Arch. Biol. Sci ., Belgrade, 65 (4), 1447-1457, 2013DOI:10.2298/ABS1304447H
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ECOTOXICITY OF AG-NANOPARTICLES ON TWO MICROALGAE,
CHLORELLA VULGARIS AND DUNALIELLA TERTIOLECTA
AMAL A. HAZANI 1, MOHAMED M. IBRAHIM 2, 3, AFAF I. SHEHATA 1, GEHAN A. EL-GAALY 1, MOHAMED DAOUD 4, DALIA FOUAD 4, HUMAIRA RIZW ANA and NADINE M S MOUBAYED
1 Botany and Microbiology Department, Science College, King Saud University, Riyadh, Saudi Arabia 2
Alexandria University, Faculty of Science, Botany and Microbiology Department, Alexandria, Egypt 3
Science College, King Saud University, Botany and Microbiology Department, Riyadh, Saudi Arabia 4
Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
Abstract – The increasing application of nanotechnology highlights the need to clarify and understand it. In this work, the subacute toxicity of Ag-NPs to the fresh water microalga Chlorella vulgaris and marine microalga Dunaliella tertiolecta were assessed. The effect of Ag-NPs was induced by exposing both algae to increasing concentrations of Ag-NPs (0, 10, 50, 100 and 200 mg/L). Cellular viability and reactive oxygen species (ROS) formation were determined to evaluate the toxic effect of Ag-NPs on algal growth. Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities and lipid peroxidation (MDA) levels in the algal cells varied with the concentration of Ag-NPs suspensions and exposure times (up to 8 d). As a result, 100 and 200 mg/L Ag-NPs caused a statistically significant decrease in cell viability, as well as SOD, CAT and POD activities, and a significant increase in ROS formation and MDA levels in tissues (P <0.05), suggesting that the algal cells exposed to these two concentrations of Ag-NPs suffered from oxidative stress. The extent of depletion of antioxidant enzyme activities and the elevation of MDA in Dunaliella tertiolecta was the greatest, indicating that Dunaliella tertiolecta might be the most susceptible to Ag-NP exposure. These results indicated a potential risk from Ag-NPs released into the aqueous environment.
Key words: Antioxidant, Chlorella, Dunaliella , nanoparticles, oxidative stress
INTRODUCTION
Nanotechnology manipulates matter at a nanoscale (1-100 nm), producing nanoproducts and nanoma-terials (NMs) that can have novel and size-related physicochemical properties differing significantly from those of larger particles (Nel et al., 2006). The novel properties of NMs have been exploited widely for use in medicine (Barnett et al., 2007; Dong and Feng, 2007), cosmetics (Lens, 2009), renewable en-ergies (Wei et al., 2008), environmental remediation (Tungittiplakorn et al., 2004) and electronic devices
(Kachynski et al., 2008). Silver is considered relative-ly harmless to humans. Indeed, silver’s bactericidal properties have been exploited by certain groups commercializing colloidal silver suspensions as ‘health supplements’. The last decade is distinguished by the dramatic growth in production and use of manufactured nanoparticles (NPs). NPs of metal ox-ides such as ZnO and TiO2 are already widely used in personal care products (e.g., sunscreens), coatings and paints; CuO is used in gas sensors, photovoltaic cells, in catalyst applications and in heat transfer na-nofluids. Subsequently, the risk of natural water con-
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tamination by synthetic NPs continuously increases (Klaine et al., 2008).
The environmental impacts of Ag-NPs are as yet unknown. However, previous knowledge on the environmental and physiological implications of exposure to dissolved silver ions and silver salts in fresh- and seawater organisms provides a baseline for assessment and a reason for concern; from this baseline, the potential effects and impacts of Ag-NPs to organisms and ecosystems can be developed. Prior to the interest in NPs, the silver ion (Ag + (aq)) was considered the most toxic form of silver in wa-ter (Ratte, 1999). As with all metals, the chemistry of the surrounding environment affects the association of silver ions with various ligands, in turn influenc-ing bioavailability and toxicity (Luoma et al., 2008; Adams and Kramer, 1998; Erickson et al., 1998). For instance, in freshwater systems organic matter and sulfide, with a high silver affinity, probably dominate Ag speciation and reduce silver bioavailability. In seawater systems the silver chloro complex is highly bioavailable and it is the primary form in waters with a salinity greater than about 3 (Luoma, 2008; Luoma et al., 1995).
Algae species vary widely in their responses to different toxic chemicals (Boyle, 1984). Park et al. (2010) reported that Ag-NPs have selective inhibi-tory effects on the harmful cyanobacterium Micro-cystis aeruginosa, and this alga was more sensitive to Ag-NPs than green algae. Klaine et al. (2008) re-ported that major differences exist in the chemical behavior of nanoparticles in seawater compared to freshwater that will impact on the behavior of na-noparticles and thereby the habitats or organisms being exposed.
As nanoparticles have a large surface area-to-volume ratio, it is thought that Ag-NPs react strong-ly with compartments within and outside of the cell, which may cause problems such as an increase in free radical production, causing oxidative stress that may fatally damage the cells. Nanoparticles’ toxic-ity could be due to the algae cell wall. Perreault et al. (2011) compared aggregate formation in wild-
type Chlamydomonas reinhardtii to its cell wall-deficient mutant exposed 48 h to glycodendrimer-coated gold NPs. They observed that the wild-type Chlamydomonas reinhardtii formed large aggregates while no aggregates were observed when the cell wall was lacking. It was reported that SiO 2 and TiO 2 NPs were able to interact directly with the algal cell surface through adsorption to the cell walls (Van Hoecke et al., 2008; Sadiq et al., 2011). Aggregate formation might reduce the light available to algal cells and thus inhibit their growth (Navarro et al., 2008; Perreault et al., 2011), or alter the cellular ac-quisition of essential nutrients by clogging the walls (Wei et al., 2010).
Nanoparticles affect different microorganisms and induce the generation of free radicals, which re-sults in the deleterious effect on cellular functions. Oxidative stress is an important factor in nanopar-ticle-induced toxicity. Antioxidant enzyme activities such as SOD, CAT and POD, are very sensitive to the stress of pollutants and can be used as an oxidative-stress signal for an early warning of environmental pollution (Nel et al., 2006).
Lipid peroxidation can be defined as the oxida-tive deterioration of cell membrane lipids and has been used extensively as a marker of oxidative stress (Sayeed et al., 2003).
In the present study, we aimed to assess the ef-fects of exposure of freshwater microalgae Chlorella vulgaris and marine microalgae Dunaliella tertiolecta to Ag-NPs. The subacute toxicity effects on the algae were examined, including physiological responses, by documenting the induction of ROS formation and antioxidant enzymes in addition to the lipid per-oxidation (MDA) level.
MATERIALS AND METHODS
Algal culture
The fresh water microalgae Chlorella vulgaris and ma-rine green alga Dunaliella tertiolecta were obtained from the Culture Collection for Algae and Protozoa
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(United Kingdom). The microalga Chlorella vulgaris was grown in sterile BG-11 liquid medium and the stock culture was aerated with bubbling air (Rippka et al., 1979); Dunaliella tertiolecta was cultivated in a seawater growth medium according to McLachlan (1960). The cells were grown under continuous and constant light intensity (100 mmol m _2 s _1, GRO-LUX Aquarium Wide Spectrum fluorescent lamps at 25 oC). An aliquot of algal samples was used when cellular cultures were in their exponential growth phase. We note here that Dunaliella tertiolecta is a cell-wall-lacking alga (Oliveira et al., 1980).
Ag-NP characterization
Silver nanoparticles (Ag-NPs, particle size 50 nm, surface area (30 ± 10) m 2/g, and crystal structure, with a purity >97.0%) were purchased from Sigma-Aldrich Co., Ltd., USA. Ag-NPs suspensions were sonicated for 20 min in a bath-type sonicator (100 W , 40 kHz) to disperse the particles. To investigate the suspension stability of Ag-NPs in water, differ-ent concentrations (5, 10, 25, 50, 100, 200 and 400 mg/l) of Ag-NP suspensions were prepared in tripli-cate. The Ag-NP concentrations in aqueous solution were determined every day for 8 days according to the method introduced by Zhang et al. (2006).
Oxidative stress parameters analysis
According to preliminary test results, the algae were exposed to 10, 50, 100 and 200 mg/L Ag-NPs for 8 days using a semi-static exposure test. The experi-ment was designed to allow for sublethal physiologi-cal effects over the exposure period. The exposure time of 8 days was chosen to enable some physiologi-cal or biochemical responses to the exposure. Algae from three flasks per treatment were randomly col-lected at day 2, 4, 6 and 8, respectively, for biochemi-cal analysis. Algal cells were immediately snap-frozen in liquid nitrogen and stored at –20°C until needed.The viability of cells, as well as reactive oxygen spe-cies formation, was measured and the lipid peroxi-dation level was also measured for the content of malondialdehyde (MDA). All assays were preformed in triplicate.
The frozen cells were rinsed in 9-fold chilled 100 mmol/L, pH 7.8 sodium phosphate buffer solution and homogenized by a hand-driven glass homoge-nizer. The homogenates were centrifuged at 10000 × g at 4°C for 20 min and the supernatant was stored in Eppendorf tubes at 4°C. The prepared supernatants were analyzed for antioxidant enzyme, i.e., superox-ide dismutase (SOD), catalase (CAT) and peroxidase (POD), activities to determine possible effects on oxidative stress and antioxidant defense.
SOD activity was estimated based on its ability to inhibit the reduction of nitroblue tetrazolium (NBT) by superoxide radicals generated by riboflavin ac-cording to the method of Beauchamp and Fridovich (1971). One unit of SOD activity was defined as the quantity of SOD required to produce a 50% inhibi-tion of NBT reduction under the experimental con-ditions.
CAT activity was determined using the method of Beaumont et al. (1990) by measuring the initial rate of the decrease in absorbance at 240 nm as a con-sequence of H 2O 2 consumption over 1 min. Activity was expressed as a unit (one activity unit defined as a 0.01 change in absorbance at 240 nm per min).POD activity was assayed using guaiacol as a hy-drogen donor by measuring the change at 470 nm over 1 min as reported previously by Chance and Maehly (1955). Enzyme activity is defined as one unit (one unit of activity is defined as a 0.01 change in ab-sorbance at 470 nm per min), per gram fresh weight of tissue. Lipid peroxidation was measured using the thiobarbituric acid (TBA) assay by the method of Buege and Aust (1978). The level of lipid peroxida-tion was expressed as μmol MDA/g fresh tissue.
Determination of viable cells
Fluorescein diacetate (FDA) is a non-polar ester that passes through cell membranes. Once inside the cell, FDA is hydrolyzed by esterase (an enzyme present in viable cells) to produce fluorescein, which accumu-lates inside viable cell walls and fluoresces under UV light (Regel et al., 2002). Viability of algal cells was
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estimated using the FDA method (Mayeretal, 1997). Each Ag-NP treatment and the control was treated with 5 mM of FDA in 1 mL of solution. The fluores-cence was measured using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Determination of reactive oxygen species (ROS)
formation 2′,7′-Dichlorodihydrofluorescein diacetate is a cell-permeable non-fluorescent probe. It is de-esterified intracellularly and turns to highly fluo-rescent 2′,7′-dichlorofluorescein upon oxidation; 2′,7′-Dichlorodihydrofluorescein diacetate is used for the rapid quantitation of ROS in response to oxi-dative metabolism. ROS formation was measured using the cell permeable indicator 2′,7′-dichlorodihy-drofluorescein diacetate (Gerber and Dubery, 2003). Cellular esterases hydrolyze the probe to the non-fluorescent 2′, 7′-dichlorodihydrofluorescein, which is better retained in the cells. In the presence of ROS and cellular peroxidases, 2′, 7′-dichlorodihydrofluo-rescein diacetate is transformed to the highly fluo-rescent 2′,7′- dichlorofluorescein (DCF). Each Ag-NP treatment and control was treated with 5 mM of 2′,7′-dichlorodihydrofluorescein diacetate in 1 mL of solution. The DCF fluorescence was measured using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. All the fluorescence data were collected using a fluorescence plate reader.
Statistical analysis
Each treatment was replicated three times for sta-tistical analysis. The results were expressed as mean±standard deviation. The differences between the experimental and control groups were tested for significance using one way analysis of variance (ANOV A). Differences were considered significant at P <0.05.
RESULTS
ROS after exposure to Ag-NP
Exposure of Chlorella vulgaris and Dunaliella terti-
olecta culture to Ag-NPs for 8 days induced an in-crease in intracellular ROS concentrations through-out the experimental period. After 8 days of exposure, ROS formation increased in Chlorella vulgaris and Dunaliella tertiolecta by 68% and73%, respectively, compared to the control (p≤0.05) at 50 mM Ag-NPs (Fig.1). At 100 and 200 mg/L Ag-NPs, ROS forma-tion increased 3.2- and 4.3-fold in Chlorella vulgaris, and 4- and 6-fold in Dunaliella tertiolecta compared to control at the end of experimental period (p≤0.05). Therefore, AgNPs can lead to toxicological injury through the production of cellular viability and reac-tive oxygen species (ROS).
Viable cells
Algal cell viability for Chlorella vulgaris and Dunaliel-la tertiolecta was evaluated by fluorescein diacetate indicator (FDA), revealing that exposure of the algae to 10-200 mg/L Ag-NPs resulted in a highly signifi-cant reduction of viable cells compared to the control (p<0.05) (Fig. 2). A great reduction in viable cells was observed throughout the experimental period, espe-cially with higher concentrations of Ag-NPs, 100 and 200 mg/L. After 2 days of exposure, the frequency of oxidative stress indices in the exposed algae was elevated gradually with increasing Ag-NP concen-trations, and a significant difference can be observed for the exposure to more than 100 mg/L Ag-NPs compared to the control (Fig. 2). Exposure of algae to 50 mg/L Ag-NPs for 8 days induced a 68 % and 73% decrease of viable cells for Chlorella vulgaris and Dunaliella tertiolecta , respectively, compared to the control (p<0.05). Reduction of viable cells reached 91% and 95% at 200 mg/L Ag-NPs for Chlorella vul-garis and Dunaliella tertiolecta , respectively, after the same duration of Ag-NP exposure.
Oxidative stress and antioxidant defense In our study, there was obvious change in the anti-oxidant enzymatic activities of Chlorella vulgaris and Dunaliella tertiolecta after their exposure to the Ag-NP concentration of 100 mg/L or higher. SOD activities in Chlorella vulgaris and Dunaliella terti-olecta treated with various Ag-NP concentrations
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and exposure times are expressed in Figs. 3A and B. Exposed to 10 mg/L Ag-NP , SOD activities were stimulated and showed a significant increase, which might be due to the synthesis of new enzymes and/or the enhancement of a pre-existing enzyme under lower concentrations. At 50 mg/L, SOD activity ini-tially increased and peaked on day 2 in both treat-ed cells, and on day 6 decreased close to that of the control. This trend in the reduction of SOD activity might be an indication that the antioxidant defense systems of these algae were under stress. However, at 100 and 200 mg/L Ag-NPs, there was a small rise at the beginning and then a sharp decrease in SOD activity, indicating that due to an overproduction of ROS and decreased defense capability, the SOD ac-tivity was inhibited.
CAT and POD are also key enzymes in anti-oxidant defense systems for the conversion of the resulting free radicals H 2O 2 to water and oxygen. According to our results, CAT and POD activi-ties in the two studied algal species fluctuated with concentration and exposure time, respectively, as shown in Figs. 3C-F. After exposure to 10 mg/L Ag-NPs, CAT activity showed a slight decrease up to day 2 and then a remarkable increase. At 50 mg/L, CAT activity slowed down until day 4 and then in-creased close to control level until the end of ex-posure time. However, 100 and 200 mg/L Ag-NPs caused a substantial decrease in CAT activity up to day 6 in both studied species. Results indicated that under the stress CAT activity was inhibited, and ROS scavenging weakened and accumulated gradu-
Fig. 1. Effect of various concentrations of Ag-NPs on reactive oxygen species level/cell viability of Chlorella vulgaris (A) and Dunaliella tertiolecta (B) during 8 days of treatment.
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ally in the cells (Figs. 3 C & D). Like SOD, POD ac-tivities followed a similar pattern in different tissues with a remarkable increase at lower concentrations of Ag-NPs and a considerable reduction at higher concentrations (Figs. 3 E & F). In addition, the CAT and POD activities in Chlorella vulgaris were 2-3-fold and 5-10-fold higher than that in Dunaliella tertiolecta at the same exposure concentration, re-spectively.
The lipid peroxidation indicated by MDA con-tent in both studied species was not obviously differ-ent from that in the control after exposure to 10 and 50 mg/L Ag-NPs, however, a significant increase in
MDA level was found after 6 and 8 d of exposure to 100 and 200 mg/L Ag-NPs (Fig. 4).
DISCUSSION
Knowledge about nanoparticle physicochemistry, as well as biotic and abiotic factors affecting NP behav-ior over time, is necessary for a fuller understanding of the mode of action of Ag-NPs on cells. Indeed, ini-tial results have shown a size- and shape-dependent interaction with prokaryotic organisms, with small (10 nm) and truncated Ag-NPs being the particles most likely to be taken up by microorganisms and affect cellular viability (Choi et al., 2010; Shrivastava
Fig. 2. Effect of various concentrations of Ag-NPs on cell viability of Chlorella vulgaris (A) and Dunaliella tertiolecta (B) during 8 days of treatment.
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et al., 2007). Other sizes and shapes appear to be less damaging to prokaryotes, suggesting that differences in shape and aggregate size could be responsible for some of the variability in observed toxicity, as the metal composition remains the same.
The toxicity of silver nanoparticles (Ag-NPs) to fresh microalgae Chlorella vulgaris and marine microalgae Dunaliella tertiolecta has been examined by measuring some oxidative stress indices such as reactive oxygen species levels and lipid peroxida-
Fig. 3. The effect of various concentrations of Ag-NPs on antioxidant enzymes activities of Chlorella vulgaris and Dunaliella tertiolecta ; SOD, A& B; CAT, C & D; POD, E & F during 8 days of treatment.
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tion indicated by malondialdehyde content, together with the measurement of cell viability. Reactive oxy-gen species (ROS) have been reported to aff ect the physiology, growth and survival of algae (Di et al., 2013). Handy et al. (2008) reported that an aggrega-tion of nanoparticles in seawater is more likely than in freshwater and that the pH of the water may also infl uence the aggregation rate depending on the sur-face charge of the particles involved.
Th e frequency of oxidative stress indices ex-pressed by reactive oxygen species level as well as the cell viability in our studied algae were elevated gradually with increasing Ag-NP concentration, and a signifi cant diff erence can be observed for the ex-posure to more than 100 mg/L Ag-NPs compared to the control. Chlorella vulgaris and Dunaliella terti-olecta cells exposed to the highest Ag-NP concentra-tions exhibited the signs of death aft er a short dura-
tion. Th e phenomenon indicated that physiological changes of algal cells were aff ected by higher Ag-NP concentrations. Similarly, the abnormal behavioral changes in Chattonella marina exposed to Ag-NPs were reported previously (Di et al., 2012).
Reduction of viable cells reached 91% and 95% at 200 mg/L Ag-NPs, respectively, for Chlorella vulgaris and Dunaliella tertiolecta aft er the same duration of Ag-NP exposure. Other studies also showed the low toxicity of Ag-NPs caused a highly signifi cant re-duction in the viability of algal cells (Abdallah et al., 2012)
Algae, like higher plants, possess well-developed antioxidant defense systems for neutralizing the tox-ic eff ects of ROS (Pandey et al., 2003). Th ese defense systems include antioxidant enzymes (e.g., SOD, CAT and POD) and low-molecular weight, non-
Fig. 4: Th e e ff ects of various concentrations of Ag-NPs on malondialdehyde content (MAD) of Chlorella vulgaris (A) and Dunaliella tertiolecta (B) during 8 days of treatment.
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enzymatic antioxidants (e.g., glutathione, GSH and ascorbic acid, ASA) (Vander et al., 2003; Ibrahim and Bafeel 2011).
The SOD-CAT-POD system provides the first defense against oxidative toxicity at a cellular level (Fang and Zheng, 2002). SOD is considered the first enzyme to deal with oxyradicals and to catalyze the dismutation of superoxide radicals O 2.? to O 2 and H 2O 2. The depletion of SOD activity is used as an indication of free radical scavenging ability, showing that the antioxidant defense system is overwhelmed by ROS, and that oxidative stress has occurred (Patra et al., 2009; Krishnaraj et al., 2012).
According to our results, the SOD activity in Chlorella vulgaris were 4-9-fold that in Dunaliel-la tertiolecta at the same exposure concentration, showing that Dunaliella tertiolecta might be the algae more sensitive to Ag-NP exposure. Our results are consistent with the results obtained by Christian et al., 2013, who reported that SOD activity was inhib-ited significantly in wheat plant after Ag-NP treat-ment and caused oxidative stress.
Variations of CAT and POD activities were not the same, but they were coordinated with each other and jointly played roles in the antioxidant defense systems. The significant response of CAT and POD in Dunaliella tertiolecta again indicated that Dunaliella tertiolecta might be the susceptible algae to Ag-NP exposure. Similarly, Renault et al., 2008 showed that CAT activity in two freshwater algal species was sig-nificantly induced after exposure to various concen-trations of gold nanoparticles.
The oxidative deterioration of cell membrane lip-ids has been used extensively as a marker of oxidative stress and estimated by measuring the malondialde-hyde content. Our results indicated that Chlorella vulgaris and Dunaliella tertiolecta , after exposure to different concentrations of Ag-NPs, underwent oxi-dative stress, which was consistent with our results of higher concentration of Ag-NPs exhibiting more potent effects on the antioxidant defense systems in algal cells. Similarly, Di et al., 2012 observed that Ag-
NPs increase reactive oxygen species and cause an orchestrated sequence of synergistic oxidative stress effect in algae.
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How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.
英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn
一般过去式 时间状语：yesterday just now (刚刚) the day before three days ag0 a week ago in 1880 last month last year 1. I was in the classroom yesterday. I was not in the classroom yesterday. Were you in the classroom yesterday. 2. They went to see the film the day before. Did they go to see the film the day before. They did go to see the film the day before. 3. The man beat his wife yesterday. The man didn’t beat his wife yesterday. 4. I was a high student three years ago. 5. She became a teacher in 2009. 6. They began to study english a week ago 7. My mother brought a book from Canada last year. 8.My parents build a house to me four years ago . 9.He was husband ago. She was a cooker last mouth. My father was in the Xinjiang half a year ago. 10.My grandfather was a famer six years ago. 11.He burned in 1991
关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快
1.How to build a business that lasts100years 0:11Imagine that you are a product designer.And you've designed a product,a new type of product,called the human immune system.You're pitching this product to a skeptical,strictly no-nonsense manager.Let's call him Bob.I think we all know at least one Bob,right?How would that go? 0:34Bob,I've got this incredible idea for a completely new type of personal health product.It's called the human immune system.I can see from your face that you're having some problems with this.Don't worry.I know it's very complicated.I don't want to take you through the gory details,I just want to tell you about some of the amazing features of this product.First of all,it cleverly uses redundancy by having millions of copies of each component--leukocytes,white blood cells--before they're actually needed,to create a massive buffer against the unexpected.And it cleverly leverages diversity by having not just leukocytes but B cells,T cells,natural killer cells,antibodies.The components don't really matter.The point is that together,this diversity of different approaches can cope with more or less anything that evolution has been able to throw up.And the design is completely modular.You have the surface barrier of the human skin,you have the very rapidly reacting innate immune system and then you have the highly targeted adaptive immune system.The point is,that if one system fails,another can take over,creating a virtually foolproof system. 1:54I can see I'm losing you,Bob,but stay with me,because here is the really killer feature.The product is completely adaptive.It's able to actually develop targeted antibodies to threats that it's never even met before.It actually also does this with incredible prudence,detecting and reacting to every tiny threat,and furthermore, remembering every previous threat,in case they are ever encountered again.What I'm pitching you today is actually not a stand-alone product.The product is embedded in the larger system of the human body,and it works in complete harmony with that system,to create this unprecedented level of biological protection.So Bob,just tell me honestly,what do you think of my product? 2:47And Bob may say something like,I sincerely appreciate the effort and passion that have gone into your presentation,blah blah blah-- 2:56(Laughter) 2:58But honestly,it's total nonsense.You seem to be saying that the key selling points of your product are that it is inefficient and complex.Didn't they teach you 80-20?And furthermore,you're saying that this product is siloed.It overreacts, makes things up as it goes along and is actually designed for somebody else's benefit. I'm sorry to break it to you,but I don't think this one is a winner.
关于工作的优秀英语演讲稿 Different people have various ambitions. Some want to be engineers or doctors in the future. Some want to be scientists or businessmen. Still some wish to be teachers or lawers when they grow up in the days to come. Unlike other people, I prefer to be a farmer. However, it is not easy to be a farmer for Iwill be looked upon by others. Anyway,what I am trying to do is to make great contributions to agriculture. It is well known that farming is the basic of the country. Above all, farming is not only a challenge but also a good opportunity for the young. We can also make a big profit by growing vegetables and food in a scientific way. Besides we can apply what we have learned in school to farming. Thus our countryside will become more and more properous. I believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. All the working position can provide him with a good chance to become a talent. 1 ————来源网络整理，仅供供参考
●I wonder if it’s because I have been at school for so long that I’ve grown so crazy about going home. ●It is because she wasn’t well that she fell far behind her classmates this semester. ●I can well remember that there was a time when I took it for granted that friends should do everything for me. ●In order to make a difference to society, they spent almost all of their spare time in raising money for the charity. ●It’s no pleasure eating at school any longer because the food is not so tasty as that at home. ●He happened to be hit by a new idea when he was walking along the riverbank. ●I wonder if I can cope with stressful situations in life independently. ●It is because I take things for granted that I make so many mistakes. ●The treasure is so rare that a growing number of people are looking for it. ●He picks on the weak mn in order that we may pay attention to him. ●It’s no pleasure being disturbed whena I settle down to my work. ●I can well remember that when I was a child, I always made mistakes on purpose for fun. ●It’s no pleasure accompany her hanging out on the street on such a rainy day. ●I can well remember that there was a time when I threw my whole self into study in order to live up to my parents’ expectation and enter my dream university. ●I can well remember that she stuck with me all the time and helped me regain my confidence during my tough time five years ago. ●It is because he makes it a priority to study that he always gets good grades. ●I wonder if we should abandon this idea because there is no point in doing so. ●I wonder if it was because I ate ice-cream that I had an upset student this morning. ●It is because she refused to die that she became incredibly successful. ●She is so considerate that many of us turn to her for comfort. ●I can well remember that once I underestimated the power of words and hurt my friend. ●He works extremely hard in order to live up to his expectations. ●I happened to see a butterfly settle on the beautiful flower. ●It’s no pleasure making fun of others. ●It was the first time in the new semester that I had burned the midnight oil to study. ●It’s no pleasure taking everything into account when you long to have the relaxing life. ●I wonder if it was because he abandoned himself to despair that he was killed in a car accident when he was driving. ●Jack is always picking on younger children in order to show off his power. ●It is because he always burns the midnight oil that he oversleeps sometimes. ●I happened to find some pictures to do with my grandfather when I was going through the drawer. ●It was because I didn’t dare look at the failure face to face that I failed again. ●I tell my friend that failure is not scary in order that she can rebound from failure. ●I throw my whole self to study in order to pass the final exam. ●It was the first time that I had made a speech in public and enjoyed the thunder of applause. ●Alice happened to be on the street when a UFO landed right in front of her. ●It was the first time that I had kept myself open and talked sincerely with my parents. ●It was a beautiful sunny day. The weather was so comfortable that I settled myself into the
尊敬的领导，老师，亲爱的同学们， 大家好！我是5班的梁浩东。今天早上我坐车来学校的路上，我仔细观察了路上形形色色的人，有开着小车衣着精致的叔叔阿姨，有市场带着倦容的卖各种早点的阿姨，还有偶尔穿梭于人群中衣衫褴褛的乞丐。于是我问自己，十几年后我会成为怎样的自己，想成为社会成功人士还是碌碌无为的人呢，答案肯定是前者。那么十几年后我怎样才能如愿以偿呢，成为一个受人尊重，有价值的人呢？正如我今天演讲的题目是：自主管理。 大家都知道爱玩是我们孩子的天性，学习也是我们的责任和义务。要怎样处理好这些矛盾，提高自主管理呢？ 首先，我们要有小主人翁思想，自己做自己的主人，要认识到我们学习，生活这一切都是我们自己走自己的人生路，并不是为了报答父母，更不是为了敷衍老师。 我认为自主管理又可以理解为自我管理，在学习和生活中无处不在，比如通过老师，小组长来管理约束行为和同学们对自身行为的管理都属于自我管理。比如我们到一个旅游景点，看到一块大石头，有的同学特别兴奋，会想在上面刻上：某某某到此一游话。这时你就需要自我管理，你需要提醒自己，这样做会破坏景点，而且是一种素质低下的表现。你设想一下，如果别人家小孩去你家墙上乱涂乱画，你是何种感受。同样我们把自主管理放到学习上，在我们想偷懒，想逃避，想放弃的时候，我们可以通过自主管理来避免这些，通过他人或者自己的力量来完成。例如我会制定作息时间计划表，里面包括学习，运动，玩耍等内容的完成时间。那些学校学习尖子，他们学习好是智商高于我们吗，其实不然，在我所了解的哪些优秀的学霸传授经验里，就提到要能够自我管理，规范好学习时间的分分秒秒，只有辛勤的付出，才能取得优异成绩。 在现实生活中，无数成功人士告诉我们自主管理的重要性。十几年后我想成为一位优秀的，为国家多做贡献的人。亲爱的同学们，你们们？让我们从现在开始重视和执行自主管理，十几年后成为那个你想成为的人。 谢谢大家！
关于工作的英语演讲稿 【篇一：关于工作的英语演讲稿】 关于工作的英语演讲稿 different people have various ambitions. some want to be engineers or doctors in the future. some want to be scientists or businessmen. still some wish to be teachers or lawers when they grow up in the days to come. unlike other people, i prefer to be a farmer. however, it is not easy to be a farmer for iwill be looked upon by others. anyway,what i am trying to do is to make great contributions to agriculture. it is well known that farming is the basic of the country. above all, farming is not only a challenge but also a good opportunity for the young. we can also make a big profit by growing vegetables and food in a scientific way. besides we can apply what we have learned in school to farming. thus our countryside will become more and more properous. i believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. all the working position can provide him with a good chance to become a talent. 【篇二：关于责任感的英语演讲稿】 im grateful that ive been given this opportunity to stand here as a spokesman. facing all of you on the stage, i have the exciting feeling of participating in this speech competition. the topic today is what we cannot afford to lose. if you ask me this question, i must tell you that i think the answer is a word---- responsibility. in my elementary years, there was a little girl in the class who worked very hard, however she could never do satisfactorily in her lessons. the teacher asked me to help her, and it was obvious that she expected a lot from me. but as a young boy, i was so restless and thoughtless, i always tried to get more time to play and enjoy myself. so she was always slighted over by me. one day before the final exam, she came up to me and said, could you please explain this to me? i can not understand it. i
高中英语~词性~句子成分~语法构成 第一章节：英语句子中的词性 1.名词：n. 名词是指事物的名称，在句子中主要作主语.宾语.表语.同位语。 2.形容词;adj. 形容词是指对名词进行修饰~限定~描述~的成份，主要作定语.表语.。形容词在汉语中是（的）.其标志是: ous. Al .ful .ive。. 3.动词：vt. 动词是指主语发出的一个动作，一般用来作谓语。 4.副词：adv. 副词是指表示动作发生的地点. 时间. 条件. 方式. 原因. 目的. 结果.伴随让步. 一般用来修饰动词. 形容词。副词在汉语中是（地）.其标志是：ly。 5.代词：pron. 代词是指用来代替名词的词，名词所能担任的作用，代词也同样.代词主要用来作主语. 宾语. 表语. 同位语。 6.介词：prep.介词是指表示动词和名次关系的词，例如：in on at of about with for to。其特征：
介词后的动词要用—ing形式。介词加代词时，代词要用宾格。例如：give up her（him）这种形式是正确的，而give up she（he）这种形式是错误的。 7.冠词：冠词是指修饰名词，表名词泛指或特指。冠词有a an the 。 8.叹词：叹词表示一种语气。例如：OH. Ya 等 9.连词：连词是指连接两个并列的成分，这两个并列的成分可以是两个词也可以是两个句子。例如：and but or so 。 10.数词：数词是指表示数量关系词，一般分为基数词和序数词 第二章节：英语句子成分 主语：动作的发出者，一般放在动词前或句首。由名词. 代词. 数词. 不定时. 动名词. 或从句充当。 谓语：指主语发出来的动作，只能由动词充当，一般紧跟在主语后面。 宾语：指动作的承受着，一般由代词. 名词. 数词. 不定时. 动名词. 或从句充当. 介词后面的成分也叫介词宾语。 定语：只对名词起限定修饰的成分，一般由形容
Dear teacher and colleagues: my topic is on “spare time”. It is a huge blessing that we can work 996. Jack Ma said at an Ali's internal communication activity, That means we should work at 9am to 9pm, 6 days a week .I question the entire premise of this piece. but I'm always interested in hearing what successful and especially rich people come up with time .So I finally found out Jack Ma also had said :”i f you don’t put out more time and energy than others ,how can you achieve the success you want? If you do not do 996 when you are young ,when will you ?”I quite agree with the idea that young people should fight for success .But there are a lot of survival activities to do in a day ,I want to focus on how much time they take from us and what can we do with the rest of the time. As all we known ,There are 168 hours in a week .We sleep roughly seven-and-a-half and eight hours a day .so around 56 hours a week . maybe it is slightly different for someone . We do our personal things like eating and bathing and maybe looking after kids -about three hours a day .so around 21 hours a week .And if you are working a full time job ,so 40 hours a week , Oh! Maybe it is impossible for us at