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Research ArticleToxic Effects of Ethyl Cinnamate on the Photosynthesis andPhysiological Characteristics of Chlorella vulgaris Based onChlorophyll Fluorescence and Flow Cytometry Analysis

Yang Jiao Hui-Ling Ouyang Yu-Jiao Jiang Xiang-Zhen Kong Wei He Wen-Xiu LiuBin Yang and Fu-Liu Xu

MOE Laboratory for Earth Surface Processes College of Urban amp Environmental Sciences Peking University Beijing 100871 China

Correspondence should be addressed to Fu-Liu Xu xuflurbanpkueducn

Received 29 January 2015 Revised 10 April 2015 Accepted 7 May 2015

Academic Editor Huansheng Cao

Copyright copy 2015 Yang Jiao et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The toxic effects of ethyl cinnamate on the photosynthetic and physiological characteristics of Chlorella vulgariswere studied basedon chlorophyll fluorescence and flow cytometry analysis Parameters including biomass119865V119865119898 (maximal photochemical efficiencyof PSII)BPSII (actual photochemical efficiency of PSII in the light) FDA and PI staining fluorescence were measured The resultsshowed the following (1) The inhibition on biomass increased as the exposure concentration increased 1mgL ethyl cinnamatewas sufficient to reduce the total biomass of C vulgaris The 48-h and 72-h EC50 values were 207mgL (194ndash220) and 189mgL(182ndash197) (2) After 24 h of exposure to 2ndash4mgL ethyl cinnamate the photosynthesis of C vulgaris almost ceased manifestingin BPSII being close to zero After 72 h of exposure to 4mgL ethyl cinnamate the 119865V119865119898 of C vulgaris dropped to zero (3) Ethylcinnamate also affected the cellular physiology of C vulgaris but these effects resulted in the inhibition of cell yield rather than celldeath Exposure to ethyl cinnamate resulted in decreased esterase activities in C vulgaris increased average cell size and alteredintensities of chlorophyll a fluorescence Overall esterase activity was the most sensitive variable

1 Introduction

Algae are the main primary producers in aquatic ecosystemsand are often used as environmental quality indicators [1] andexperimental species for aquatic ecotoxicological research[2 3] Algal bloom is an important cause of the degradation ofaquatic ecosystems in eutrophic water bodies [4] Traditionalchemical methods can effectively remove algae but canalso cause secondary pollutions [5] Meanwhile traditionalphysical technology and biotechnology for bloommitigationalso have some disadvantages such as long operating timeoperating difficulty and high costs [6] In recent years theuse of allelochemicals to inhibit algal blooms has become acost-effective method with broad application prospects [7]

Allelopathy was first introduced by Molisch in 1937 andit means that creatures including microorganisms exerta favorable or unfavorable influence on others throughbiochemical substances Rice constrained allelopathy in hisdefinition in 1979 holding the view that allelopathy merely

meant that plants have adverse effects on other plantsand the biochemical substances were released by organisms[8] Studies have shown that allelopathy is ubiquitous inaquatic ecosystems and that almost all primary producerscan produce and release allelochemicals [9] For practicalapplications there are generally three methods to inhibitalgae using allelopathy of plants (I) Cultivate aquatic plantsin water and utilize the substances that are released by plantsto inhibit algae (II) Add dead plants into the water andutilize the substances that are released during the rot processto inhibit algae (III) Add allelochemicals that have beenextracted from plants into water [6]

In recent years research on the utilization of allelochemi-cals to inhibit algae has become increasingly popular Variousallelochemicals have been extracted and identified such assesquiterpene lactones organic acids and phenols Amongthese allelochemicals phenolic acids (such as ethyl cinna-mate) have strong activities [8ndash11] Allelochemicals couldaffect many photosynthetic and physiological characteristics

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 107823 12 pageshttpdxdoiorg1011552015107823

2 The Scientific World Journal

of algae such as cell membrane permeability reproduc-tion photosynthesis and respiration protein synthesis andresistance to diseases [12ndash20] Allelochemicals affect algaeselectively therefore different species of algae have variedinhibition responses [21ndash24]

Currently research on allelopathy mainly focuses onaquatic plants and rarely on terrestrial plants especiallyxylophyta Compared with hydrophytes terrestrial plantsare more widely distributed In addition terrestrial plantsare easy to cultivate break and process Many species ofherbage and xylophyta are rich in allelochemicals and asa consequence have better prospects in inhibiting algalblooms and the emergency response to algal blooms [25]Theallelochemicals that are produced and released by terrestrialplants can bewashed from land intowater by rain and interactwith organisms in the water Therefore research on theallelochemicals such as ethyl cinnamate that are produced byterrestrial plants has more practical significance in inhibitionof algae However current research on the effects of cinnamicacid and its derivatives on algal inhibition is still rare andmostly utilizes traditional methods of algal toxicology Rel-evant research mainly focuses on the algal reproduction theactivities of algal antioxidant enzymes the lipid peroxidationof cell membrane and the interaction with nutrients suchas N and K [26ndash29] However these traditional methodscannot distinguish between dead and living cells duringcultivation potentially causing variations that deviate fromthe real results [30] In addition traditional researchmethodsof algal toxicity focus more on the toxicity of substances andless on the response of other algal physiological processessuch as photosynthesis It is impossible to understand themechanism of allelopathy comprehensively

Chlorophyll fluorescence analysis and flow cytometry arenewmethods that have been used to study the photosynthetictoxicity and cytotoxicity of heavy metals and other pollutantson algae in recent years [2 3] Chlorophyll fluorescenceanalysis is based on the photosynthetic theory using chloro-phyll fluorescence in vivo as a probe to study the photosyn-thetic physiological state of organisms under the influencesof various external factors [31] Chlorophyll fluorescenceanalysis is rapid and sensitive and it can also detect thephotosynthetic toxicitywith nodamage to algae [31ndash34] Flowcytometry (FCM) an automated cell or biological particlesanalysis technique is capable of rapid multiple parameterdetections of a single cell and has been extensively usedin algal toxicity studies in recent years [35ndash37] Comparedwith traditional methods flow cytometry is conducive toobtaining algal responses under stress conditions on cellularlevel The application of flow cytometry allows for algaltoxicological research on cellular level and this techniquecontributes to understanding the actionmechanismof toxins

In this study experiments were performed to studythe toxic effects of ethyl cinnamate on Chlorella vulgarisa dominant species of green algae This study based ontraditional algal toxicological researchmethods used chloro-phyll fluorescence analysis and flow cytometry to study howethyl cinnamate affected the growth photosynthesis and cellphysiology of C vulgaris in order to understand the effectsand the action mechanism of allelochemicals further

2 Materials and Methods

21 Experimental Materials

211 Algal Strain In this study Chlorella vulgaris wasselected as algal material for its two advantages C vulgarisrequires relatively simple growth conditions and has a strongenvironmental tolerance and a high reproductive rate Inaddition many domestic and overseas researchers have alsoselected C vulgaris as test algal material [38] which makescomparing results convenient C vulgaris was purchasedfrom the Freshwater Algae Culture Collection of the Instituteof Hydrobiology Chinese Academy of Sciences

212 Chemical Materials Ethyl cinnamate (991 purity)was purchased from Alfa Aesar (America) and used in expo-sure experiments of Chlorella vulgaris Dimethyl sulfoxide(analytical grade) was purchased from Beijing ChemicalWorks (China) for the solubilization of ethyl cinnamateMain components in BG11 medium were all purchased fromSinopharm Chemical Reagent Co Ltd (China) for culti-vation of C vulgaris Fluorescein diacetate (FDA) was pur-chased from Sigma (America) for staining algal cells in flowcytometric analysis Acetone was purchased from BeijingChemical Works (China) for the solubilization of FDAPropidium iodide (PI) was purchased from Sigma (America)for staining algal cells in flow cytometric analysis

22 Experimental Methods

221 Experimental Conditions

(1) Algal Culture Conditions Chlorella vulgaris was cultivatedin BG11 medium at 24 plusmn 1∘C with a cycle of light (14 h4000 Lux) and dark (10 h 0 Lux) in a GXZ-280B illuminationcultivation cabinet (China) C vulgaris was cultivated stati-cally and was not aerated during the cultivationThe cultureswere shaken two to three times daily and their positions werechanged randomly

(2) Exposure Experiments Chlorella vulgaris was cultivatedin 50mL BG11 medium in 150-mL Erlenmeyer flasks (theinitial cell density of C vulgaris was approximately 2 times106 cellsmL) C vulgaris was exposed to ethyl cinnamate atfinal total concentrations of 05mgL 1mgL 2mgL 3mgLand 4mgL for 96 h In the experiment 01mL dimethylsulfoxide was added to each sample (50mL in total) for thesolubilization of ethyl cinnamate Preliminary results showedthat 02 dimethyl sulfoxide (01mL in 50mL medium) hadno significant impact on the photosynthetic and physiologicalcharacteristics inC vulgaris Exposure tests and controlswereset up in triplicate The biomass chlorophyll fluorescenceand flow cytometry were measured every 24 h during theexperiments

222 Measurements of the Indicators

(1) Biomass Analysis Optical density of algal samples at450 nm correlated with algal cell density determined by

The Scientific World Journal 3

hemocytometer (cell density (106 cellsmL) = 276 times OD450and 1198772 = 09994) Therefore biomass was obtained by mea-suring the optical density of algal samples at 450 nm using amicroplate reader (Model-680 Bio-Rad) The growth inhibi-tion rate (119875

1) of Chlorella vulgaris was calculated using

1198751 =119873119905control minus 119873119905treatment119873119905control minus 1198730

(1)

In formula (1) 119873119905control and 119873

119905treatment represent theoptical density of the blank control and the treatment attime 119905119873

0represents the initial optical density The medium

effective concentration (EC50) inhibiting 50 the biomassat time 119905 was calculated by fitting 119875

1and ethyl cinnamate

concentration with a linear regression

(2) Chlorophyll Fluorescence Analysis In this study chloro-phyll fluorescence was used to measure the photosyntheticactivity of Chlorella vulgaris Values of chlorophyll fluores-cence are related with the switch state of reaction center ofPSII Chlorophyll fluorescence cannot be detected from deadcellsThus chlorophyll fluorescence can be used as a probe tomonitor the photosynthesis of C vulgaris

The parameters including 1198650 (minimal fluorescence)119865119898(maximal fluorescence) andBPSII (actual photochemical

efficiency of PSII in the light) were measured by MAXI-Imaging-PAM (Walz Germany) after adaptation in the darkfor 25minThe 119865V119865119898 or (119865119898minus1198650)119865119898 (maximal photochem-ical efficiency of PSII) was obtained by calculation What isnoteworthy is that when algal cells are in normal state thenumber of cells has little effect on 119865V119865119898 andBPSII

The parameter ETR (photosynthetic electron transportrate) can be calculated automatically by the instrumentaccording to the formula as follows

ETR = Yieldtimes 084 times 05 timesPAR (2)

Yield is actual photochemical efficiency of PSII in the light(BPSII) 084 is empirical constant (absorption rate of lightfrom blade) 05 is due to photosynthetic system includingtwo centers and electron transport in one center needing twophotons PAR is photosynthetically active radiation

(3) Flow Cytometric Analysis The flow cytometer BD FAC-SCalibur was used to measure the parameters of algal cellsThe negative control (heat-treated cells at 100∘C for 10min)was set before the experiments Before the measurementthe voltage level of each detection channel was adjustedaccording to the fluorescent intensity of the heat-treated cellsand normal cells to make the fluorescent intensity of thenormal algal cells in the middle level while reserving enoughspace for affected cells And during the measurement thevoltage level was constant

Fluorescein diacetate (FDA) was used to assess theesterase activities during the experiments [30 35 37] TheFDA (dissolved in acetone to a final concentration of25 120583molL) stained the algal cells for 8min in the dark andthen the fluorescence in the FL1 channel (515ndash545 nm) wasmeasured An acetone control was added to eliminate effectsresulting fromacetoneThe results demonstrated that acetonehad no effect on the esterase activities of Chlorella vulgaris

0000

0100

0200

0300

0400

0 24 48 72 96Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL

OD450

nm

Figure 1 The effects of ethyl cinnamate on the biomass of Chlorellavulgaris (The values represent the means plusmn 1 standard deviation and119899 = 3)

Propidium iodide (PI) was used to assess the integrity ofthe cell membrane [36 37 39] The PI (final concentration10 120583molL) stained the algal cells for 15min in the dark andthen the fluorescence in the FL2 channel (564ndash606 nm) wasmeasured

Cellular autofluorescence (chlorophyll a fluorescence)was detected in the FL3 channel (gt650 nm) while measuringthe FDA and PI staining fluorescence

23 Data Analysis Using Excel 2010 for statistical calcula-tions the results were expressed as the triplicate arithmeticmean plusmn the standard deviation ANOVA and two-factoranalysis of variance were performed by SPSS 200 Significantdifference levels were set to 005 119901 gt 005 and 119901 lt005 respectively representing no significant difference andsignificant difference

Summit 50 software was used for the data that wasobtained by the flow cytometry the fluorescent analysisof algal cells using histograms and the scatter plots Thefluorescent intensity in channel FL3 could help distinguishbetween algal cells and impurities to reduce the interferenceTo analyze the data the Gate should be set in accordance withthe fluorescent intensity in channel FL3 and the fluorescentintensity of the sample in the Gate could be used to calculatethe mean fluorescent intensity of the algal cells (MFI) and theratio of normal algal cells

3 Results and Discussion

31 Growth Inhibition The effects of ethyl cinnamate on thegrowth of Chlorella vulgaris are shown in Figure 1 Afterexposure to 05mgL ethyl cinnamate and a blank controlfor 96 h the biomass decreased compared with that afterexposure for 72 hHowever under the 1mgL ethyl cinnamatetreatment the biomass increased sustainably The blankcontrol and the 05mgL ethyl cinnamate treatmentmay haveextra interference possibly causing the biomass ofC vulgaris


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Figure 2The photoinduced curves of Chlorella vulgaris after 24 h of exposure to ethyl cinnamate ((a) Blank control (b) 05mgL treatment(c) 1mgL treatment (d) 2mgL treatment (e) 3mgL treatment and (f) 4mgL treatment)


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to be stagnant or even suppressed As a consequence after72 h 05mgL ethyl cinnamate had no significant effect onthe biomass of C vulgaris (119901 gt 005) However with theincreasing concentrations of ethyl cinnamate the degree ofalgal cell yield inhibition gradually increased The exposureconcentrations exposure duration and interaction of the twofactors significantly influenced the biomass of C vulgaris(119901 lt 005) The 48-h and 72-h EC50 of ethyl cinnamate were207mgL (194ndash220) and 189mgL (182ndash197)

The study results of Pinheiro et al [40] on the effectsof microcystin-LR (MC-LR) and cylindrospermopsin (CYN)(they are also regarded by some researchers as allelopathicsubstances) on C vulgaris indicated that MC-LR and CYNat environmentally occurring concentrations were unableto affect negatively growth of C vulgaris This could bedue to the fact that these molecules played roles otherthan allelopathy in natural ecosystems or the selectivity ofallelochemicals

32 Photosynthetic Toxicity The photoinduced curves ofChlorella vulgaris after exposure to ethyl cinnamate for24 h are shown in Figure 2 After exposure to the 05mgLethyl cinnamate for 24 h the 119865

0 119865119898 and 119865

1198981015840 decreased

by 41 69 and 09 respectively (Figure 2(b)) Thedecrease degree of chlorophyll fluorescence increased withthe concentration of ethyl cinnamate When the concentra-tion of ethyl cinnamate reached 2mgL the 119865

0 119865119898 and 119865

1198981015840

decreased by 121 356 and 234 respectivelyWhen theconcentrations of ethyl cinnamate were more than 2mgLthe chlorophyll fluorescence of C vulgaris was significantlyinhibited Although there was potential for photosynthesisthe actual photosynthetic activities were low with fluctua-tions of chlorophyll fluorescence curves close to 0 for specificperformance (Figures 2(e) and 2(f))

The photoinduced curves of C vulgaris under the blankcontrol and ethyl cinnamate treatment after 96 h are shownin Figure 3 05mgL and 1mgL ethyl cinnamate had littleimpact on the chlorophyll fluorescence of C vulgaris Com-pared with those of blank control the 119865

0 119865119898 and 119865

1198981015840 of

C vulgaris exposed to 05mgL ethyl cinnamate decreasedby 43 02 and 33 respectively Although the 119865

0of

C vulgaris under 1mgL treatments decreased by 32 the119865119898

and 1198651198981015840 increased by 49 and 13 respectively The

2mgL ethyl cinnamate affected the chlorophyll fluorescenceof C vulgaris significantly for 119865

0 119865119898 and 119865

1198981015840 decreasing

by 635 730 and 671 respectively The photoinducedcurves of C vulgaris were nearly linear under the 3mgLand 4mgL treatments indicating that photosynthesis wascompletely inhibited in these two treatment groups (Figures3(e) and 3(f))

The 119865V119865119898 (maximal photochemical efficiency of PSII)BPSII (actual photochemical efficiency of PSII in the light)ETR (photosynthetic electron transport rate) and chloro-phyll a fluorescence were used tomanifest the photosynthetictoxicity of ethyl cinnamate onC vulgaris Values ofmaximumphotochemical efficiency (119865V119865119898) reflect the potential quan-tum efficiency of PSII and the decline in values will be seenwhen plants are under stress [31] As a consequence 119865V119865119898could be a sensitive indicator for photosynthesis Actual

0 1

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FFm

ΦPSII

Figure 4 The fluorescent imaging of C vulgaris under the stress ofethyl cinnamate

photochemical efficiencymeasures the proportion of the lightabsorbed by chlorophyll associated with PSII that is used inphotochemistry also getting lower when plants are understress [31] In the experiment the chlorophyll fluorescentimaging of C vulgaris under the stress of ethyl cinnamate isshown in Figure 4 and the effects of ethyl cinnamate on the119865V119865119898BPSII and ETR of C vulgaris are shown in Figure 5

05mgL ethyl cinnamate had little impact on the 119865V119865119898of C vulgaris Although 1mgL ethyl cinnamate affected the119865V119865119898 in the first 48 h there was no difference betweenthe 119865V119865119898 of blank control and that of the ethyl cinnamatetreatment group after exposure for 72 h and 96 h 2ndash4mgLethyl cinnamate significantly affected the119865V119865119898 ofC vulgarisand the effects increasedwith the exposure concentration andtime After exposure to 4mgL ethyl cinnamate for 72 h and96 h the photosynthesis of C vulgariswas completely (100)inhibited

Compared with the 119865V119865119898 ethyl cinnamate inhibited theBPSII and ETR of C vulgaris much more After exposure to2mgL ethyl cinnamate for 24 hC vulgaris barely performedphotosynthesis with a slight recovery occurring after 96 hTheBPSII andETRofC vulgarisunder the 3mgL and 4mgLethyl cinnamate treatments were 0 during the entire exposureprocess

Chlorophyll a fluorescence reflects chlorophyll a concen-tration The decrease of values of chlorophyll a fluorescenceoften indicates that the photosynthesis of plants is inhibitedThis parameter could also be a sensitive indicator for pho-tosynthesis Regarding the ratio of normal fluorescent cells05ndash4mgL ethyl cinnamate had no significant impact on theFL3 fluorescence (the autofluorescence of chlorophyll a) (119901 gt005) but affected the mean FL3 fluorescence (the mean of


(02)

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24h48h

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(c)

Figure 5 The effects of ethyl cinnamate on the 119865V119865119898 BPSII and ETR of C vulgaris ((a) 119865V119865119898 (b) BPSII (c) ETR) (The relative value ofchlorophyll fluorescence parameters treatment-to-blank control ratio of chlorophyll fluorescence parameters)

FL3 fluorescence value for all tested algal cells) in the differenttreatment groups as shown in Figure 6 Studies have shownthat the inhibition of the electron acceptor of PSII reactioncenters led to an increase in the chlorophyll a fluorescence[41 42] while the inhibition of the electronic supply resultedin a decrease [41] In this study chlorophyll content did notreduce significantly (according to the measurement resultsof the fluorescence of chlorophyll a) and the photosyntheticactivities were inhibited Based on the results it was specu-lated that ethyl cinnamate inhibited the photosynthetic rates

of C vulgaris mainly through electron transfer chain ratherthan the structure of the chloroplasts

The study results of Gao et al [26] on the effects of ethylcinnamate on Chlorella pyrenoidosa revealed a decrease ofchlorophyll a and inhibition of growth consistent with theresults in this study The study results of Bruckner et al[43] on the effects of ragweed inflorescence extract on thegrowth of C vulgaris and Chlamydomonas sp showed theallelochemicals in the extract decreased chlorophyll a contentof C vulgaris significantly In this study ethyl cinnamate had


00

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0mgL05mgL1mgL

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Figure 6 The effects of ethyl cinnamate on the fluorescence ofchlorophyll a

no significant impact on the FL3 fluorescence but affectedthe mean FL3 fluorescence consistent with the results ofBruckner et al to some extent

33 Cellular Physiological Toxicity FDA and PI stainingfluorescence as well as FSC fluorescence were measuredto provide information in toxic effects of ethyl cinnamateon cellular physiology of Chlorella vulgaris FDA stainingfluorescence represents esterase activities of cells and FDAonly stains living cells Thus FDA staining fluorescence isa sensitive indicator for distinguishing between living cellsand dead ones Higher values of FDA staining fluorescenceindicate higher esterase activities Correspondingly low val-ues of FDA staining fluorescencemean low esterase activitiesin other words the cells are inhibited The effects of ethylcinnamate on the FDA staining fluorescence of Chlorellavulgaris are shown in Figure 7 The blue area of the imagerepresents the normal algal cells the area of which can beused to calculate the ratio of normal fluorescent cellsThe redarea demonstrates that the esterase activities of C vulgariswere affected therefore the FDA staining fluorescence isoutside the normal range The 05mgL and 1mgL ethylcinnamate caused the ratio of normal fluorescent cells to belower than that of the blank control nevertheless this ratioincreased with the exposure time After exposure for 96 hthere were no prominent distinctions between the ratios ofnormal fluorescent cells of the blank control and that of theethyl cinnamate treatment groups Under the 2ndash4mgL ethylcinnamate treatments the ratios of algal cells of which thefluorescence was affected were much higher than normalcells In addition as the exposure time increased so did theratio of affected algal cells

The effects of ethyl cinnamate on the esterase activitiesof C vulgaris are shown in Figure 8 The 05ndash4mgL ethylcinnamate treatments decreased the esterase activities of Cvulgaris which could be observed even after 24 h indicatingthat both the ratios of normal fluorescent cells and the FL1-MFI were lower than those of the blank control In addition

the effects of ethyl cinnamate on the esterase activities ofC vulgaris were concentration-dependent Under low ethylcinnamate concentrations (05 and 1mgL) as the exposuretime increased the degree of esterase activity inhibitiondecreased The ratios of normal fluorescent cells were notmuch different from blank control after 96 h neverthelessthe FL1 fluorescence indicated that the esterase activities werestill lower than blank control Under high ethyl cinnamateconcentrations (2 3 and 4mgL) the degree of esterase activ-ity inhibition increased with the exposure time indicatingthat the ratios of normal fluorescent cells decreased continu-ously

PI staining fluorescence could reflect in the integrity ofthe cell membrane and PI only stains dead cells As a resultthe values of this parameter also represent mortality Highmortalitymeans high values of PI staining fluorescence Fromthe results of PI staining cells of C vulgaris under treatmentand blank control were in the same range while no cells werefound in the range of affected cells This demonstrated thatethyl cinnamate had no significant effect on the integrity ofthe cell membrane ofC vulgaris (data not shown) Comparedwith the results in Section 31 it was possible that the effectsof ethyl cinnamate on the growth of C vulgaris were mainlythe suppression of the agamogony According to the studyresults of Gao et al [26] ethyl cinnamate induced the overac-cumulation of ROS and the increase of MDA suggesting thatethyl cinnamate could lead to the damage of cell membranesystem However according to this study ethyl cinnamatehad no significant effects on cell membrane integrity of Cvulgaris demonstrating allelochemicals had diverse effectson different species of algae in other words selectivity Thisperhaps also demonstrated Chlorella pyrenoidosa was moresensitive to allelochemicals

The values of FSC fluorescence are relevant to the sizeof cells The values of FSC fluorescence get higher with thesize of cells being bigger Ethyl cinnamate also significantlyaffected the size of the C vulgaris cells As shown in Figure 9ethyl cinnamate increased the size of the C vulgaris cells

4 Conclusions

Ethyl cinnamate could significantly affect the growth photo-synthesis and cellular physiology ofChlorella vulgaris 1mgLethyl cinnamate effectively inhibited the growth ofC vulgarisThe 48-h and 72-h EC50 values were 207mgL (194ndash220)and 189mgL (182ndash197) respectively Ethyl cinnamate sig-nificantly inhibited the 119865V119865119898 of C vulgaris at concentrationsof 2 3 and 4mgL with an even greater inhibition onthe BPSII and ETR Based on the results it was speculatedthat ethyl cinnamate inhibited the photosynthetic rate of Cvulgaris mainly through affecting the electron transfer chainrather than the structure of the chloroplasts On individualcell basis ethyl cinnamate decreased esterase activities in thecell but did not alter the integrality of cell membrane It alsoresulted in increasing average cell size

Disclosure

Hui-Ling Ouyang is the co-first author


The n

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FL1 fluorescence (AU)

t = 24h t = 48h t = 72h t = 96h

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Figure 7 The effects of ethyl cinnamate on the FDA staining fluorescence of Chlorella vulgaris


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(a)

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Figure 8 The effects of ethyl cinnamate on the esterase activities of C vulgaris ((a) The ratio of normal fluorescent cells and (b) the meanFL1 fluorescence)

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Figure 9The effects of ethyl cinnamate on the size of theC vulgariscells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The funding for this study was provided by National Projectfor Water Pollution Control (2012ZX07103-002) and the

National Science Foundation of China (NSFC) (4127146241030529)This work is also supported by a grant from the 111Project (B14001) the undergraduate student research trainingprogram of the Ministry of Education and Mao YugangUndergraduate Research Grant Peking University

References

[1] Y-J Jiang W He W-X Liu et al ldquoThe seasonal and spatialvariations of phytoplankton community and their correlationwith environmental factors in a large eutrophic Chinese lake(Lake Chaohu)rdquo Ecological Indicators vol 40 pp 58ndash67 2014

[2] H L Ouyang X Z Kong W He et al ldquoEffects of five heavymetals at sub-lethal concentrations on the reproduction andphotosynthesis of Chlorella vulgarisrdquo Chinese Science Bulletinvol 57 no 10 pp 785ndash793 2012

[3] H L Ou-Yang X Z Kong M Lavoie et al ldquoPhotosyntheticand cellular toxicity of cadmium in Chlorella vulgarisrdquo Environ-mental Toxicology and Chemistry vol 32 no 12 pp 2762ndash27702013

[4] F L Xu ldquoExergy and structural exergy as ecological indicatorsfor the development state of the Lake Chaohu ecosystemrdquoEcological Modelling vol 99 no 1 pp 41ndash49 1997

[5] Y C Wang and K H Lu ldquoThe harm of cyanobacterial bloomsand governancerdquo Chinese Journal of Fisheries vol 17 no 1 pp90ndash93 2004

[6] H Y Hu Y J Men and F M Li ldquoReview on inhibition of plantallelopathy of algae reproductionrdquo Ecology and Environmentvol 15 no 1 pp 153ndash157 2006


[7] FM Li andH YHu ldquoMechanismof phyto-allelochemicals andits application for harmful algae control in nature water bodyrdquoWater ampWastewater Engineering vol 30 no 2 pp 1ndash4 2004

[8] E L Rice Allelopathy Academic Press London UK 2nd edi-tion 1984

[9] E M Gross D Erhard and E Ivanyi ldquoAllelopathic activity ofCeratophyllum demersum L and Najas marina ssp intermedia(Wolfgang) Casperrdquo Hydrobiologia vol 506-509 no 1ndash3 pp583ndash589 2003

[10] S Nakai Y Inoue and M Hosomi ldquoAlgal reproduction inhibi-tion effects and inducement modes by plant-producing phe-nolsrdquoWater Research vol 35 no 7 pp 1855ndash1859 2001

[11] H Kato-Noguchi ldquoAllelopathic substances in Pueraria thunber-gianardquo Phytochemistry vol 63 no 5 pp 577ndash580 2003

[12] N T Doan R W Rickards J M Rothschild and G D SmithldquoAllelopathic actions of the alkaloid 12-epi-hapalindole E iso-nitrile and calothrixin A from cyanobacteria of the genera Fis-cherella and Calothrixrdquo Journal of Applied Phycology vol 12 no3ndash5 pp 409ndash416 2000

[13] T Igarashi M Satake and T Yasumoto ldquoPrymnesin-2 a potentichthyotoxic and hemolytic glycoside isolated from the red tidealga Prymnesium parvumrdquo Journal of the American ChemicalSociety vol 118 no 2 pp 479ndash480 1996

[14] K D Kearns and M D Hunter ldquoToxin-producing Anabaenaflos-aquae induces settling of Chlamydomonas reinhardtii acompetingmotile algardquoMicrobial Ecology vol 42 no 1 pp 80ndash86 2001

[15] R Pratt and J Fong ldquoStudies on Chlorella vulgaris II Furtherevidence that Chlorella cells form a reproduction-inhibitingsubstancerdquo American Journal of Botany vol 27 pp 431ndash4361940

[16] C K Schmitt K C Meysick and A D OrsquoBrien ldquoBacterialtoxins friends or foesrdquo Emerging Infectious Diseases vol 5 no2 pp 224ndash234 1999

[17] G D Smith and N T Doan ldquoCyanobacterial metabolites withbioactivity against photosynthesis in cyanobacteria algae andhigher plantsrdquo Journal of Applied Phycology vol 11 no 4 pp337ndash344 1999

[18] S Suikkanen G O Fistarol and E Graneli ldquoEffects of cyano-bacterial allelochemicals on a natural plankton communityrdquoMarine Ecology Progress Series vol 287 pp 1ndash9 2005

[19] S Suikkanen J Engstrom-Ost J Jokela K Sivonen and MViitasalo ldquoAllelopathy of Baltic Sea cyanobacteria no evidencefor the role of nodularinrdquo Journal of Plankton Research vol 28no 6 pp 543ndash550 2006

[20] T Yasumoto B Underdal T Aune V Hormazabal O M Skul-berg and Y Oshima ldquoScreening for hemolytic and ichthyotoxiccomponents of Chrysochromulina polylepis and Gyrodiniumaureolum from Norwegian coastal watersrdquo in Toxic MarinePhytoplankton pp 436ndash440 Elsevier New York NY USA1990

[21] S Korner and A Nicklisch ldquoAllelopathic growth inhibitionof selected phytoplankton species by submerged macrophytesrdquoJournal of Phycology vol 38 no 5 pp 862ndash871 2002

[22] G Mulderij E van Donk and J G M Roelofs ldquoDifferentialsensitivity of green algae to allelopathic substances fromCharardquoHydrobiologia vol 491 no 1ndash3 pp 261ndash271 2003

[23] K K Schrader N P Dhammika Nanayakkara C S Tucker AM Rimando M Ganzera and B T Schaneberg ldquoNovel deriva-tives of 910-anthraquinone are selective algicides against themusty-odor cyanobacterium Oscillatoria perornatardquo Applied

and Environmental Microbiology vol 69 no 9 pp 5319ndash53272003

[24] X-X Sun J-K Choi and E-K Kim ldquoA preliminary studyon the mechanism of harmful algal bloom mitigation by useof sophorolipid treatmentrdquo Journal of Experimental MarineBiology and Ecology vol 304 no 1 pp 35ndash49 2004

[25] G G Bian ldquoReview on inhibition of terrestrial plant allelopathyof algae reproductionrdquo Environmental Science amp Technologyvol 35 no 2 pp 90ndash95 2012

[26] L L Gao P Y Guo G M Su and Y F Wei ldquoEffects of alle-lochemicals ethyl cinnamate on the growth and physiologicalcharacteristics ofChlorella pyrenoidosardquo Environmental Sciencevol 34 no 1 pp 156ndash162 2013

[27] M He T Zhang A Wu and L Nie ldquoInhibition of cinnamicacid onMicrocystis aeruginosa K and Scenedesmus arcuatus LrdquoChinese Journal of Applied amp Environmental Biology vol 14 no6 pp 774ndash778 2008

[28] K K Schrader M Q De Regt P R Tidwell C S Tuckerand S O Duke ldquoSelective growth inhibition of the musty-odor producing Cyanobacterium Oscillatoria cf chalybea bynatural compoundsrdquo Bulletin of Environmental Contaminationand Toxicology vol 60 no 4 pp 651ndash658 1998

[29] G B Williamson E M Obee and J D Weidenhamer ldquoInhibi-tion of Schizachyrium scoparium (poaceae) by the allelochem-ical hydrocinnamic acidrdquo Journal of Chemical Ecology vol 18no 11 pp 2095ndash2105 1992

[30] J L Stauber N M Franklin and M S Adams ldquoApplications offlow cytometry to ecotoxicity testing using microalgaerdquo Trendsin Biotechnology vol 20 no 4 pp 141ndash143 2002

[31] K Maxwell and G N Johnson ldquoChlorophyll fluorescencemdashapractical guiderdquo Journal of Experimental Botany vol 51 no 345pp 659ndash668 2000

[32] e Govindjee ldquoSixty-three years since Kautsky chlorophyll afluorescencerdquoAustralian Journal of Plant Physiology vol 22 no2 pp 131ndash160 1995

[33] G H Krause ldquoPhotoinhibition of photosynthesis An eval-uation of damaging and protective mechanismsrdquo PhysiologiaPlantarum vol 74 no 3 pp 566ndash574 1988

[34] D Lazar ldquoChlorophyll a fluorescence inductionrdquo Biochimica etBiophysica Acta vol 1412 no 1 pp 1ndash28 1999

[35] N M Franklin J L Stauber and R P Lim ldquoDevelopment offlow cytometry-based algal bioassays for assessing toxicity ofcopper in natural watersrdquo Environmental Toxicology and Chem-istry vol 20 no 1 pp 160ndash170 2001

[36] N M Franklin M S Adams J L Stauber and R P LimldquoDevelopment of an improved rapid enzyme inhibition bioassaywith marine and freshwater microalgae using flow cytometryrdquoArchives of Environmental Contamination and Toxicology vol40 no 4 pp 469ndash480 2001

[37] Y Yu F Kong M Wang L Qian and X Shi ldquoDeterminationof short-term copper toxicity in a multispecies microalgal pop-ulation using flow cytometryrdquo Ecotoxicology and EnvironmentalSafety vol 66 no 1 pp 49ndash56 2007

[38] F Li G Wu S Hu Z Fan and Q Gao ldquoGrowth behaviorand physiological characteristics of Chlorella vulgaris in thepresence of deicing saltrdquo Procedia Environmental Sciences vol18 pp 20ndash25 2013

[39] W Liu S Chen X Quan and Y-H Jin ldquoToxic effect ofserial perfluorosulfonic and perfluorocarboxylic acids on themembrane system of a freshwater alga measured by flowcytometryrdquo Environmental Toxicology and Chemistry vol 27no 7 pp 1597ndash1604 2008


[40] C Pinheiro J Azevedo A Campos S Loureiro and V Vas-concelos ldquoAbsence of negative allelopathic effects of cylin-drospermopsin and microcystin-LR on selected marine andfreshwater phytoplankton speciesrdquo Hydrobiologia vol 705 no1 pp 27ndash42 2013

[41] G Samson J Claude Morisette and R Popovic ldquoCopperquenching of the variable fluorescence in Dunaliella tertiolectaNew evidence for a copper inhibition effect on PSII photochem-istryrdquo Photochemistry and Photobiology vol 48 no 3 pp 329ndash332 1988

[42] I Yruela M Alfonso I O De Zarate G Montoya and RPicorel ldquoPrecise location of the Cu(II)-inhibitory binding sitein higher plant and bacterial photosynthetic reaction centersas probed by light-induced absorption changesrdquoThe Journal ofBiological Chemistry vol 268 no 3 pp 1684ndash1689 1993

[43] D J Bruckner A Lepossa and Z Herpai ldquoInhibitory effect ofragweed (Ambrosia artemisiifolia L)-inflorescence extract onthe germination ofAmaranthus hypochondriacus L and growthof two soil algaerdquo Chemosphere vol 51 no 6 pp 515ndash519 2003

Submit your manuscripts athttpwwwhindawicom

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Environmental and Public Health

Journal of



EcosystemsJournal of


MeteorologyAdvances in

EcologyInternational Journal of


Marine BiologyJournal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Advances in


Environmental Chemistry

Atmospheric SciencesInternational Journal of



Waste ManagementJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal of

Geophysics


Geological ResearchJournal of

EarthquakesJournal of


BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OceanographyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of Computational Environmental SciencesHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


ClimatologyJournal of


of algae such as cell membrane permeability reproduc-tion photosynthesis and respiration protein synthesis andresistance to diseases [12ndash20] Allelochemicals affect algaeselectively therefore different species of algae have variedinhibition responses [21ndash24]

Currently research on allelopathy mainly focuses onaquatic plants and rarely on terrestrial plants especiallyxylophyta Compared with hydrophytes terrestrial plantsare more widely distributed In addition terrestrial plantsare easy to cultivate break and process Many species ofherbage and xylophyta are rich in allelochemicals and asa consequence have better prospects in inhibiting algalblooms and the emergency response to algal blooms [25]Theallelochemicals that are produced and released by terrestrialplants can bewashed from land intowater by rain and interactwith organisms in the water Therefore research on theallelochemicals such as ethyl cinnamate that are produced byterrestrial plants has more practical significance in inhibitionof algae However current research on the effects of cinnamicacid and its derivatives on algal inhibition is still rare andmostly utilizes traditional methods of algal toxicology Rel-evant research mainly focuses on the algal reproduction theactivities of algal antioxidant enzymes the lipid peroxidationof cell membrane and the interaction with nutrients suchas N and K [26ndash29] However these traditional methodscannot distinguish between dead and living cells duringcultivation potentially causing variations that deviate fromthe real results [30] In addition traditional researchmethodsof algal toxicity focus more on the toxicity of substances andless on the response of other algal physiological processessuch as photosynthesis It is impossible to understand themechanism of allelopathy comprehensively

Chlorophyll fluorescence analysis and flow cytometry arenewmethods that have been used to study the photosynthetictoxicity and cytotoxicity of heavy metals and other pollutantson algae in recent years [2 3] Chlorophyll fluorescenceanalysis is based on the photosynthetic theory using chloro-phyll fluorescence in vivo as a probe to study the photosyn-thetic physiological state of organisms under the influencesof various external factors [31] Chlorophyll fluorescenceanalysis is rapid and sensitive and it can also detect thephotosynthetic toxicitywith nodamage to algae [31ndash34] Flowcytometry (FCM) an automated cell or biological particlesanalysis technique is capable of rapid multiple parameterdetections of a single cell and has been extensively usedin algal toxicity studies in recent years [35ndash37] Comparedwith traditional methods flow cytometry is conducive toobtaining algal responses under stress conditions on cellularlevel The application of flow cytometry allows for algaltoxicological research on cellular level and this techniquecontributes to understanding the actionmechanismof toxins

In this study experiments were performed to studythe toxic effects of ethyl cinnamate on Chlorella vulgarisa dominant species of green algae This study based ontraditional algal toxicological researchmethods used chloro-phyll fluorescence analysis and flow cytometry to study howethyl cinnamate affected the growth photosynthesis and cellphysiology of C vulgaris in order to understand the effectsand the action mechanism of allelochemicals further

2 Materials and Methods

21 Experimental Materials

211 Algal Strain In this study Chlorella vulgaris wasselected as algal material for its two advantages C vulgarisrequires relatively simple growth conditions and has a strongenvironmental tolerance and a high reproductive rate Inaddition many domestic and overseas researchers have alsoselected C vulgaris as test algal material [38] which makescomparing results convenient C vulgaris was purchasedfrom the Freshwater Algae Culture Collection of the Instituteof Hydrobiology Chinese Academy of Sciences

212 Chemical Materials Ethyl cinnamate (991 purity)was purchased from Alfa Aesar (America) and used in expo-sure experiments of Chlorella vulgaris Dimethyl sulfoxide(analytical grade) was purchased from Beijing ChemicalWorks (China) for the solubilization of ethyl cinnamateMain components in BG11 medium were all purchased fromSinopharm Chemical Reagent Co Ltd (China) for culti-vation of C vulgaris Fluorescein diacetate (FDA) was pur-chased from Sigma (America) for staining algal cells in flowcytometric analysis Acetone was purchased from BeijingChemical Works (China) for the solubilization of FDAPropidium iodide (PI) was purchased from Sigma (America)for staining algal cells in flow cytometric analysis

22 Experimental Methods

221 Experimental Conditions

(1) Algal Culture Conditions Chlorella vulgaris was cultivatedin BG11 medium at 24 plusmn 1∘C with a cycle of light (14 h4000 Lux) and dark (10 h 0 Lux) in a GXZ-280B illuminationcultivation cabinet (China) C vulgaris was cultivated stati-cally and was not aerated during the cultivationThe cultureswere shaken two to three times daily and their positions werechanged randomly

(2) Exposure Experiments Chlorella vulgaris was cultivatedin 50mL BG11 medium in 150-mL Erlenmeyer flasks (theinitial cell density of C vulgaris was approximately 2 times106 cellsmL) C vulgaris was exposed to ethyl cinnamate atfinal total concentrations of 05mgL 1mgL 2mgL 3mgLand 4mgL for 96 h In the experiment 01mL dimethylsulfoxide was added to each sample (50mL in total) for thesolubilization of ethyl cinnamate Preliminary results showedthat 02 dimethyl sulfoxide (01mL in 50mL medium) hadno significant impact on the photosynthetic and physiologicalcharacteristics inC vulgaris Exposure tests and controlswereset up in triplicate The biomass chlorophyll fluorescenceand flow cytometry were measured every 24 h during theexperiments

222 Measurements of the Indicators

(1) Biomass Analysis Optical density of algal samples at450 nm correlated with algal cell density determined by





(1)















0000

0100

0200

0300

0400


0mgL05mgL1mgL

2mgL3mgL4mgL

OD450

nm









Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

Fm

Fm998400

F0

(a)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

FmFm998400

(b)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

Fm Fm998400

F0

(c)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

Fm

Fm998400

(d)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

FmFm998400

(e)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

Fm

Fm998400

(f)



Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U) Fm

Fm998400

F0

(a)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm

Fm998400

(b)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

Fm998400

Fm

F0

(c)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm998400

Fm

(d)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(e)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(f)






0 119865119898 and 119865

1198981015840 decreased


0 119865119898 and 119865

1198981015840



0 119865119898 and 119865

1198981015840 of


0of




0 119865119898 and 119865




0 1

Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


FFm

ΦPSII







(02)

00

02

04

06

08

10

12

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(a)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(b)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


24h48h

72h96h

(c)






00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics


















(1)















0000

0100

0200

0300

0400


0mgL05mgL1mgL

2mgL3mgL4mgL

OD450

nm









Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

Fm

Fm998400

F0

(a)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

FmFm998400

(b)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

Fm Fm998400

F0

(c)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

Fm

Fm998400

(d)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

FmFm998400

(e)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

Fm

Fm998400

(f)



Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U) Fm

Fm998400

F0

(a)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm

Fm998400

(b)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

Fm998400

Fm

F0

(c)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm998400

Fm

(d)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(e)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(f)






0 119865119898 and 119865

1198981015840 decreased


0 119865119898 and 119865

1198981015840



0 119865119898 and 119865

1198981015840 of


0of




0 119865119898 and 119865




0 1

Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


FFm

ΦPSII







(02)

00

02

04

06

08

10

12

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(a)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(b)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


24h48h

72h96h

(c)






00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics















Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

Fm

Fm998400

F0

(a)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

FmFm998400

(b)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

Fm Fm998400

F0

(c)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

Fm

Fm998400

(d)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

FmFm998400

(e)

Chlo

roph

yll fl

uore

scen

ce (A

U)

Time (s)

0

01

04

03

02

0 100 200 300 400

F0

Fm

Fm998400

(f)



Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U) Fm

Fm998400

F0

(a)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm

Fm998400

(b)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

Fm998400

Fm

F0

(c)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm998400

Fm

(d)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(e)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(f)






0 119865119898 and 119865

1198981015840 decreased


0 119865119898 and 119865

1198981015840



0 119865119898 and 119865

1198981015840 of


0of




0 119865119898 and 119865




0 1

Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


FFm

ΦPSII







(02)

00

02

04

06

08

10

12

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(a)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(b)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


24h48h

72h96h

(c)






00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics















Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U) Fm

Fm998400

F0

(a)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm

Fm998400

(b)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

Fm998400

Fm

F0

(c)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

F0

Fm998400

Fm

(d)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(e)

Time (s)

0

01

04

03

02

0 100 200 300 400

Chlo

roph

yll fl

uore

scen

ce (A

U)

(f)






0 119865119898 and 119865

1198981015840 decreased


0 119865119898 and 119865

1198981015840



0 119865119898 and 119865

1198981015840 of


0of




0 119865119898 and 119865




0 1

Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


FFm

ΦPSII







(02)

00

02

04

06

08

10

12

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(a)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(b)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


24h48h

72h96h

(c)






00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics


















0 119865119898 and 119865

1198981015840 decreased


0 119865119898 and 119865

1198981015840



0 119865119898 and 119865

1198981015840 of


0of




0 119865119898 and 119865




0 1

Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


Expo

sure

conc

entr

atio

ns (m

gL)

0

05

1

2

3

4


FFm

ΦPSII







(02)

00

02

04

06

08

10

12

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(a)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(b)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


24h48h

72h96h

(c)






00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics















(02)

00

02

04

06

08

10

12

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(a)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


(b)

(02)

00

02

04

06

08

10

12

14

0 05 1 15 2 25 3 35 4 45

Relat

ive v

alue

of c

hlor

ophy

ll flu

ores

cenc

e par

amet

ers


24h48h

72h96h

(c)






00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics















00

200

400

600

800

1000

1200

0 24 48 72 96

Relat

ive v

alue

of F

L3-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL








4 Conclusions


Disclosure



The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics















The n

umbe

r of c

ells


t = 24h t = 48h t = 72h t = 96h

100

101

102

103

10410

010

110

210

310

410

010

110

210

310

410

010

110

210

310

4

0mgL

05mgL

10mgL

20mgL

30mgL

40mgL



10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics















10

100

1000

10000

0 24 48 72 96

Ratio

of n

orm

al fl

uore

scen

t cel

ls (

)

Exposure time (h)


(a)

10

100

1000

10000

0 24 48 72 96

Relat

ive v

alue

of F

L1-M

FIExposure time (h)


(b)


950

1000

1050

1100

1150

1200

1250

0 24 48 72 96

Relat

ive v

alue

of F

SC-M

FI

Exposure time (h)

0mgL05mgL1mgL

2mgL3mgL4mgL




Acknowledgments



References



















































Journal of












Volume 2014

Advances in









Geophysics


























































Journal of












Volume 2014

Advances in









Geophysics














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