Abstract This study was conducted to investigate the effects of different mass concentrations of nitrobeneze on the growth， soluble sugar content， soluble protein content， chlorophyll a content and algal cell conductivity of Nitzschia hantzschia. The results showed that as the concentration of nitrobenzene increased， the growth of N. hantzschia was inhibited， and the algal culture liquids gradually changed from dark yellow to light yellow after 5 d of treatment; the soluble sugar content increased after 2 d; and the soluble protein content of the 100 mg/L nitrobenzene treatment group was 89.1% of the control group on day 1， which was the lowest value， and then showed a gradual upward trend. The low-mass concentration of nitrobenzene promoted the chlorophyll a content of algal cells， the medium and high mass concentrations had an inhibitory effect， and the chlorophyll a content of the 50 mg/L treatment gradually recovered after 3 d. The electrical conductivity of algal cells gradually increased with the increase of the mass concentration of nitrobenzene. The electrical conductivity gradually recovered after 3 d of the low mass concentration treatment， while the high mass concentration harmed the algae cells， causing N. hantzschia to gradually die.
　　Key words Nitrobenzene; Nitzschia hantzschia; Physiology; Electrical conductivity; Environmental stress
　　Nitrobenzene compounds are widely used chemical raw materials. According to statistics， more than 30 000 t of pollutants produced by nitrobenzene compounds flow into the environment every year[1]， but the demand for such compounds is showing a clear upward trend with economic development. An investigation by the US Environmental Protection Agency （EPA） found that the nitrobenzene content in the wastewater of organic chemical plants can reach up to 190 mg/L， and more than 3% of the factory wastewater contains more than 100 mg/L[2]. In 2005， domestic accidents such as the explosion of the nitrobenzene workshop of the Longteng Chemical Plant， the explosion of the nitrobenzene device of the Haimen Port Chemical Plant， and the explosion of the Benzene Plant of PetroChina Jilin Petrochemical Company led to the direct leakage of nitrobenzene[3-4]. Nitrobenzene is a highly toxic substance that can harm organisms through contact or enrichment in the food chain[5-6]. Domestic and foreign researches on nitrobenzene compounds mainly focus on their enrichment in organisms and their toxicity and toxicological mechanisms[7-11]. Algae are important primary producers of aquatic ecosystems. Nitzschia hantzschia is a widely adaptive species in freshwater ecosystems， and the stress response of nitrobenzene on its physiological characteristics has not been reported. In this study， the physiological indicators of N. hantzschia in aquatic ecological environment with nitrobenzene pollution were measured to explore the physiological response mechanism of N. hantzschia to nitrobenzenes， aiming to provide a theoretical basis for exploring the environmental impact of nitrobenzene pollutants. 　　Materials and Methods
　　Materials and experimental design
　　N. hantzschia was provided by the Freshwater Alga Germplasm Bank of the Institute of Hydrobiology， Chinese Academy of Sciences. N. hantzschia in the logarithmic growth phase was inoculated into 250 ml Erlenmeyer flasks， which were added with 100 ml HB-D1 medium for culture. The alga was cultured at a temperature （25±1） ℃ with 24 h continuous light， the intensity of which was about 2 000 lx[40 μmol/（m2·S）]， and the alga was placed in an incubator and shaken regularly： 5 times/d. Then， 10， 50 and 100 mg/L nitrobenzene were added on a clean bench in sequence， and three parallel samples were set for each treatment group. Samples were taken at 0， 24， 48， 72， 96， and 120 h to determine the physiological indicators.
　　Determination items and methods
　　Effects of nitrobenzene treatment on the growth of algal cells
　　The absorbance was measured with an ultraviolet spectrophotometer since the transfer culture of the alga. The absorbance of the nitrobenzene-treated algal solutions was measured every 24 h for 5 d continuously， and the average of the parallel samples was taken.
　　Determination of soluble protein content
　　After centrifuging a 5 ml sample， 2 ml of deionized water was added to ultrasonically break the cells， and centrifugation was performed at 4 ℃， obtaining a supernatant for testing. The soluble protein content was determined using the Coomassie Brilliant Blue G-250 method and referring to the method of Bradford et al.[12-13].
　　Determination of soluble sugar content
　　The soluble sugar content was determined by anthrone colorimetry.
　　Determination of chlorophyll a content
　　After centrifuging a certain amount of algal solution at 4 ℃， 95% ethanol by volume was added to extract chlorophyll a for 24 h at 4 ℃ in the dark. Centrifugation was performed at 4 ℃ at a rotation speed of 8 000 r/min. The supernatant was measured for absorbance with an ultraviolet spectrophotometer. According to the formula Ca=1 395D665nm-6.88D649nm， the content of chlorophyll was calculated[14].
　　Measurement of algal cell conductivity
　　A certain amount of algal solution （8 ml） was washed with deionized water at 5 000 r/min for 3 times at room temperature， added with 16 ml of deionized water， and placed in the dark at room temperature for 24 h. At a constant temperature of 20-25 ℃， the conductivity of the solution was measured with a conductivity meter. After measuring the conductivity， it was put in a boiling water bath at 100 ℃ for 15 min， and then measured for its boiling conductivity at a room temperature of 20-25 ℃ after cooling. The damage rate was calculated according to following formula： Damage rate=Treatment conductivity/Boiling conductivity×100%[15]. 　　Results and Analysis
　　Effect of nitrobenzene on the growth of algal cells
　　The growth kinetic curves of N. hantzschia under different nitrobenzene mass concentrations are shown in Fig. 1. The low-mass concentration of 10 mg/L nitrobenzene treatment had no obvious inhibitory effect on the growth of N. hantzschia; the 50 mg/L treatment showed mild growth inhibition within 2 d， and gradually resumed growth after 2 d; and the 100 mg/L nitrobenzene stress significantly inhibited the growth of N. hantzschia. After 5 d， the algal liquids changed from dark yellow to light yellow and then approached transparent. The specific situation is shown in Fig. 2. Compared with the control， the color change effects of the algal solutions were obvious. The effect of nitrobenzene on the growth of N. hantzschia showed a more obvious dose-effect relationship， which is consistent with the effect of Chlamydomonas reinhardtii[15]， while the difference is that under 50 mg/L nitrobenzene stress， N. hantzschia gradually resumed growth after 2 d， while C. reinhardtii gradually resumed growth after 4 d. N. hantzschia showed stronger tolerance under the same stress conditions.
　　Effect of nitrobenzene on soluble sugar content
　　It can be seen from Fig. 3 that the nitrobenzene treatments caused the soluble sugar content of N. hantzschia to drop to the lowest point within 2 d， and then the soluble sugar content in the algal cells showed a gradient increase with the increase of the nitrobenzene mass concentration. It might be because that N. hantzschia's own substances were degraded into soluble sugars to alleviate the damage to algal cells. In the experimental treatment groups， the faster the soluble sugar content of algal cells increased， the higher the mass concentration of nitrobenzene was. At day 5， the 50 and 100 mg/L mass concentration treatment groups were higher than the control， and the 10 mg/L mass concentration treatment group was lower than the control. It might be that under the stress of 50 and 100 mg/L nitrobenzene， the algal cells decomposed their own substances in response to the unfavorable environment， while under the stress of 10 mg/L， the mass concentration of the algal cells was weaker than the control and the damage was weaker， so the algal cells decomposed less carbohydrates.
　　Effect of nitrobenzene on soluble protein content
　　It can be seen from Fig. 4 that on the 1st day， the 10， 50， and 100 mg/L nitrobenzene treatment groups all showed the lowest soluble protein contents， which were 63.7%， 71.7%， and 93.6% of the control content in sequence， and it showed a clear upward trend in 1-3 d. After 3 d， there was no significant difference between the 10 mg/L nitrobenzene treatment and the control. After 4 d， the soluble protein contents of the 10 and 50 mg/L mass concentration treatment groups and the control were in a stable state， and the 100 mg/L mass concentration treatment showed an upward trend， indicating that the algal cells gradually adapted to the environmental stress. 　　Effect of nitrobenzene on the content of chlorophyll a
　　It can be seen from Fig. 5 that the treatment with nitrobenzene at a mass concentration of 10 mg/L had a promoting effect on the chlorophyll a content of N. hantzschia; 50 mg/L nitrobenzene had an inhibitory effect in the first 2 d after the treatment， and showed an increasing trend in chlorophyll a content after 3 d; and 100 mg/L nitrobenzene had an obvious inhibitory effect on the chlorophyll a content of N. hantzschia， and there was no sign of recovery， which was consistent with the light yellow growth of N. hantzschia.
　　Conductivity of nitrobenzene to algal cells
　　Under normal circumstances， biological cell membranes have the ability to selectively permeate substances. When under nitrobenzene stress， the algal cell membranes of N. hantzschia were destroyed， and the membrane permeability became larger， resulting in exosmosis of extracellular cytoplasm， which was directly manifested in the increase of the conductivity of the cell extract. The increase of the conductivity of the cell extract was closely related to the increase of membrane permeability， and the increase of membrane permeability was directly related to the intensity of environmental stress. It can be seen from Fig. 6 that after N. hantzschia cells were treated， compared with the control， as the mass concentration of nitrobenzene increased， the damage rate to the alga showed a gradient increase in each treatment group. In the 10 mg/L nitrobenzene treatment， the algal cells basically returned to normal state after 3 d; the 50 mg/L nitrobenzene treatment gradually recovered after 3 d， and basically returned to normal state at 5 d after treatment; and in the 100 mg/L nitrobenzene treatment， the damage to N. hantzschia cells was not recovered， which was consistent with the situation obtained from the previous growth impact determination.
　　Discussion
　　The response of the physiological characteristics of N. hantzschia to nitrobenzene stress was closely related to the stress concentration. 10 mg/L nitrobenzene had no obvious effect on the growth of N. hantzschia， while 100 mg/L nitrobenzene had a significant inhibitory effect on the physiological growth of N. hantzschia until algal cell death was caused. Although the concentration of nitrobenzene in the natural water environment is usually low， it is still possible for exposure of algal cells to nitrobenzene at a mass concentration of 100 mg/L [16-17]. Because nitrobenzene is widely used， the pollutant nitrobenzene is widespread in major river systems around the world[18-21]， and when nitrobenzene pollution incidents break out， the pollution of nitrobenzene is often higher than 100 mg/L in small environments， and algae may suffer from higher concentration of nitrobenzene stress， thereby inhibiting their growth， further affecting the stability of freshwater ecosystems. 　　This study showed that 10 mg/L nitrobenzene had almost no inhibitory effect on the absorbance of N. hantzschia， while it was difficult to detect live algae cells under the microscope after 6 d at a nitrobenzene mass concentration of 100 mg/L， which is similar to the results on C. reinhardtii. Through the 50 mg/L nitrobenzene treatment， it was found that N. hantzschia cells could adapt to the environment by releasing soluble sugars， soluble proteins and other organic matter under stress. The decrease in soluble sugar content of N. hantzschia indicated that high-mass concentration of nitrobenzene can affect the energy metabolism and utilization of algae， and high-mass concentration stress may reduce the chlorophyll a content of N. hantzschia cells and affect the photosynthesis performance[15].
　　The study on the stress of nitrobenzene on N. hantzschia found that the algal cells might have a lower utilization of nutrient substances in the medium due to environmental stress within 48 h of treatment， which was manifested in the continuous reduction in the soluble sugar content of N. hantzschia cells within 48 h after transfer culture. Compared with the control group at 48 h， the soluble sugar contents of N. hantzschia in the treatment groups were 81.82%， 67.93% and 62.64% of the control group. After 48 h of continuous cultivation， especially after 3 d， the soluble sugar content of algal cells recovered the fastest， indicating that for N. hantzschia under the stress condition of 100 mg/L nitrobenzene， 48 h was the critical point for its gradual recovery of physiological growth. After 4 d， the soluble sugar content rose to about 170 mg/L and was gradually stabilized， indicating that the algal cells could utilize the nutrients in the culture medium and effectively synthesized substances such as sugars after adaptation. It might be because that the ability of the cells to absorb nutrients in the culture medium was reduced in the late stage of algal cell growth， and another factor might be that the nutrients in the culture medium were gradually reduced and restricted. Comparing N. hantzschia and C. reinhardtii under the stress of nitrobenzene[15]， N. hantzschia showed strong and rapid adaptability， and under 100 mg/L nitrobenzene stress， C. reinhardtii gradually and rapidly died， almost no surviving algal cells could be detected at 6 d， but N. hantzschia passively adapted to adversity stress by decomposing its own cytoplasm such as soluble sugar and soluble protein， and gradually recovered physiological growth after 2 d. The study also showed that the soluble protein content of N. hantzschia cells under the stress of low， medium， and high concentration of nitrobenzene within 24 h was significantly lower than that of the control， and after 3 d， each mass concentration treatment group basically returned to a stable state. Soluble proteins and soluble sugars are the main cytoplasm in algal cells， and under nitrobenzene stress， N. hantzschia secreted soluble proteins out of the cell through its own substances to play a good buffering and protective role. If the stress environment is further enhanced， as the secretion of protein from algal cells reaches the tolerance limit， it will directly affect the physiological functions of algal cells， which will hinder the growth of N. hantzschia until death due to cell damage. The photosynthesis site of N. hantzschia is in chloroplasts， and photosynthetic pigments are the material basis. Studies have shown that the content of chlorophyll a among photosynthetic pigments in N. hantzschia responds differently to nitrobenzene stress at different mass concentrations. The content of photosynthetic pigment is significantly reduced under the stress of a higher mass concentration of nitrobenzene， which may be due to the destruction of the cell membrane system function under the stress of nitrobenzene. Studies have shown that 25 mg/L nitrobenzene can damage the plant membrane system[9]， which may be because that the nitro functional group on nitrobenzene binds to the protein in the chloroplast light-harvesting complex system[22]， which leads to the inhibition of the light-harvesting complex in the chloroplast of N. hantzschia， reducing the chlorophyll a content and the photosynthesis capacity and thus affecting the photosynthetic pigment content of N. hantzschia， which is similar to the effects on Euglena gracilis and C. reinhardtii[23]. The photosynthetic pigment content of algal cells and the absorbance during the growth of algal cells also showed good consistency. This study showed that the toxicity of nitrobenzene to the alga might be reduced by first decomposing its own soluble sugars and proteins， and when the concentration of nitrobenzene was high， the alga itself decomposed more of their own substances and denatured the proteins in some of the organelles， thus losing the possibility of continued growth. 　　The damage to the cell membrane of N. hantzschia was aggravated by the high mass concentration of nitrobenzene. The increase in its selective permeability led to increased electrolyte leakage of the cells， resulting in an increase in the damage rate， so the cells had no physiological signs of returning to normal within the test time.
　　Conclusions
　　① After exposing N. hantzschia to nitrobenzene at a mass concentration of 100 mg/L for 1 to 6 d， the damage to the algal cells was difficult to recover， that is， cell death was caused. ② When the mass concentration of nitrobenzene was 10 or 50 mg/L， the soluble protein content of N. hantzschia dropped to the lowest point after 1 d of treatment， the soluble sugar content dropped to the lowest point at 2 d， and then they returned to a stable value. This trend of change reflected the response mechanism of N. hantzschia to nitrobenzene stress. ③ By studying the physiological and biochemical response modes of N. hantzschia cell growth， soluble sugar content， soluble protein content， chlorophyll a content， and electrical conductivity under nitrobenzene stress， the results showed that 10 and 50 mg/L nitrobenzene stress affected the normal physiological state of N. hantzschia cells， which could gradually return to normal physiological growth after response， while the damage of 100 mg/L nitrobenzene stress to algal cells was difficult to recover.
　　References
　　[1]HANKENSON K， SCHAEFFER DJ. Microtox assay of trinitrotoluene， diaminonitrotoluene and dinitromethylaniline mixtures[J]. Bulletin of Environmental Contamination and Toxicology， 1991， 46（4）： 550-553.
　　[2]CAI ZQ， FUJ， DU PH， et al. Reduction of nitrobenzene in aqueous and soil phases using carboxymethyl cellulose stabilized zero-valent iron nanoparticles[J]. Chemical Engineering Journal， 2018（332）： 227-236.
　　[3]WANG C， FENG YJ， ZHAO S， et al. Adynamic contaminant fate model of organic compound： a case study of nitrobenzene pollution in Songhua River， China[J].Chemosphere， 2012， 88（1）： 69-76.
　　[4]ZHU LY， MA BL， LEI Z， et al. The study of distribution and fate of nitrobenzene in a watersediment microcosm[J]. Chemosphere， 2007， 69（10）： 1579-1585.
　　[5]HOLDER JW. Nitrobenzene carcinogenicity in animals and human hazard evaluation[J].Toxicology and Industrial Health， 1999， 15（5）： 445-457.
　　[6]BEAUCHAMP RO， RICHARD DI， DOUGLAS ER， et al. Acritical review of the literature on nitrobenzene toxicity[J]. CRC Critical Reviewsin Toxicology， 1982， 11（1）： 33-84. 　　[7]FLETCHER JS， MCFARLANE JC， PFLEEGER T， et al. Influence of root exposure concentration on the fate of nitrobenzenein soybean[J]. Chemosphere， 1990， 20（5）： 513-523.
　　[8]LIU W， LI PJ， ZHOU QX， et al. Effect of stress from chlorobenzenes on SOD activity of broadbean seedlings[J]. Journal of Agro-Environment Science， 2004， 23（3）： 432-436. （in Chinese）
　　[9]XU YM， YUAN ZH， LI JX， et al. Effects of nitrobenzene and chlorobenzene on seed germination and biological characters of wheat[J]. Journal of Irrigation and Drainage， 2004， 23（5）： 6-9. （in Chinese）
　　[10]LI XX， SHAO H， WANG Z， et al. Effects of nitrobenzene stress on seed germination and seedling physiological characteristic of rice[J]. Seed， 2007， 26（2）： 39-40. （in Chinese）
　　[11]DUNNIVANT FM， SCHWARZENBACH RP， MACALADY DL. Reduction of substituted nitrobenzenes in aqueous solutions containing natural organic matter[J].Environmental Science&Technology， 1992， 26（11）： 2133-2141.
　　[12]BRADFORD MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding[J].Analytical Biochemistry， 1976， 72（2）： 248-254.
　　[13]HUANG WM， XING W， LI DH， et al. Effects of exogenous ascorbic acid on the growth and senescence of tobacco BY-2 suspension cells[J]. Journal of Agro-Environment Science， 2006， 25（5）： 1157-1161. （in Chinese）
　　[14]LI HS. Principles and techniques of plant physiological biochemical experiment[M]. Beijing： Higher Education Press， 2000： 134-137. （in Chinese）
　　[15]DU QC， ZHANG DL， WANG GH， et al. The response of growth and photosynthesis in Chlamydomonas reinhardtii to nitrobenzene[J]. Journal of Northwest Normal University： Natural Science， 2007， 43（3）： 71-74. （in Chinese）
　　[16]ZHONG JQ， ZHANG LY， SUN LB， et al. In-situ remediation of simulated groundwater polluted by high concentration of nitrobenzene[J]. Journal of Agro-Environment Science， 2007， 26（3）： 920-924. （in Chinese）
　　[17]LU GL， GUO GL， WANG SJ， et al. Screening and biodegradation of anaerobic microorganisms for nitrobenzene in water[J]. Journal of Agro-Environment Science， 2010， 29（3）： 556-562. （in Chinese）
　　[18]YURAWECZ M P， BART JP. Identification of chlorinated nitrobenzene residues in Mississippi river fish[J]. Journal of AOAC International， 1983， 66（6）： 1345-1352.
　　[19]YAMAGISHI T， MIYAZAKI T， HORII S， et al. Identification of musk xylene and musk ketone in freshwater fish collected from the Tama River， Tokyo[J].Bulletin of Environmental Contamination and Toxicology， 1981， 26（5）： 656-662.
　　[20]NOORDSIJ A， PUYKER LM， GAAG MD. The quality of drinking water prepared from bank filtered riverwater in the Netherlands[J]. Science of the Total Environment， 1985（47）： 273-292.
　　[21]HE MC， YAN S， LI XR， et al. Distribution patterns of nitrobenzenes and polychlorinated biphenyls in water， suspended particulate matter and sediment from mid- and down-stream of the Yellow River（China）[J].Chemosphere， 2006， 65（3）： 365-374.
　　[22]LIU BY， ZHOU PJ， LI JJ， et al. Effects of γ-HCH on the growth and photosystem and lipid peroxidation of Scenedesmus obliqnus kütz[J]. Journal of Agro-Environment Science， 2006， 25（1）： 204-207. （in Chinese）
　　[23]LU GH， ZHAO YH， YANG J， et al. Bioconcentration and elimination of nitrobenzenes by the algae （Scenedesmus obliquus）[J]. Research of Environmental Sciences， 2001， 14（3）： 4-5. （in Chinese）