Nonylphenol (NP) is a typical environmental endogenous disrupter with low concentration and high toxicity. This paper describes the mechanism of NP degradation in solution by strong ionization dielectric barrier discharge (SIDBD). Furthermore, the degradation performance of NP by SIDBD was tested by changing the equipment voltage, the initial concentration of NP in aqueous solution, pH, and inorganic ions. Degradation pathways of NP were detected using a high-performance liquid chromatography-mass spectrometer. The biological effects of NP degradation were assessed by detecting indicators of embryonic development in zebrafish (survival rate, fetal movement, heartbeat, the body length, behavior, deformity) and adult fish (sex differentiation, weight, ovarian testes pathological section analysis). The results showed when the input O2 was 5 L/min and the voltage was 3.2 kV, the degradation efficiency of NP can reach 99.0% after 60 min of experiment. Equipment voltage, initial concentration of NP in solution, pH, inorganic ions and other factors can influence the degradation efficiency of NP by DBD. At the higher concentration of NP, the greater influence on embryonic development in zebrafish was noticed. Although the effects of NP on zebrafish sex differentiation were not obvious, it showed significant male weight inhibition and decrease in sperm number.
The possible pathways and mechanisms for degradation of NP by strong ionization discharge were studied.
The higher formation of ·OH was favorable for NP degradation by strong ionization discharge.
Zebrafish was used as a biological model to evaluate the toxicity of strong ionization discharge degraded water.
In recent years, with the development of industry, more and more endogenous disrupting substances have received widespread attention and attracted researchers to mitigate their possible adverse effects on human health and reproduction. Nonylphenol (NP) is recognized as an endogenous disruptor in the environment. It is not easy to degrade and has stable chemical properties. It has estrogen-like effects, which can accumulate in the environment and mutate organisms. Its structure is similar to estradiol, so it can simulate natural estrogen, affect the sex differentiation and reproduction of organisms, and destroy liver, kidney and gonadal tissues of organisms (Ben et al. 2017; Chen et al. 2018).
It has been reported that NP has been detected in surface water of reservoirs and rivers in China, and the concentration of NP has reached 30.05–288.75 μg/L, which is far higher than the standard of 6.6 μg/L. After entering the wastewater treatment plant, a major part of NP in the influents could be removed with an efficiency of about 78.0–90.0% (Fauser et al. 2003). Kuramitz et al. carried out electrochemical experiments on NP with a glass electrode (GE) in the laboratory. Through this method, the removal efficiency of NP in 60 min of electrochemical oxidation process reached 90.0% (Kuramitz et al. 2002). Neamtu et al. studied the photocatalytic degradation of NP in water, and the results showed that the degradation efficiency could reach more than 80.0% (Neamtu & Frimmel 2006). Lu et al. confirmed that NP was degraded by microorganisms in an aerobic environment at 16 °C. However, it was difficult to degrade in the absence of O2 (Lu et al. 2008). In order to solve the problems of traditional degradation methods such as long degradation time, high requirement for facility, complex operation, and incomplete products requiring further treatment, plasma water treatment technology as a high-tech technology had gradually attracted the attention of the research community. The union technology of advanced oxidation technology or new plasma technology with biological methods to deal with estrone (E1), estradiol (E2), ethynyl estradiol (EE2), and estriol (E3) are more reasonable and efficient than other treatment methods (Yang et al. 2011). Gao et al. used dielectric barrier discharge (DBD) plasma to degrade E2. When the concentration of E2 was 100 μg/L, the discharge voltage was 12 kV, and the pH was 5.6, the degradation rate of E2 was 100% after 30 min of reaction (Gao et al. 2013). Low-temperature plasma technology does not require reagents or catalysts, and the degradation process is highly efficient and non-selective, showing great prospects in the treatment of refractory organic compounds. However, there have been few studies on the use of low-temperature plasma technology to treat NP wastewater. This gave us a new idea to treat NP wastewater.
Several studies have reported that reproductive disorders occur in wild milter in rivers that receive sewage. Because the wastewater from sewage treatment plant is a very important point source of NP in a water environment, it is of great significance to study the biochemical relationship of NP in wastewater. Understanding this relationship will help to reveal the estrogenic effects and environmental behavior of NP in wastewater in aquatic environments. In recent years, studies have found that estrogen can affect the mortality, hatchability, heart rate, spontaneous embryo movement and morphology of biological zebrafish in water, and has certain toxicity to zebrafish (Fraysse et al. 2006). NP is a type of estrogen, and has been shown to inhibit testicular growth and cause spinal curvature in zebrafish (Van Landeghem et al. 2012). Quantification of motor activity in zebrafish larvae has been used to assess the neurobehavioral toxicity of chemicals (Selderslaghs et al. 2013). Various studies have focused on the combination of pollutant degradation and toxicology evaluation (Becker et al. 2016). Some studies have evaluated the ecological toxicity of DMP, lincomycin, polyethylene and others by bioluminescent bacteria, algae and zebrafish (Zhao 2014; Jiang & Shan 2015; Zhu et al. 2021). However, there have been few studies on the combination of zebrafish and degradation of NP by DBD. Therefore, we had a new idea to evaluate biotoxicity before and after degradation of NP.
Dielectric barrier discharge devices contain an insulating material in the discharge space known as a ‘barrier’. Its structure is relatively simple and its discharge state is relatively stable. Compared with other ionizing discharge methods, such as microwave discharge, glow discharge and sliding arc discharge, DBD can produce uniform and stable streamer discharge and has a stronger degree of ionization of particles (Wang et al. 2007; Du et al. 2010). The energy of active particles produced by DBD is much higher than that of similar discharge modes (Li et al. 2017; Yi et al. 2021a, 2021b). Therefore, this study adopted DBD to degrade NP wastewater through changing the voltage of equipment, the initial concentration of NP solution, pH of NP solution, inorganic ions and other factors to determine the impacts of DBD on NP degradation. The degradation pathways of NP were detected by high-performance liquid chromatography-mass spectrometry (LC-MS). Because the effects on aquatic organisms of treated water discharged into waterbodies were uncertain, therefore zebrafish was selected as a model organism to reveal the effects of NP on aquatic life, considering factors such as fetal movement, heart rate, body length, behavior, deformity rate, gender differentiation, and body weight.
MATERIALS AND METHODS
NP (99.0% purity, CAS no. 84852-15-3) was purchased from Aladdin (Shanghai Aladdin Biochemical Technology Co, Ltd, China). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO, 97.0% purity) was purchased from Tokyo Chemical Industry Plant Type Association. Methanol (CH3OH), NH3.H2O (27%, CAS no. 1336-21-6), NaOH, HCl, Na2CO3, NaHCO3 and NaCl were purchased from Sinopharm Group, all analytically pure, and all aqueous solutions were prepared in pure water obtained from ultra-pure water systems (UP PLUS-100 L, LICHEN, China).
Strong ionization degradation was performed using the DBD device, as shown in Figure 1, and samples at 0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min were selected for electron paramagnetic resonance spectrometer (ESR) detection without adding any substance. Next, 20 mm of free radical trap DMPO was added to the 1 mL syringe, and 1 mL water sample was absorbed at the sampling port with the syringe for detection. A capillary tube was used to absorb appropriate water samples, and its bottom was sealed with clay, the residual liquid of the pipe wall was wiped out before inserting it into a 5-mm diameter sample pipe for measurement. The first scan parameters are set as: single run, scan times were 20 times, scan points were 2,000 points, scan range was 3,250 G to 3,650 G, microwave energy was set at 15 MW, modulation coil amplitude was set at 2 G, digital gain was set to 12 dB, and scan was started after tuning.
The maximum absorption, wavelength and concentration of the degradation aqueous solution at different times were determined by scanning the whole band with ultraviolet visible spectrophotometer (UV-Vis). The input voltage was set at 3.2 kV and O2 flow rate was set at 5 L/min in the experiment. The degradation efficiency of NP was determined by changing the initial concentration of the NP solution, equipment voltage, pH and inorganic ions contained in the solution. The degradation efficiency of the study, which was shown by Ct/Co (the concentration of NP at t min/the initial concentration of NP), for the specific process of NP degradation pathways was also necessary in the process of NP oxidation. Therefore, the intermediates and final products were analyzed by LC-MS. The specific conditions of LC-MS were as follows: The chromatographic column: a Symmetry® C18 column, 4.6 mm × 250 mm, 5 mm. Mobile phase: methanol (A) and 0.1% ammonia solution (B); gradient elution: 0–2.5 min, 30.9% A-90.0% A; 2.5–3 min, 90.0%–95.0% A; 3–7 min, 95.0% A; 7–10 min, 95.0%–30.0% A; 10–11 min, 30.0% A; Flow rate: 0.8 mL/min; Column temperature: 30°C.
NP was prepared in egg water at an effective concentration of 5 mg/L and stored at 4 °C. The egg water was prepared according to the zebrafish book standard: 0.137 mmol/L NaCl, 5.4 mmol/L KCl, 0.25 mmol/L Na2HPO4, 0.44 mmol/L K2HPO4, 1.3 mmol/L CaCl2, 1.0 mmol/L MgSO4, 4.2 mmol/L NaHCO3. The chemical properties of NaHCO3 aqueous solution are very unstable, and it will decompose into CO2, NaCO3 and H2O at room temperature. Zebrafish embryos were collected within 1 hpf and then randomly assigned to 60 mm dishes (30 embryos per well) with 5 mL of egg water per well. The final exposure concentrations were 2 μg/L, 200 μg/L and 5 mg/L, respectively, with Control (egg water), Control+ (egg water plus 1/50,000 methanol) and 5 mg/L water for 1 h as controls. Each group was repeated in triplicate and grown in a thermostatic incubator of 28.5 °C. Zebrafish embryo survival was measured at 24 hpf and 96 hpf, fetal motility at 24 hpf, heart rate at 48 hpf, hatchability at 96 hpf, and behavioral analysis at 96 hpf. A new treatment plan was changed daily and the dead embryos were removed in time. After treatment with NP, morphological changes in zebrafish embryos were visualized under a stereomicroscope. The tail swing frequency (24 hpf), the survival rates (24 hpf and 96 hpf), the incubation rate (48 hpf) and malformations (96 hpf) of zebrafish embryos development were measured. Then, after washing twice with egg water, the larvae were transferred to a 96-well plate (1 well) and the same volume of egg water was added. Their total distance and mean speed of swimming were monitored in the dark for 10 min, in the bright for 10 min and in the dark for 10 min.
The experiment selected 180 zebrafish with normal development, disease-free and consistent size for 20 days. Then these zebrafish were assigned to six groups: Control (blank groups), Control+ (methanol concentration of 1/50,000), 2 μg/L (tap water), 200 μg/L (maximum tolerance concentration of sexually mature zebrafish as reported in the μg/L (related literature), 5 mg/L and 1 h after degradation (NP had not been detected). Three replicates (10 L glass tanks, 10 fish each) were set for a 70 d exposure.
RESULTS AND DISCUSSION
NP degradation analysis
Detection of free radicals
5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was added to the water samples to capture the radicals produced during the experiment, DMPO reacts with ·OH to form DMPO-·OH, the rate constant for the DMPO-·OH is 109 L/(mol·s) (Kohno 2011). The most commonly used ·OH inhibitor is tert-Butanol. Tert-Butanol is a tertiary alcohol, the oxygen atoms on the hydroxyl group are affected by three power supply groups, electron cloud density is large, hydrogen atoms and oxygen atoms bind firmly, solidly, and are connected to the hydrogen atoms. Thus, tert-Butanol is not easy to oxidize nor dehydrogenate. The results showed that the reaction rate constant of tert-Butanol and ·OH was 5 × 108 L/(mol·s), which was faster than the binding rate of DMPO and ·OH. Therefore, tert-Butanol was used as an inhibitor in this paper.
The distance between AN and AHβ was compared using the peaks obtained by ESR detection with the standard peaks of DMPO-·OH, as shown in Figure 2. To determine the presence of ·OH in the samples, tert-Butanol was added to the aqueous solution at a concentration of 30 mg/L, and it was observed that the hydroxyl radical concentration was reduced without a significant peak. Therefore, it was obvious that the hydroxyl radicals were present in the solution.
NP UV full-band scan
It was found that the maximum absorption wavelength of NP was 275 nm by scanning the NP wastewater at different degradation times using a UV-Vis spectrophotometer. The peak height dropped as time passed, and the concentration of NP lowered. NP degradation efficiency had reached about 95.0% at 30 min, NP characteristic peaks were absent at 60 min, and complete degradation had occurred by default, as shown in Figure 3.
Effects of the initial concentration
The initial concentration of pollutant is always one of the key factors affecting the efficiency of pollutant degradation. This section explores these changes in NP degradation efficiency at different initial concentrations. This was achieved by setting an input voltage of 3.2 kV and O2 flow rate of 5 L/min, at an initial concentration of 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, and the change in degradation efficiency (Ct/Co) over time was measured as shown in Figure 4 below. As the initial concentration decreased, Ct/Co decreased. The content of Ct/Co after 30 min of aqueous NP solution degradation was 47.9%. At this time, the initial concentration of 5 mg/L NP solution Ct/Co was 5.0%, degradation efficiency had exceeded 95.0%. This can be attributed to the fact that when the applied voltage O2 flow and other factors were constant, the output of active particles in the equipment was constant. With the increasing of initial concentration, the competition of NP molecules for active particles also intensified. At the same concentration, the degradation efficiency of NP decreased with the extension of degradation time. It can be speculated that a series of intermediates and by-products generated by NP would also participate in the competition of oxidants during the process of oxidation. In addition, the degradation efficiency of 20 mg/L NP water sample was lower than other groups after 5 min degradation, and the degradation concentration was lower than 10.0% of the original concentration. Therefore, it was speculated that the degradation efficiency was related to the solubility of NP. The solubility of pure NP was only 5.4–8 mg/L. Although methanol was selected as an auxiliary solvent, which improved the dissolution ability to a certain extent, it was difficult to completely dissolve when NP concentration exceeded 15 mg/L in a short time, and NP was not completely dissolved into the water, so the concentration did not change much. When the treatment time was extended to 60 min, the degradation efficiency of each concentration group was higher than 90.0%, which showed that the active particles generating and accumulating by the strong ionization discharge water treatment system can continuously act on the degradation of NP and its by-products.
Effects of voltage
Experimental conditions were set as follows: initial concentration of O2 flow rate was 5 L/min, NP was 15 mg/L, water treatment was 60 L, and circulating water flow rate was 800 L/h. Under different voltages at 2.2 kV, 2.8 kV, 3.2 kV and 3.6 kV, the degradation treatment time was 0 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, respectively. As can be seen from Figure 5, the degradation curve gradually slowed down under the same voltage, indicating that the intermediates generated in the degradation process are degraded simultaneously with NP. Among different voltages, as the input voltage increased, the Ct/Co of the NP decreased, and the degradation efficiency increased. The Ct/Co of NP at 2.2 kV, 2.8 kV, 3.2 kV, 3.6 kV after degradation 60 min was 42.9%, 16.4%, 4.8%, 2.9%, and the degradation efficiency was 57.1%, 83.6%, 95.2%, and 97.2%, respectively. It can be seen that when the applied voltage increased, the electric field intensity became higher, the excited ionization of O2 molecule was enough, and the yield of active particles was higher, and Ct/Co also decreased. However, with the same increase of 0.4 kV, the decrease of Ct/Co in NP from 3.2 kV to 3.6 kV is significantly less than that from 2.8 kV to 3.2 kV. This can be the reason that when the voltage rose to a certain extent, because of the O2 flowed and the presence of O2 molecules in the discharge gap, the external voltage was no longer the limiting factor of the production of active particles. At this time, the yield of active particles became stable and an increase in yield no longer occurred, therefore the degradation efficiency of NP was not greatly improved. Conversely, when the input voltage was 3.2 kV, discharge type was strong ionization discharge, the energy between the plates can ionize O2 into active particles with high-energy state O2, which greatly improved the degradation efficiency.
Effects of the initial pH
Effects of inorganic ions
The relatively representative Cl−, HCO3− and CO32− were selected as variables to investigate the effects of inorganic ions on degradation efficiency during degradation by strong ionization discharge and NP. The degradation results of NP alone and with the presence of these inorganic ions were plotted when the initial concentration of NP aqueous solution was set to 15 mg/L, input voltage was 3.2 kV, and all the inorganic ion concentrations were 5 mg/L.
Mineralization efficiency is an important indicator to measure the degradation capacity of the water treatment process. To some extent, it can also reflect the change of products in the degradation process of pollutants. This section has examined the changes of total organic carbon (TOC) of NP water samples during 60 min degradation, and the curves are plotted in Figure 8. When the initial concentration of NP was 15 mg·L−1 with an applied voltage of 3.2 kV and an O2 flow rate of 5 L·min−1, the degradation efficiency of NP can reach 97.67%. Whereas the TOC removal efficiency is 44.81% when the reaction to 60 min, removal efficiency is stable. This shows that NP is completely degraded but not completely mineralized into H2O, CO2 and small molecule inorganic salts. However, compared to other studies with the TOC removal efficiency of NP using O3, degradation was 26.5%, the TOC removal obtained in the article by SIDBD degradation was better able to degrade NP (Gyu 2017). It may be that because the benzene ring is difficult to degrade, so further analysis of the product structure is needed. Depending on the degradation process, a product containing a benzene ring may be present at 60 min. To determine the specific products present, LC-MS was used for the analysis.
Because the simulated wastewater itself was made up of tap water-dissolved NP, it contained certain impurities, and the treatment system was an open system, and some uncontrollable events in the detection process could occur. Therefore, it was necessary to exclude the impurity in the degradation products in the mass spectrometry analysis. Potential intermediates inferred from the mass spectrometry were detected and compared with past literature data as shown in Figure 9.
According to the analysis of intermediate products, the degradation by the strong ionization dielectric barrier discharge (SIDBD) device mainly occurred in three ways: the carbon bond on the NP alkyl chain is broken by active particles such as O3 and hydroxyl radicals, and hydrogen ions in water to produce C6H4O2. The same process continues to form the alkyl chain, and eventually becomes C6H5O2 to form C6H4O4. Another part of the NP is attacked by ·OH, the C-H bond on the alkyl breaks, and the hydrogen ion is replaced by the hydroxyl group to produce the hydroxyl compound. In addition, benzoquinone in the degradation of aqueous NP solution was not found in this experiment and it was speculated that benzoquinone itself is not stable enough to convert into phenol with the presence of phenol in LC-MS (Garoma & Matsumoto 2009). Finally, in the action of O3 and ·OH, the benzene ring is opened, and the last open ring is oxidized to small molecules carboxylic acid and carbon dioxide (Hu & Xie 2002).
This paper focuses on studying the degradation of NP and the 60 min water-like zebrafish bio-evaluation. Nonylphenol has many intermediates, and no more detailed path analysis was performed in this paper. Therefore, the water samples of NP degradation after 0 and 20 and 60 min were analyzed. Then, the toxicity of 60 min degraded water samples was comprehensively evaluated by zebrafish. At 0 min the material was mainly NP. As shown in Figure 9, the main products of NP degradation at 20 min are C9H12O, C6H4O2, C9H19O, C6H6O, C6H6O2, C9H18 and C9H19O2. At 60 min, nonylphenol and intermediates are degraded simultaneously, and the degradation products are mainly small molecules carboxylic acid, water and carbon dioxide. This result also demonstrates the effect of TOC degradation in Figure 8. Thus, nonylphenol and intermediates do not cause toxic effects on the environment.
Effects of NP on zebrafish embryos and adult fish
Effects of NP on embryonic survival, fetal, heartbeat, and the incubation rate in zebrafish
Many studies had been conducted to check the effects of the cardiovascular development of juvenile zebrafish embryos by detecting phenotypic end points such as heart rate and cardiac rhythm (Zhu et al. 2014). When testing the toxicity of wastewater containing many types of drugs, Jiang et al. found that various drug components contained in the original wastewater would cause abnormal heart rates and other abnormalities of zebrafish embryo, pericardial edema, and seriously affect its incubation rate (Jiang et al. 2013). Carlsson et al. tested the toxicity of a sewage treatment plant collecting wastewater effluent from about 90 drug producing units in the southern Indian city of Hyderabad, showing that fluoroquinolone antibiotics contained in wastewater produced cardiovascular toxicity in zebrafish embryos (Carlsson et al. 2009).
There was no significant difference among all the groups in zebrafish embryos at 24 hpf survival, as shown in Figure 10, but at 96 hpf the experimental groups plummeted, the concentration was lower and the incubation was significantly lower than the control groups. There was no obvious difference between fetal movement and heartbeat at 24 hpf, 48 hpf, but the data in the experimental groups were significantly lower than the control groups, and at the higher concentration, more obvious on the fetal movement and heartbeat. The experimental data showed that the higher the concentration of NP, the greater the impact on zebrafish embryo development, and there was no obvious toxicity to zebrafish embryos 1 h after degradation.
Effects of NP on malformations in zebrafish at 96 hpf
Exposure of zebrafish embryos to NP resulted in significant twisting or kinking of the notochord at 96 hpf. NP might act specifically on the basement membrane at the onset of the chorda mesoderm differentiation thereby weakening the membrane structures at specific regions resulting in distortions or kinks in the notochord. The notochord of the control group (Control), the solvent control group (Control+) and the water-like embryos obtained after 1 h degradation were normal, with no significant difference, as shown in Figure 11. The degree of notochord distortion in embryos with NP concentration of 2 μg/L and 5 mg/L were more severe than that in embryos with a NP concentration of 200 μg/L. It can be concluded that NP had an impact on the notochord development of zebrafish juvenile, and the higher the concentration of NP, the greater the impact on zebrafish juveniles. A 1 h degradation solution had no significant toxicity on zebrafish juveniles, indicating that NP had been basically degraded.
Effects of NP on behavior of zebrafish at 96 hpf
Behavior is the cumulative manifestation of biochemical, physiological and environmental cues, reflecting the consequence of an animal's integrated physiological response and biochemical alterations caused by contamination (Peakall 1996; Sharma et al. 2015). Since behavior links physiological function with ecological processes for a given species, it might provide a useful indicator or biomarker for detecting harmful chemical pollutants (Ciotfelter & Rodriguez 2006). Lu et al. found that high concentrations of marine and locust base caused a significant reduction in autonomous movement of zebrafish embryos at 22 to 30 hpf stages and inhibited the swimming behavior of zebrafish after hatching, when studying the neurodevelopmental toxicity of marine alkaloids (Lu et al. 2014). Cocaine, ethanol, nicotine, and atrazine were found to have effects on zebrafish motility (Lopez-Patino et al. 2008).
In this study, the behavioral effects of methanol and treated ethonylphenol solutions on zebrafish were observed by comparing zebrafish fish treated with Control + and NP solutions with the Control. Only the total action distance and mean velocity in the control groups were compared at 96 hpf, because all the zebrafish embryos in the experimental groups essentially failed to hatch. There was no significant change in behavior in zebrafish embryos among controls groups, which is obvious from Figure 12.
Effects of NP on the sex differentiation of zebrafish
Recent studies had shown that NP may cause damage to the reproductive system of living organisms. Matina et al. found that endocrine disruptors (EDCs) interfered with the estrogen receptors in zebrafish and affected the endocrine and reproductive systems (Matina & Segner 2004). Chronic toxicity resulting from chronic exposure to parental zebrafish is inherited to offspring through reproduction (Yu et al. 2011).
This was a little higher in each experimental groups compared with Control, and Control + , considering that the blank control ratio was 15:13 (none was significant except for the 20 μg/L NP treatment group), as shown in Figure 13. NP exposure at 20 μg/L significantly increased the proportion of zebrafish female (75.0%), while a high concentration of NP (200 g/L) had no significant effects. The results showed that NP had certain effects on sex differentiation of zebrafish, and the effect of low concentration of NP was greater than that of high concentration.
Effects of NP on the body length of zebrafish (A) and body weight of zebrafish (B)
Length and weight are an important sign of zebrafish development, and the growth and development of zebrafish are important signs of the reproductive health. Zhang et al. characterised these factors for 180 days of female and male zebrafish in a circulating exposure system (Zhang et al. 2012). The results showed that zebrafish decreased their body weight, body length and liver body index in both genders. Yu et al. also found that chronic toxicity resulting from prolonged exposure to the parental zebrafish was passed on to offspring through reproduction (Yu et al. 2011). NP can exert a range of effects on zebrafish body length (A) and body weight (B) of zebrafish. This paper makes some records of the zebrafish weight of each group, and the conclusions are shown in Figure 14.
Body weight of zebrafish in all groups are shown in Figure 13 there were no data in the Control+ group compared with the Control, so the solvent group was set as a control. Group comparisons revealed a slight gain in body weight in 20 μg/L compared to Control + , but the effects were not significant, but in both 200 μg/L and 30 min groups compared with Control + , there was apparently a significant inhibition on growth and development in zebrafish. For groups of male and females, it can be clearly found that male body weight was less than female, and male size was less than female, this is a common phenomenon in nature, especially in insects and fish it is more common, from the observed data smaller males in different concentrations of NP aqueous solution were larger, which showed the effects of NP on male growth and development more than female.
Pathological analysis of the ovarian tissue sections
The ovaries are the reproductive organs of females, and follicle formation, maturation of fertilized eggs, steroid production, and steroid hormone synthesis all occur there (Zala & Penn 2004). Ovarian sections of female zebrafish in each group showed oocyte development at each period, with normal development without significant pathological changes as shown in Figure 15. The effect of NP on ovarian development was not significant.
Histopathological analysis was also performed on the male zebrafish gonads, whose gonadal slice shown in Figure 16 showed abundant sperm in the Control with Control+ with spermatid germ cells in each spermatocyte. Although the experimental group also had germ cells in each period, the number of sperm was significantly decreased, and the cell gap appeared. Especially the testicular sections at 200 μg/L were degraded at 30 min, the number of spermatocyte sperm was significantly reduced, fragmented, and large numbers of intercellular spaces appeared. Studies had reported that sex hormones can directly regulate spermatogenesis in fish and maintain a balance at the T and E2 ratios (Hannon & Flaws 2015). Imbalance in T and E2 ratios had been reported to interfere with gonadal development, sex differentiation and reproductive function in fish (Liu et al. 2018). In this study, the ratio of T to E2 in both zebrafish groups was significantly decreased at 200 μg/L and degraded at 30 min, which may also be one of the reasons for the pathology of zebrafish nests. This indicated that the presence of NP had great effects on zebrafish sperm, and the degradation of NP was not complete after 30 min, and still had toxic effects on zebrafish.
NP aqueous solution was degraded by SIDBD, a micro ESR test proved that active particles such as ·OH by SIDBD were produced. When determining the influence of various factors on the degradation efficiency, it was found that the lower initial concentration of NP and lower applied voltages resulted in lower Ct/Co and higher degradation efficiency. The change of pH had little effects on the final degradation efficiency. When Cl−, HCO3− and CO32− were added to the NP solution, it was found that CO32− had the greatest inhibitory effects on NP aqueous solution degradation, while Cl− had the fewest inhibitory effects on NP solution degradation. NP degradation produced seven intermediates, including C9H12O, C6H4O2, C9H19O, C6H6O, C6H6O2, C9H18 and C9H19O2. Ultimately their ring structure was attached and broken and they were finally oxidized into small molecule carboxylic acids and CO2. The degradation efficiency of NP was 95.0% at 30 min, and the degradation efficiency was more than 99.0% when it was degraded for 60 min inferring no toxicity and complete degradation. In the toxicological experiments of NP, it was found that the higher the concentration of NP, the higher the influence on the survival rate, fetal movement, heartbeat, the body length and the malformation rate of zebrafish embryos, but the influence was not significant among the control groups, indicating that water treated for 60 min had no significant toxic effects on zebrafish embryos. NP had no apparent effects on male and female differentiation of juvenile zebrafish, but it showed obvious inhibition of male body weight, testis pathology and sperm number reduction. This experiment showed that the degradation efficiency of NP aqueous solution by DBD became higher and higher with the increasing of time, and even can be completely degraded after 60 min, so there is almost no toxicity after NP degradation which had no effects on zebrafish.
Thanks to the Practice Innovation Program of Jiangsu Province (No. KYCX18_2272), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment for their support of this work.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST STATEMENT
The authors declare there is no conflict.