Chemosphere 131 (2015) 139–148

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Temporal variation and interaction of full size spectrum Alcian bluestainable materials and water quality parameters in a reservoir

http://dx.doi.org/10.1016/j.chemosphere.2015.03.0230045-6535/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +886 3 571 2121x55507; fax: +886 3 572 5958.E-mail address: cphuang@mail.nctu.edu.tw (C. Huang).

Nguyen Thi Thuy a, Justin Chun-Te Lin b, Yaju Juang a, Chihpin Huang a,⇑a Institute of Environmental Engineering, National Chiao Tung University, Hsinchu, Taiwanb Disaster Prevention and Water Environmental Research Center, National Chiao Tung University, Hsinchu, Taiwan

h i g h l i g h t s

�We study Alcian blue (AB) stainedfractions in freshwater for one year,intensively during phytoplanktonbloom.� Sampling frequency and species

composition are important for linkingAB stained material andphytoplankton.� AB stained particles and pTEP are

generated directly from somephytoplankton species instead of byabiotic pathway.� FlowCAM is a promising technique

for AB stained particles visualization.

g r a p h i c a l a b s t r a c t

Visualization Alcian blue stained material

Quantification

Strong blue color particle Internal stained cell

Mucilage sheath of cell (e.g. Gloeocystis sp.)

Dark-blue particle

Apr May Jun Jul

Par

ticle

x10

3 mL-1

0

5

10AB stained particles

mgL

-1

0

1

2

678

dAPS cTEP pTEP

a r t i c l e i n f o

Article history:Received 4 January 2015Received in revised form 28 February 2015Accepted 8 March 2015

Handling Editor: Keith Maruya

Keywords:Transparent exopolymer particlesPhytoplanktonAcid polysaccharideAlcian blueFlowCAM

a b s t r a c t

This paper reports on the fate of different fractions of Alcian blue (AB) stainable material in Pao-Shanreservoir, Taiwan, in a one-year study (2013–2014) and an intensive study during phytoplankton bloom(2014). The interactions between the fractions, including AB stained particles, particle and colloidal trans-parent exopolymer particles (pTEP and cTEP), dissolved acid polysaccharide (dAPS), and their relationshipto other water quality parameters were analyzed. The Flow Cytometer and Microscope (FlowCAM) wasfor first time used to characterize AB stained particles. The results of the one-year study likely showedrelationships of pTEP concentration to phytoplankton count and chlorophyll a, while in the intensivestudy, AB stained particles abundance and pTEP concentration were correlated neither phytoplanktoncount nor chlorophyll a, but strongly positively correlated with some phytoplankton species’ abundance.The difference indicates that sampling frequency and phytoplankton composition should be addressedfor studying the links between AB stained fractions and phytoplankton. The interaction between differentAB stained fractions further suggests that the majority of AB stained particles and pTEP would be directlygenerated by some phytoplankton species, whereas their abiotic generation by cTEP or dAPS may onlyhave contributed partly to their formation. This differs from previous studies which generally positedthat pTEP are mainly formed abiotically from dissolved precursors. Successful application of FlowCAMfor visualization of AB stained particles recommends this technique by which particle morphologiescan be conserved and morphological features of particle can be simultaneously elucidated.

� 2015 Elsevier Ltd. All rights reserved.

140 N.T. Thuy et al. / Chemosphere 131 (2015) 139–148

1. Introduction

There are several terminologies referring to materials which arestainable with Alcian blue (AB), a dye specific for anionic carboxylor halfester-sulfate groups of acid polysaccharides (Passow andAlldredge, 1995). These materials can be termed transparentexopolymer particles (TEP) (Passow and Alldredge, 1994), AB-stained particles (Worm and Søndergaard, 1998), particle TEP(pTEP) and colloidal TEP (cTEP) (Villacorte et al., 2009a), and acidicpolysaccharides (APS) (Thornton et al., 2007). The main differencesbetween them are their size and their methods of analysis. TEP, asthe most frequently used term, is collected by 0.2 or 0.4-lmmembrane filters before staining with AB, and can then bemeasured by microscopic enumeration for abundance and size(Logan et al., 1994; Passow and Alldredge, 1994), and can also beanalyzed by colorimetric method for concentration (Passow andAlldredge, 1995). Villacorte et al. (2009a) extended the conceptof TEP into two types: pTEP and cTEP in which pTEP is the sameas the TEP of Passow and Alldredge (1995), while cTEP is the TEPcollected on a 0.05-lm filter after passing through 0.4-lm filters.Instead of targeting TEP, Thornton et al. (2007) introduced amethod for APS measurement either in whole water samples orin filtrate from 0.4-lm filter. In contrast with TEP measurementthat collects TEP on filters, APS is measured in its own solution.Thornton et al. (2007) then claimed that in whole water samples,Alcian blue staining pools of APS are the total amount of APSwithin the water sample, including TEP, dissolved APS, APS asso-ciated with the external coating of organisms, and internal poolsof APS within cells.

This study proposes that ‘‘Alcian blue stainable material’’ is amore appropriate term that would cover most of the terms intro-duced above. In terms of particle size, the materials includeparticle fractions (covering AB stained particles, pTEP, and cTEP)and dissolved fractions (covering APS in filtrate). While manystudies have dealt with pTEP, few studies have focused on cTEP(Villacorte et al., 2009a,b, 2010; Van Nevel et al., 2012), which isgenerally more abundant than pTEP. Also, dissolved fractions ofAB stainable material have rarely been described through APS inwhole water sample and APS in 0.4-lm filtrate (Thornton et al.,2007; Thornton, 2009; Thornton and Visser, 2009). To the best ofour knowledge, the interaction between these fractions has notpreviously been reported on.

Most of studies on the temporal variability of TEP have beencarried out in seawater (Radic et al., 2005; Villacorte et al., 2010;Taylor et al., 2014). There are only two reports on the temporalrelationship between TEP and phytoplankton (biomass andcomposition): a 3-year study in seawater (Radic et al., 2005), anda study on sea surface water during a spring diatom bloom(Taylor et al., 2014). Interactions between TEP and phytoplankton,as a major source of exudate TEP precursors, and other waterquality parameters are widely described in the literature, althoughmostly for cultures (Corzo et al., 2000; Fukao et al., 2010), meso-cosm laboratory experiments (Mari et al., 2005), or natural watersources related to the sea, coast or estuary (Radic et al., 2005;Prieto et al., 2006; Ortega-Retuerta et al., 2009; Wetz et al., 2009;Villacorte et al., 2010; Taylor et al., 2014). For freshwaters, TEPhas been described only in discrete studies (Kennedy et al., 2009;Villacorte et al., 2009a,b; Berman and Parparova, 2010; deVicente et al., 2010; Berman et al., 2011; Van Nevel et al., 2012),and the interactions between TEP and other water parameters havebeen reported only once to date (de Vicente et al., 2010). There wasthus a need for a systematic investigation of the variation andinteraction of different AB stained fractions in freshwater sources.

Together with diverse biochemical roles of TEP in aquaticenvironments, its role in water industry field as potential agents

for membrane fouling was firstly suggested by Berman andHolenberg (2005). Later, reports in its involvement in membranefouling have been increased (Discart et al., 2015) and its reductionby existing treatment processes has been summarized (Villacorteet al., 2015b). Therefore, understanding of TEP formation path-ways, size and their dynamics in feedwater sources would beuseful in management of derived fouling in membrane technolo-gies (Bar-Zeev et al., 2015). In this study, we describe the firstextensive investigation on the temporal variation of AB stainablefactions including AB stained particles, pTEP, cTEP and dissolvedAPS (dAPS, in the filtrate collected after 0.05-lm filter), coveringthe full size range, in Pao-Shan reservoir – a water supply source– in Taiwan. The interaction among these fractions, theirrelationship with the phytoplankton community structure, andother biological and chemical factors were analyzed. It is also thefirst time that FlowCAM was applied to characterize AB stainedparticles.

2. Materials and methods

2.1. Materials, apparatus and solution preparation

Alcian blue 8GX (AB, analytical reagent grade), gum xanthan(GX, from Xanthomonas campestris) and 37% formaldehyde solutionwere sourced from Sigma–Aldrich Co. LLC. 0.4 and 0.05-lm mem-brane filters (47 mm diameter, polycarbonate) were sourced fromWhatman Plc. The filtration set-up consists of a vacuum pump(Rocker 600, Rocker Scientific Co., Ltd. Taiwan) and an adjustablevalve to control pressure precisely at 150 mm Hg for a stainlesssteel filter holder (Gelman Sciences, Inc., Ann Arbor, MI). AnUV–Vis spectrophotometer (SP 8001, Metertech Inc., Taiwan) wasused to measure absorbance. A tailor-made tissue grinder(100 mL, Sunway Scientific Co., Taiwan) was used to prepare GXsolution. A microbalance (Model CP2P-F, Sartorius AG, Germany)was used to measure GX weight on filters. A centrifuge (AvantiJ-E centrifuge, Beckman Coulter) with centrifugal tubes (50 mL,polycarbonate, Beckman Coulter) was used for APS analysis.FlowCAM Imaging Particle Analysis Technology (Portable seriesFlowCAM, Fluid Imaging Technoglogies, Inc.) was used forcharacterizing AB stained particles (hereafter referred to asFlowCAM.).

Temperature and pH were measured by a benchtop pH meter(FiveEasy Plus, Mettler-Toledo, Switzerland). Turbidity wasmeasured using a Hach 2100P Portable Turbidmeter. A totalorganic carbon analyzer (TOC-L, Shimadzu Corp.) was used toanalyze TOC and DOC. A light microscope (Olympus EX51), camera(Olympus DP 20), at 400� and 1000� magnification, togetherwith a hemacytometer counting chamber (Neubauer-improved,Marienfeld) were used for phytoplankton count and identification.A scanning electron microscope (SEM) (SM-6700F SEM, JEOL Co,Tokyo, Japan) was also used for species identification.

A 0.02% Alcian blue solution (adjusted to pH 2.5 using aceticacid) was prepared for TEP, dAPS and AB stained particles analyses.The 0.05-lm and 0.4-lm pre-filtered AB solutions were used incase of TEP and AB stained particles, respectively, while theunfiltered solution was used for dAPS.

2.2. Water collection and storage

In the one-year study, raw water was sampled monthly fromApril 2013 to March 2014 at Pao-Shan reservoir I (E 255756.00,N 2737648.00), located in Baoshan Township, Hsinchu County,Taiwan. This reservoir, which was described previously by Liuet al. (2009), has an effective capacity of 5 � 106 m3, mainly

N.T. Thuy et al. / Chemosphere 131 (2015) 139–148 141

offering public water supply. A sample volume of 5 L was taken inan acid – washed bottle from the surface water, at the monitoringstation situated at about 100 m far from the shore of the reservoir.In the intensive study, sampling in the same reservoir was carriedout mostly weekly from April to July 2014 in anticipation of aplankton bloom, which in 2013 had occurred during May.

2.3. TEP and APS measurement

Samples for analyses of pTEP (in whole study period), cTEP anddAPS (during the bloom in 2014) were either tested immediatelyor preserved with 2% formalin at 3 �C until analysis. pTEP andcTEP were measured according to the protocols proposed byPassow and Alldredge (1995) and Villacorte et al. (2009a), respec-tively. At least three replicate filters for each sample were preparedfor each pTEP or cTEP analysis. dAPS was analyzed by a modifiedmethod from two methods given by Arruda Fatibello et al. (2004)and Thornton et al. (2007). 30 mL filtrate collected after 0.05-lmfiltration were placed in the 50 mL centrifuge tubes. 5 mL of theunfiltered AB solution were added, and final pH was adjustedapproximately to 2.5 by 1.3 mL acetic acid. Four replicate cen-trifuge tubes were prepared for each sample. The centrifuge tubeswere mixed gently for 1 min, then centrifuged for 30 min at10000 rpm (12096 � g). The supernatants were taken to measureabsorbance at 610 nm.

The model polysaccharides, GX was used to calibrate TEP andAPS. TEP calibration was following Passow and Alldredge (1995)with four replicate filters for each volume of standard solution.For APS calibration, a GX stock solution was prepared by mixing20 mg GX into 200 mL DI water, ground with a tissue grinder,mixed for 30 min, and ground again. The stock solution was dilutedto five concentrations (1, 5, 10, 15, 20 lg mL�1). Four replicates ofeach dilution (30 mL) were placed in centrifuge tubes and followedthe same procedure noted above for APS analysis. Calibrationcurves were drawn based on the relationship between the GXconcentration and the supernatant absorbance.

2.4. Characterization of AB stained particles (>5-lm) by FlowCAM

AB stained particles in the water sample (taken in the 2014bloom) were analyzed by FlowCAM using the auto-image modeat a constant rate of 20 frames s�1, 100�magnification (10� objec-tive and 100-lm flow cell). The flow rate was set at 0.15 mL min�1

with a fluid volume imaged of 0.2500 ± 0.0001 mL. Both raw waterand AB stained samples were analyzed for comparison. To preparethe AB stained sample, 4 mL of the 0.4-lm pre-filtered AB solutionand 1 mL acetic acid were added into 50 mL of raw water sample ina volume metric flask. By the adding acetic acid, pH values of theAB stained samples were about 2.6 ± 0.1. These samples were thenmixed gently up and down several times prior to analysis, whilethey were gently stirred during analysis. Two replicates of eachwater sample were prepared for FlowCAM analysis. After theanalysis, images of particles with the size from 5-lm werecollected. Images of AB stained particles were selected manuallyfrom the AB stained samples’ images, based on their specific bluecolor and the comparison with the raw samples’ images. Theselecting process was easier with the application of ‘‘sort by color’’(average blue) since a significant change in blue intensity of thesestained particles was found after staining of sample with AB.Particle count and volume fractions of the selected particles wereautomatically provided, in which volume fractions (in ppm) ofthe particles were calculated based on the volume of particle asfollowing equation:

ppm ¼ Total volume of particles using ESD ðlm3Þ

� 106=total volume imaged ðlm3Þ

For characterization of AB stained particles, particles size dis-tribution (by equivalent spherical diameter, ESD) and aspect ratiowere considered.

2.5. Other water quality parameters measurement

Raw water quality parameters such as temperature, pH,turbidity, chlorophyll a (chl a), TOC and phytoplankton count weremonitored during whole study period. Chl a was measured by fol-lowing the standard method of EPA, Taiwan (NIEA E508.00B).During the 2014 bloom, DOC, phytoplankton species’ identificationand count were additionally measured. Note that the samples forDOC analysis were prepared by filtering the raw water throughthe 0.4-lm polycarbonate filter. The phytoplankton count wasconducted using light microscope (LM). Species identificationwas based on their morphology using LM with the supportingmeans of SEM. Unit of phytoplankton counting was natural unitcount (units mL�1).

2.6. Statistical analysis

Regression analysis was used to assess the potential correlationbetween different parameters gained during the phytoplanktonbloom of 2014, performed by IBM SPSS Statistic 21.0 software.Data were Log transformed except where indicated.

3. Results and discussion

3.1. One-year study of pTEP and other water quality parameters(2013–2014)

Variations of pTEP concentration and other water qualityparameters in Pao-Shan reservoir were investigated monthly forone year, from April 2013 to March 2014. Clearly, temperature,phytoplankton count, chl a, and pTEP concentration showed thesame trends from April to July 2013 (detailed information of thevariations is given in Fig. A1 in supplementary data). A phytoplank-ton bloom occurred during May 2013, as the beginning of summer,with the phytoplankton count up to 22200 units mL�1 and chl a of15.4 lg L�1. At the same time, temperature, pH and pTEP reachedtheir maximum values. On the other hand, the trends of TOC andturbidity seemed not to correlate with pTEP. Unfortunately, thechanges of pTEP and other parameters at the time of the bloomwere not measured, since the frequency of sampling was only onceper month. Therefore, the fluctuations of these parameters wereintensively measured during the bloom period in 2014, asexplained in the following section. During the summer season,from July to September 2013, the temperature remained at highlevels, while pTEP concentration, chl a, phytoplankton count, andpH tended to decrease, perhaps as a result of the depletion ofnutrients in the reservoir (Corzo et al., 2000; Mari et al., 2005;Wetz et al., 2009). From October to December 2013, temperaturesdecreased sharply, indicative of the winter season, and thenremained at low values up to March 2014. Chl a, phytoplanktoncount and pTEP concentration were likely not linked during thisperiod. For instance, in October, when the phytoplankton countdecreased, chl a doubled and pTEP concentration remained almostunchanged compared to the previous month. This temporalrelationship between pTEP and chl a is consistent with the resultsof Prieto et al. (2006), who conducted the experiment in verticalprofile off the Strait of Gibraltar and found a correlation betweenTEP and chl a in the summer, but not in the winter.

142 N.T. Thuy et al. / Chemosphere 131 (2015) 139–148

3.2. Intensive study of AB stained fractions and other waterparameters during the phytoplankton bloom in 2014

3.2.1. Composition and abundance of phytoplankton species and otherwater quality parameters

The results of the one-year study reported in Section 3.1 sug-gested the occurrence of a phytoplankton bloom in early each sum-mer. Thus, intensive sampling was conducted in the early summerin 2014, from April to July, in order to investigate in greater detailthe variations of AB stained fractions and other water parametersduring a phytoplankton bloom period. As the release ofexopolysaccharides by microorganisms depends on the species,the physiological state of the organisms and environmental growthconditions (Passow, 2002b), careful monitoring of speciescomposition was suggested in TEP dynamic study in aquaticenvironment during phytoplankton bloom (Fukao et al., 2010).Hence, beside chl a, identification and count of phytoplankton spe-cies were additionally monitored for this bloom period.

Fig. 1. Fluctuation during phytoplankton bloom in 2014 of (a) temperature, pH

Fig. 1 shows the composition and abundance of phytoplanktonspecies and other water quality parameters in Pao-Shan reservoirduring the phytoplankton bloom in 2014. Temperature graduallyincreased from 20 �C to 33 �C, while turbidity fluctuated from1.68 to 5.04 NTU. TOC and DOC changed slightly, ranging from1.01 to 2.55 and 1.00 to 2.06 mg L�1, respectively and pH was8.69 ± 0.16. In whole study period, Aphanocapsa delicatissima andCyclotella spp. were the most frequently found and remained themost abundant species. The first phytoplankton bloom with thedominance of A. delicatissima was found during April 2014 (up to56100 units mL�1 on 16 April), followed by the secondphytoplankton bloom around 03 June 2014. In the second bloom,phytoplankton composition was more complicated, with a consis-tently high number of A. delicatissima, as well as three other species(Cyclotella spp., Gloeocystis sp. and Achnanthes minutissima) whichwere found in quite large numbers. After that, individual bloomsof Cyclotella spp., Gloeocystis sp. and A. minutissima appearedsimultaneously, reaching their highest counts on 13 June, at

, DOC, TOC, and turbidity; and (b) chl a and phytoplankton species’ count.

Fig. 2. AB stained particles: (a) variation in concentration and volume ratio; (b)typical FlowCAM images arranged using ‘‘soft by color’’ (average blue). (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

N.T. Thuy et al. / Chemosphere 131 (2015) 139–148 143

16000, 5300, and 2350 units mL�1, respectively. On 26 June,corpses of some previous observed species appeared (such asCyclotella spp., Desmodesmus grahneisii, and A. minutissima) indicat-ing that these species were going into senescence. Typical imagesof the phytoplankton species can be seen in Fig. A2 in supplemen-tary data.

Chl a and total phytoplankton count variations were significantlydifferent, with a very simple trend of chl a compared to that of thephytoplankton count. Chl a concentration ranged from 0.9 to13.9 lg L�1, from the beginning of the bloom until senescence.Interestingly, the sample with the highest chl a concentration didnot correspond to the first bloom which contained the highestphytoplankton counts, but was coupled with the second bloom.Similar observations have been made in other studies (Dolan et al.,1978; Felip and Catalan, 2000). The reason may be accounted forby the variation in composition of phytoplankton between twoblooms, since chl a within a single cell varies among taxa and theirphysiological condition, temperature, nutrient concentration andlight intensity (Felip and Catalan, 2000). In the second bloom,phytoplankton species were more diverse, including cyanobacteria,chlorophyta, bacillariophyceae, while in the first bloom,cyanobacteria species were mainly found. Hence, estimatingcommunities with a single metric, such as chl a may not be sufficient.

We further found that frequency of sampling was a factoraffecting the results of phytoplankton variation. For instance, whensampling was done once per month (Section 3.1), there seemed tobe relationships between the changes of chl a and phytoplanktoncount, both peaking at the end of May, 2013. However, whensampling was performed more intensively during the blooms in2014, a significant difference was found between behaviors ofthese two biomass estimators, the highest value of chl a appearingin the second bloom, as noted above.

3.2.2. Characterization of AB stained particles with size larger than5-lm

Observation of TEP by traditional microscopic methods (Loganet al., 1994; Passow and Alldredge, 1994) in which samples needundergoing filtration process, may lead to distortion (Anja, 2009)and overlap of particles. Moreover, due to the flexibility of TEP,filtration pressure may result in passing and therefore under-estimating these particles (Passow and Alldredge, 1995). Thenotion of using a FlowCAM for AB stained particle analysis camefrom the effort to keep particle from becoming distorted duringanalysis, by retaining AB stained particle in its original solutioninstead of filtering. This solution then was directly passed throughthe FlowCAM’s flow cell for capturing images. As a rapid form ofinvestigation and automatically providing related morphologicalfeatures of particle, FlowCAM has been widely applied to enumer-ate plankton (Álvarez et al., 2011). This is the first time FlowCAMhas been used for AB stained particles, mainly containingphytoplankton-derived polysaccharides.

Fig. 2 illustrates the variation of AB stained particles in concen-tration and volume ratio, and typical images collected fromFlowCAM during the phytoplankton bloom in 2014. From Fig. 2a,concentration of AB stained particles ranged from 2400 to 12000particles mL�1, which is higher than the stained TEP (>5-lm) con-centration range reported in the literature, e.g. between 1 and 8000particles mL�1 (Passow, 2002b). Expressed as volume fraction, ABstained particles ranged from 0.75 to 7.70 ppm, which was withinreported ranges, e.g. 0.1 to >300 ppm (Passow, 2002b). In the firstbloom, though phytoplankton count was very high, AB stained par-ticles only increased slightly. AB stained particles then sharplyincreased and reached the highest value in concentration (on 13June) and in volume fraction (during 03–13 June).

AB stained particles examined by FlowCAM showed a diversityof morphological forms. When the particle images were arranged

using ‘‘soft by color’’ (average blue), several identifiable formscould be seen, typically showed in Fig. 2b: particles with a strongblue color, often appeared amorphous and cloud-like, which maycontain mainly acid polysaccharide; particles originated frommucilage surrounding or still attaching to phytoplankton cells;particles as internally stained cells; and particles as detritus par-tially stained with AB in a dark blue color.

Observation of AB stained particles’ images suggested that thecontribution of each morphological form varied during the bloomperiod. Remarkably, a predominant number of the particles origi-nated from the mucilage covers surrounding the cells ofGloeocystis sp. was observed during June 2014, exactly at the bloomof this species as presented in Section 3.2.1. AB stained particles ofGloeocystis sp. were reassured by LM and SEM as shown in Fig. 3.These particles appeared with cores of spherical cells of 3 to4-lm, surrounding by spherical mucilage sheaths of 8 to 16-lm.

The contribution of this species was confirmed by further analy-sis of morphological features of AB stained particles through parti-cles size distribution (PSD) (Fig. 4a and 4b) and aspect ratio (AR)(Fig. 4c). Clearly, the size of AB stained particles was mainly within5–20-lm. Beside a consistent high number of small size particles(around 5-lm), larger size particles (about 9–11-lm) tended torise from 27 May, created a distinguish peak on 13 June, thengradually decreased. Results of volume ratio additionally revealedthat these larger particles were dominant in volume contributioncompared to the smaller particles. By sorting the size regionaround the peak, the majority of images displayed were AB stainedparticles generated by Gloeocystis sp. From another approach basedon AR, AB stained particles with AR close to 1 varied with the sametendency as the larger particles mentioned above. Based on Fig. 4d,

Fig. 3. Gloeocystis sp. after AB staining: spherical cell with mucilage sheath by LM (a, b) and by SEM (c, d).

Fig. 4. Particle size distribution by frequency (a) and volume fraction (b); aspect ratio by frequency (c), and typical images corresponding to aspect ratio of sample taken on13 June 2014 of AB stained particles (d).

144 N.T. Thuy et al. / Chemosphere 131 (2015) 139–148

N.T. Thuy et al. / Chemosphere 131 (2015) 139–148 145

the particles with AR close to 1 were assigned again mainly to ABstained particles generated from Gloeocystis sp. Therefore,FlowCAM can provide valuable information of particles’ features,and would consequently offer different ways to assess thecontribution of various particles.

However, there are still some limitations of using FlowCAM forAB stained particles. Though taking images of particles was auto-mated, separation of images of AB stained particles from imagesof other particles was done manually based on their specific bluecolor. Creating a set of parameters (e.g. average blue, transparencyand aspect ratio, with specific ranges) used in software filterswould be an option to automatically isolate images of AB stainedparticles from other particles. Another restriction of usingFlowCAM is the accuracy in estimate the volume of AB stainedparticle since this volume was provided automatically as thevolume of whole particle, which may contain non-staining partssuch as cells, organisms and detritus. This estimate would beimproved by further considering the operating conditions ofFlowCAM such as particle segmentation (dark and white thresh-olds, and close holes) in defining a pixel as being a part of particle.

3.2.3. Variation of other AB stained fractionsIn this section, the different fractions (pTEP, cTEP and dAPS)

covering the full size range of AB stainable material weresimultaneously monitored during the phytoplankton bloom in2014 by using the colorimetric method. This differs from ABstained particles identified and quantified by microscopic visual-ization and enumeration as detailed in the previous section.Moreover, some overlaps of AB stainable substances measured asAB stained particles and pTEP are expected since the measure-ments of AB stained particles and pTEP consider the particles from5 lm and the particles bigger than 0.4 lm, respectively.

pTEP concentration ranged from 0.088 to 0.604 mg L�1, whichwas quite stable from beginning up to 14 May, then started toincrease and peaked on 13 June, and later gradually decreasing(Fig. 5). This trend of variation is similar to the variation in numeri-cal concentration of AB stained particles mentioned in Section 3.2.2.pTEP range was within pTEP values reported in the literature,e.g. 0.064–2.755 mg L�1 in lake Kinneret, Israel (Berman andViner-Mozzini, 2001), 0.036–1.462 mg L�1 in northern temperatelakes, Michigan (US) and 0.066–9.038 mg L�1 in Mediterraneanlakes (southern Spain) (de Vicente et al., 2010). On the other hand,cTEP was found to be in a wider range, from 0.258 to 2.153 mg L�1

and its trend was more complicated compared to that of pTEP.Overall, an upward trend was observed through several

Fig. 5. Fluctuations of pTEP, cTEP and dAPS concentration during phytoplanktonbloom period in 2014.

fluctuations with local maxima, followed by a decline after 26June. Since no temporal fluctuation of cTEP concentration has beenreported in the literature, data for cTEP was not available for com-parison. However, cTEP range in this study included discrete resultsfrom previous studies for surface waters, e.g. 0.21 mg L�1 in river(Villacorte et al., 2010), 0.5 mg L�1 in a lake, 0.36 mg L�1 in a canal(Villacorte et al., 2009a), 0.43 mg L�1 in a reservoir or 1.64 mg L�1 ina canal (Villacorte et al., 2009b), and 0.684 ± 94 mg L�1 in surfacewater (Van Nevel et al., 2012).

dAPS concentration was found to be notably higher comparedto pTEP and cTEP concentration, with an average of6.57 ± 0.56 mg L�1. The trend of dAPS was not obvious, with aslightly increase (11%) during 20 May to 3 June compared to thebeginning of the bloom period, and then a gradual increase from26 June and continuing to the end of the period. There has been lit-tle work on dissolved APS (Thornton et al., 2007; Thornton, 2009;Thornton and Visser, 2009; Villacorte et al., 2015a), with tworeports in freshwaters by Thornton et al. (2007) and Villacorteet al. (2015a). In Thornton et al. (2007), a concentration of APS of4.87 ± 0.42 mg L�1 in a golf course pond and 21.1 ± 0.09 mg L�1 ina garden pond were reported. Note that the APS in their studywas the APS in the 0.4-lm filtrate. Our results for dAPS (in the0.05-lm filtrate) fell within these values. Villacorte et al. (2015a)has been the latest report which considered dissolved polysaccha-ride in form of TEP and its precursor, measured by the new methodproposed in their study. Similarly, TEP and its precursor were foundsignificantly higher than pTEP alone (measured based on themethod of Passow and Alldredge (1995)). It is also noted that, whileour study measured dAPS in its own original water, Villacorte et al.(2015a) measured TEP and its precursor which were re-suspendedin ultra-pure water for minimizing the interference of dissolvedsalts. Though freshwater contains low salt concentrations, furtherstudy may be needed to confirm whether salinity in freshwatersources interferes AB staining in dAPS analysis.

3.2.4. Correlation of APS fractions to chemical, biological parameters,and phytoplankton composition

To assess possible drivers of AB stainable material in the watercolumn, the relationships among AB stained fractions, chemicaland biological variables in the 2014 bloom were statistically inves-tigated (Table 1). Temperature and pH seemed to play an impor-tant role in regulating AB stained particles, pTEP and cTEP, sincethey both had a strong positive correlation with these fractions.cTEP was also found to be correlated with TOC and DOC.Surprisingly, AB stained particles, pTEP and cTEP were not signifi-cant correlated to either phytoplankton count or chl a, while dAPSwas negatively related to phytoplankton count. This result partiallydiffers from the results of the one-year study, which likely showeda relationship of pTEP to both phytoplankton count and chl a dur-ing the bloom in 2013.

In the literature, pTEP concentration and chl a have mostly beenfound significantly correlated during the exponential growth per-iod of batch cultures or phytoplankton blooms (Corzo et al.,2000; Passow, 2002a; Mari et al., 2005; Radic et al., 2005;Ortega-Retuerta et al., 2009; Villacorte et al., 2010), but also havebeen found not to be correlated in some studies (Radic et al.,2005; Wetz et al., 2009; Taylor et al., 2014). If there was a correla-tion, it was specific for each individual bloom, but no general over-all relationship between TEP and chl a has be derived (Passow,2002b). This is likely to be true for our study as well, since the firstbloom with the highest total count, dominated by A. delicatissima,did not lead to an increase of pTEP and only a slight increase in thenumber of AB stained particles, while in the second bloom, with alower total count but with a more diverse composition, a sharplyincrease of pTEP and AB stained particles was observed. The high-est values of pTEP and AB stained particles were found at the same

Table 1Regression analysis with Pearson coefficient (r) and p values on Log-transformed data (except pH) performed between concentrations of AB stained fractions and other waterquality variables.

AB stained fractions Variables

Phytoplankton count chl a Temperature Turbidity pH TOC DOC

AB stained particles r 0.443 0.409 0.684 0.001 0.877 0.557 0.323p 0.065 0.083 0.005 0.498 0.000 0.024 0.141

pTEP r 0.061 0.375 0.547 �0.123 0.610 0.341 0.021p 0.422 0.103 0.027 0.345 0.013 0.127 0.473

cTEP r 0.093 0.106 0.859 �0.320 0.646 0.725 0.546p 0.381 0.366 0.000 0.143 0.008 0.003 0.027

dAPS r �0.569 �0.103 0.476 �0.373 �0.132 0.414 0.455p 0.021 0.369 0.050 0.105 0.333 0.080 0.059

n = 13, bold numbers indicate statistically significant correlation (p < 0.05).

Table 2Correlation matrix with Pearson coefficient (r) and p values of concentrations of ABstained fractions.

AB stained particles pTEP cTEP dAPS

AB stained particles – 0.824 0.649 0.027pTEP 0.000 – 0.486 0.081cTEP 0.008 0.046 – 0.334dAPS 0.465 0.396 0.132 –

n = 13, correlation is significant at p < 0.05 (in bold), p values: italic.

146 N.T. Thuy et al. / Chemosphere 131 (2015) 139–148

time with the individual blooms of Cyclotella spp., Gloeocytis sp.and A. minutissima. This observation suggested looking further forpotential species linked to the formation of AB stained fractionsby investigating the relationship between these fractions andphytoplankton composition during the bloom period.

The results of regression analysis of the concentrations of ABstained fractions and the abundance of individual phytoplanktonspecies are given in Table A1 in supplementary data. Clearly, ABstained particles and pTEP concentration show the strongestcorrelation with A. minutissima, Gloeocystis sp., Cyclotella spp.,followed by Sphaerocystis sp., Monoraphidium sp. and Oocystisspp. Of these, Cyclotella spp. were the most abundant, followedby Gloeocystis sp., then A. minutissima (Section 3.2.1). Variousstudies have shown that Cyclotella spp. can excrete copiousamounts of extracellular polysaccharide threads (i.e. mucilage(Stevenson et al., 1996)) for attachment in chains (Okaichi, 2004;Seckbach and Kociolek, 2011). Achnanthes sp. has also beenreported to secrete a basal pad of mucilage from one pole of theraphe-bearing valve, and the additional formation of a mucilagestalk (Nagabhushanam, 1997). Gloeocystis sp. forms a broad firmenvelope made of concentric layers of mucilage (Shephard,1987), which was clearly seen in this study. Hence, Cyclotellaspp., Gloeocystis sp. and A. minutissima could be the principalspecies that release pTEP and AB stained particles. By contrast,A. delicatissima, which was previously described as a pico-cyanobacteria appearing in colonies in diffuse mucilage (Callieriet al., 2012), showed a weak negative correlation with pTEP andAB stained particles even though this species appeared in highnumbers.

cTEP concentration had the strongest correlation with Oocystisspp. and D. grahneisii, followed by Pediastrum asymmetricum,Coelastrum sp. and Tetraedron minimum (Table A1), though theseall species appeared only in unremarkable amounts. In contrastto other AB stained fractions, dAPS was negatively correlated tophytoplankton count (Table 1), and in negative—but not signifi-cant—relationship with pH and chl a (Table 1), and several species(Table A1). A weak positive correlation between dAPS concentra-tion with P. asymmetricum was also found, but this correlationmay not play an important role because of the insignificantamount of this species.

3.2.5. Interaction among different AB stained fractionsIn natural waters, the formation pathway of TEP is still contro-

versial since simultaneous measurements of TEP and its precursorshave been scantily reported. Some studies have attempted to find alink between them, but the precursors were represented by TOC,DOC, POC, dissolved monosaccharides (DMCHO), and dissolvedpolysaccharides (DPCHO) (Ortega-Retuerta et al., 2009; de Vicenteet al., 2010). Since DOC, TOC and POC parameters cover a widerange of organic compounds, and DMCHO and DPCHO are the

measurements of both neutral and acidic dissolved carbohydrates,they may be not appropriate to represent TEP precursors thatapparently contain a more homogenous group of acidic polysaccha-rides (Ortega-Retuerta et al., 2009). In our work too, no conclusionscould be drawn, even though we also found a correlation among ABstained particles and cTEP concentration with DOC or/and TOC.Hence, monitoring of all AB stained fractions together would be abetter way to track the fate of as well as the interaction amongthem, because all these fractions are predominantly made up ofacidic polysaccharides.

Table 2 shows the correlation between different AB stainedfractions during the bloom in 2014. As can be seen, even thoughthe results of AB stained particles’ abundance and pTEP concentra-tion obtained by two different methods of measurement are notdirectly comparable, consistency between them was confirmed(r = 0.824, p < 0.001). cTEP concentration also showed a positivecorrelation with pTEP, but at a less pronounced level (r = 0.486,p = 0.046). Surprisingly, dAPS concentration was not statisticallyrelated to any other factions.

Since dAPS concentration was negatively correlated withphytoplankton abundance, and not correlated with other ABstained fractions (AB stained particles, pTEP and cTEP), it was unli-kely to be generated directly by the phytoplankton, and is probablynot the main precursor of AB stained particles and pTEP. On theother hand, the strong relationship of AB stained particles andpTEP with abundance of some phytoplankton species further indi-cates that these two fractions were predominantly releaseddirectly by these phytoplankton. Consequently, abiotic formationof these two fractions from dissolved polysaccharides (i.e. dis-solved APS) may be only partly involved. This finding contrastedwith major previous studies that have suggested that pTEP wasformed mainly abiotically from dissolved precursors (Mopperet al., 1995; Passow, 2000, 2002b), dissolved exopolymers(Alldredge et al., 1993), or DOM (Mari and Burd, 1998; Wurlet al., 2011). Nevertheless, it is noted that those previous studiesmainly conducted for marine water sources while this study basedon a single freshwater source. More extensive investigation isneeded to see whether freshwater and marine water possess dif-ferent formation pathways of these fractions.

N.T. Thuy et al. / Chemosphere 131 (2015) 139–148 147

4. Conclusions

Phytoplankton was confirmed as the major source of excretionof AB stained particles and pTEP, not through the total phytoplank-ton count or chl a, but the abundance of some individual species,including Cyclotella spp., Gloeocystis sp. and A. minutissima. Thisresult differs from the result of the one-year study, which showedan apparent relationship between pTEP concentration, phytoplank-ton count, and chl a. This difference indicates that higherfrequently sampling with phytoplankton species considerationwould be important for studying the links between AB stainedfractions and phytoplankton. From the relation between differentfractions of AB stained material, dAPS was unlikely to have beengenerated directly from phytoplankton, and therefore may not bethe main precursor of AB stained particles and pTEP. Thus, the abi-otic formation of AB stained particles and pTEP from dissolvedpolysaccharides, as suggested by many previous studies, may beinsignificant, while their formation would be dominant by thedirect release of some individual phytoplankton species.

The successful application of FlowCAM in visualizing AB stainedparticles recommends this technique by which particle morpholo-gies can be conserved and valuable morphological features of suchparticles can be simultaneously provided. Future study can focuson the optimization of the analytical conditions and programmingfor the separation automatically of images of AB stained particlesfrom other non-stained particles so that this technique can bewidely applied for various water sources.

Acknowledgement

This work was supported by National Science Council, Taiwan[NSC 101-2221-E-033-031].

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2015.03.023.

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