amendments helps plants overcome trace element induced oxidative stress and allows phytostabilisation. Chemosphere. 223, 223–231 (2019).
Wang, C., Yang, X., Zheng, Q., Moe, B. & Li, X. F. Halobenzoquinone-induced developmental toxicity, oxidative stress, and apoptosis in zebrafish embryos. Environ. Sci. Technol. 52 (18), 10590–10598 (2018a).
Velki, M. & Hackenberger, B. K. Biomarker responses in earthworm Eisenia andrei exposed to pirimiphos-methyl and deltamethrin using different toxicity tests. Chemosphere. 90 (3), 1216–1226 (2013).
Velki, M. et al. Pesticides diazinon and diuron increase glutathione levels and affect multixenobiotic resistance activity and biomarker responses in zebrafish (Danio rerio) embryos and larvae (Environ. Sci. Eur, 2019).
Gu, W. et al. Single and joint toxic effects of waterborne exposure to copper and cadmium on Coregonus ussuriensis Berg. Ecotoxicology. 32, 895–907 (2023).
Ding, N. et al. Polyethylene microplastic exposure and concurrent effect with Aeromonas hydrophila infection on zebrafish. Environ. Sci. Pollut Res. 29, 63964–63972 (2022).
Sadi, G. & Güray, T. Gene expressions of Mn-SOD and GPx-1 in streptozotocin-induced diabetes: effect of antioxidants. Mol. Cell. Biochem. 327, 127–134 (2009).
Reemtsma, T., Alder, L. & Banasiak, U. Emerging pesticide metabolites in groundwater and surface water as determined by the application of a multimethod for 150 pesticide metabolites. Water Res. 47 15, 5535–5545 (2013).
Usenko, C., Harper, S. & Tanguay, R. Fullerene C60 exposure elicits an oxidative stress response in embryonic zebrafish. Toxicol. Appl. Pharmacol. 229 (1), 44–55 (2008).
Boxall, A., Sinclair, C., Fenner, K., Kolpin, D. & Maund, S. When synthetic chemicals degrade in the environment. Environ. Sci. Technol. 38, 368–375 (2004).

No competing interests reported.

Download PDF

Editorial decision: Revision requested
25 Sep, 2024
Reviews received at journal
24 Sep, 2024
Reviews received at journal
19 Sep, 2024
Reviewers agreed at journal
13 Sep, 2024
Reviewers agreed at journal
12 Sep, 2024
Reviewers invited by journal
10 Sep, 2024
Editor assigned by journal
10 Sep, 2024
Editor invited by journal
06 Sep, 2024
Submission checks completed at journal
04 Sep, 2024
First submitted to journal
27 Aug, 2024

You are reading this latest preprint version

Comparison of the aquatic toxicity of diquat and its metabolites to zebrafish Danio rerio

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material and Methods

2.1 Materials and reagents

2.2 Fish handling and exposure

2.3 Exposure concentration verification

2.4 Contents of oxidative damage biomarkers

2.5 Activities of enzymatic antioxidants

2.6 Transcription of oxidative damage related genes

2.7 Statistical analysis

3. Results

3.1. Quantification of DQ, DQ-M, and DQ-D in the exposure solutions

3.2. Developmental toxicity to zebrafish

3.3. Contents of oxidative damage biomarkers

3.4. Activities of enzymatic antioxidants

3.5. Expression levels of antioxidative-related genes

4. Discussion

5. Conclusion

Declarations

Declaration of Competing Interest

Author Contribution

Acknowledgments

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1