Rapid effects of diverse toxic water pollutants on ... › ... › files › 2016 › 07 › Choi_etal_201… · on chlorophyll a ﬂuorescence: Variable responses among freshwater

ww.sciencedirect.com

wat e r r e s e a r c h x x x ( 2 0 1 2 ) 1e1 2

Available online at w

journal homepage: www.elsevier .com/locate/watres

Rapid effects of diverse toxic water pollutantson chlorophyll a fluorescence: Variable responsesamong freshwater microalgae

Chang Jae Choi 1, John. A. Berges, Erica. B. Young*

Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA

a r t i c l e i n f o

Article history:

Received 3 November 2011

Received in revised form

24 January 2012

Accepted 11 February 2012

Available online xxx

Keywords:

Chlorophyll a fluorescence

Algae

Toxin

Rapid response

Photosystem II

Diuron�

KCN

Glyphosate

Atrazine

Fv/FmFluorometry

Pseudokirchneriella

Diatom

Cryptophyte

Chlorophyte

Chrysophyte

Water quality

Toxic water pollutant

Toxicity assessment

Abbreviations: Chlorophyll, chl; PAM, pulsquantum yield of photosystem II.* Corresponding author. Tel.: þ1 414 229 325E-mail addresses: [email protected]

1 Present address: The University of Texas

Please cite this article in press as: Choi, CVariable responses among freshwater m

0043-1354/$ e see front matter ª 2012 Elsevdoi:10.1016/j.watres.2012.02.027

a b s t r a c t

Chlorophyll a fluorescence of microalgae is a compelling indicator of toxicity of dissolved

water contaminants, because it is easily measured and responds rapidly. While different

chl a fluorescence parameters have been examined, most studies have focused on single

species and/or a narrow range of toxins. We assessed the utility of one chl a fluorescence

parameter, the maximum quantum yield of PSII (Fv/Fm), for detecting effects of nine

environmental pollutants from a range of toxin classes on 5 commonly found freshwater

algal species, as well as the USEPA model species, Pseudokirchneriella subcapitata. Fv/Fmdeclined rapidly over <20 min in response to low concentrations of photosynthesis-specific

herbicides Diuron� and metribuzin (both <40 nM), atrazine (<460 nM) and terbuthylazine

(<400 nM). However, Fv/Fm also responded rapidly and in a dose-dependent way to toxins

glyphosate (<90 mM), and KCN (<1 mM) which have modes of action not specific to

photosynthesis. Fv/Fm was insensitive to 30e40 mM insecticides methyl parathion, carbo-

furan and malathion. Algal species varied in their sensitivity to toxins. No single species

was the most sensitive to all nine toxins, but for six toxins to which algal Fv/Fm responded

significantly, the model species P. subcapitatawas less sensitive than other taxa. In terms of

suppression of Fv/Fm within 80 min, patterns of concentration-dependence differed among

toxins; most showed MichaeliseMenten saturation kinetics, with half-saturation constant

(Km) values for the PSII inhibitors ranging from 0.14 mM for Diuron� to 6.6 mM for terbu-

thylazine, compared with a Km of 330 mM for KCN. Percent suppression of Fv/Fm by

glyphosate increased exponentially with concentration. Fv/Fm provides a sensitive and

easily-measured parameter for rapid and cost-effective detection of effects of many dis-

solved toxins. Field-portable fluorometers will facilitate field testing, however distinct

responses between different species may complicate net Fv/Fm signal from a community.

ª 2012 Elsevier Ltd. All rights reserved.

e amplitude modulated; PSII, photosystem II; Fv/Fm, maximum quantum yield of PSII; FPSII,

7; fax: þ1 414 229 3926.texas.edu (C.J. Choi), [email protected] (J.A. Berges), [email protected] (E.B. Young).at Austin, Marine Science Institute, Port Aransas, TX 78373, USA.

.J., et al., Rapid effects of diverse toxic water pollutants on chlorophyll a fluorescence:icroalgae, Water Research (2012), doi:10.1016/j.watres.2012.02.027

ier Ltd. All rights reserved.

mailto:[email protected]



www.sciencedirect.com/science/journal/00431354

http://dx.doi.org/10.1016/j.watres.2012.02.027




1. Introduction periods of 5e72 h (e.g. Brack and Frank, 1998; Juneau et al.,

Microalgae are commonly used for aquatic toxicological

testing because they are easy to grow and expose to dissolved

toxins in culture, they are sensitive to hazardous chemicals or

wastewater contaminants, and they have short generation

times that allow rapid assessment of growth responses to

toxins. United States Environmental Protection Agency

(USEPA) and the Organisation for Economic Co-operation and

Development (OECD) have standardized tests that measure

toxicity of dissolved chemicals as inhibition of growth over

3e4 days in replicate microalgal cultures, typically using

model test species, including the green alga Pseudokirchneriella

subcapitata (Korshikov) Hindak (formerly Selenastrum capri-

cornutum) (OECD, 1984; USEPA, 2002). Althoughmicroalgae and

other model organisms (including vertebrates) are important

for characterizing toxin effects under controlled conditions,

growth and maintenance of test organisms can be time

consuming and expensive, and correct end-point measure-

ments for meaningful comparisons are not easily predicted

(Hughes et al., 1988). For detection of environmental

contamination, and in water security applications, there is

a need to find cheaper and faster testing procedures which

provide reliable and ecologically-relevant detection of aquatic

toxins (Galloway et al., 2004). This has led to the development

of instrumentation for rapid testing including immobilized

algae (Bozeman et al., 1989), miniaturized microplate and

microbiotest assays using algae (Lukavsky, 1992; Muller et al.,

2008), array-chip multi-algal biosensors (Podola and

Melkonian, 2005) and in-line algal biomonitors (recent

advances in applications reviewed by Ralph et al., 2007;

Buonasera et al., 2011) These new developments offer

advantages over the USEPA standard 3e4 day growth bioas-

says, but can still require extensive set-up and extended

incubation periods.

Advances in technologies to measure chlorophyll a (chl a)

fluorescence offer good prospects for toxicity testing using

bioassays with algal species (Brack and Frank, 1998;

Buonasera et al., 2011). Chl a fluorescence provides a sensitive

integrated measure of a key photosynthetic process - the

efficiency of energy conversion at photosystem II (PSII) reac-

tion centers. Chl a fluorescence parameters respond to envi-

ronmental stresses, including nutrient limitation (Parkhill

et al., 2001; Young and Beardall, 2003), high light or UV expo-

sure (Franklin and Forster, 1997) and oxidative stress

(Drabkova et al., 2007). Rapid changes in chl a fluorescence

parameters have previously been shown to provide similar

information to suppression of photosynthetic C fixation

(Dorigo and Leboulanger, 2001), and growth inhibition over

3e4 d (e.g. Fai et al., 2007; Macedo et al., 2008; Ralph et al., 2007;

Kviderova, 2009). Pulse Amplitude Modulated (PAM) fluorom-

eters offer one approach to very high sensitivitymeasurement

of chlorophyll a fluorescence in cells for use with natural

marine or freshwater phytoplankton samples, and are

increasingly being applied to toxicity testing (Schreiber et al.,

1993, 2002). Studies have frequently focused on a small

number of toxins or a single test species (often P. subcapitata or

other species commonly used in laboratory studies) and have

examined responses of chl a fluorescence to toxins over

Please cite this article in press as: Choi, C.J., et al., Rapid effects ofVariable responses among freshwater microalgae, Water Resear

2002; Vallotton et al., 2008; Kviderova, 2009). However, chl

a fluorescence changes much more rapidly in response to

stress (including exposure to toxic chemicals (Rodriguez et al.,

2002; Macedo et al., 2008)) and so responses over minutes

rather than hours, are potentially useful for rapid toxicological

assessments (Rodriguez et al., 2002; Schreiber et al., 2002;

Rodriguez and Greenbaum, 2009).

Of a range of chl a fluorescence parameters which respond

rapidly to toxin exposure, the most commonly used is the

quantum yield of PSII, a measure of photochemical efficiency

(Ralph et al., 2007; Buonasera et al., 2011). Changes in PSII

quantum yield could be used to examine toxin exposure in

natural freshwater algal assemblages over less than 25 min,

with potential applications for real-time testing and water

security applications (Schreiber et al., 1993; Rodriguez et al.,

2002; Buonasera et al., 2011). However, successful applica-

tion of chl a fluorometry to toxicity testing in natural ecosys-

tems requires much more detailed understanding of the

variability of responses of Fv/Fm to both awider range of toxins

and in a wider range of species.

In the present study, we aimed to examine the variability of

responses of Fv/Fm in diverse algal species in response to

a range of toxins. Our specific objectives were: 1) to assess the

range of responses of different algal taxa, particularly how

sensitivities of common algal taxa compare with that of

laboratory model organisms, 2) to identify which toxins elicit

a response in Fv/Fm and over what concentration ranges, and

3) to clarify whether sensitivity of Fv/Fm responses is related to

specific toxins via examination of changes in chl a fluores-

cence parameters F0 and Fm. We selected five freshwater algal

species representing four broad taxonomic groups commonly

found in temperate freshwater ecosystems (chlorophytes,

diatoms, chrysophytes and cryptophytes), and included the

EPA model chlorophyte species P. subcapitata for comparison.

We exposed the microalgae to nine environmental pollutants

(somewith specific effects on photosynthesis, as well as those

with other modes of action) with potential for contamination

in natural waterways or reservoirs (Gilliom et al., 1999;

Rodriguez et al., 2002), andmeasured very short-term changes

in Fv/Fm.

2. Materials and methods

2.1. Test organisms and culture conditions

Freshwater algal taxa tested represented four distinct taxo-

nomic groups e Chlorophyta, Bacillariophyta, Cryptophyta

and Heterokontophyta (Wehr and Sheath, 2003). Cultures of

the algae were obtained from the Canadian Phycological

Culture Collection (CPCC, www.phycol.ca) and all cultures

were maintained axenically in the same growing conditions

(18 �C, 100 mmol photons m�2 s�1 on a 12:12 light:dark cycle,

gently stirred and bubbledwith filtered air). Triplicate cultures

of each species were diluted with growth medium every 3e5

days to keep cells in exponential growth phase. The green alga

Chlamydomonas reinhardtii Dang. (CPCC 84 syn. UTEX 89) was

grown in DY-V freshwater medium pH 7.0 (Andersen, 2005).

diverse toxic water pollutants on chlorophyll a fluorescence:ch (2012), doi:10.1016/j.watres.2012.02.027

http://www.phycol.ca



wat e r r e s e a r c h x x x ( 2 0 1 2 ) 1e1 2 3

The green alga P. subcapitata (Korshikov) Hindak (CPCC 37,

formerly known as S. capricornutum or Raphidocelis subcapitata

syn. UTEX 1648), the diatoms Navicula pelliculosa (Breb.) Hilse

(CPCC 552 syn. CCAP 1050/3c) and Asterionella formosa Hass.

(CPCC 605), the cryptophyte Cryptomonas erosa Ehrenberg

(CPCC 446) and the chrysophyte Synura petersenii Korshikov

(CPCC 442 syn. CCMP 866) were all grown in CHU-10 medium,

pH 7.0 (Andersen, 2005). DY-V and CHU-10 are artificial

freshwater media with similar constituents but we found

Chlamydomonas grew better in DY-V than CHU-10; DY-V

containsMES orMOPS but CHU-10 does not include an organic

pH buffer (Andersen, 2005). Cell growth was monitored by

changes in bulk chl a fluorescence measured in a Turner TD-

700 fluorometer with red-sensitive PM tube and a chl

a optical kit (Turner Designs, Sunnyvale, CA, USA). When

cultures reached mid log-phase, 20 mL of cultures were

transferred to 50 mL tubes for toxin exposure and chl a fluo-

rescence measurements.

2.2. Toxins

We tested nine toxins representing six chemical classes

including those with modes of action directly on photosyn-

thetic processes and photosystem II (PSII) (atrazine, metribu-

zin, terbuthylazine and Diuron�) and others with modes of

actions not directly related to photosynthesis (KCN, glyph-

osate, carbofuran, malathion and methyl parathion) (Table 1).

The toxins atrazine, metribuzin, and terbuthylazine (triazine

herbicides), carbofuran (a carbamate pesticide), malathion

and methyl parathion (organothiophosphate pesticides) were

obtained from SPEX Certiprep (Metuchen, NJ, USA), and

glyphosate (a phosphonoglycine herbicide) (Restek, Belle-

fonte, PA), all supplier-certified as 99% pure, and KCN (Fisher

Scientific, Hannover Park, IL, USA) and Diuron� (DCMU,

a phenyl-urea herbicide) (SigmaeAldrich Corp. St. Louis, MO,

USA), certified as �98% pure, were also tested. Toxins were

dissolved in water, acetone, ethanol or methanol according to

their solubility (see Table 1) and small volumes (<1% of culture

volume) of toxin solutions were added directly to the 20 mL

culture subsample and gently mixed. Final toxin concentra-

tions tested ranged from 1 nM to 30 mM. To test for effects of

the solvent without the toxin, control trials for each species

were carried out with solvents alone at the highest volume

added (0.2 mL solvent in 20 mL culture). All species-toxin

concentration combinations were tested using triplicate

cultures.

2.3. Chl a fluorescence and cell viability measurements

Chl a fluorescence was measured using a Walz Pulse Ampli-

tude Modulated XE-PAM fluorometer (Schreiber et al., 1993)

(Walz GmbH, Effeltrich, Germany). The excitation light was

filtered using a 5 mm BG39 band-pass filter (500 nm peak;

Schott Elmsford, NY, USA). Fluorescence emissionwas filtered

by a 1 mm R65 long-pass (>650 nm) dichroic filter (Balzers,

Zarentem, Belgium). Preliminary trials were carried out to

determine the effect of dark acclimation prior to addition of

toxins. Dark incubation either increased the quantum yield or

had no effect, so for all species the following exposure and

measurement protocol was used. Immediately after

Please cite this article in press as: Choi, C.J., et al., Rapid effects ofVariable responses among freshwater microalgae, Water Researc

harvesting from the culture, cells were incubated for 30min in

the dark at 18e22 �C with gentle shaking, prior to initial

measurement of chl a fluorescence. In dark-incubated cells,

the Xe-PAM fluorometer measured F0 - the minimal chl

a fluorescence output, and Fm - themaximal chl a fluorescence

output during a saturating pulse, and derived Fmax, the

maximum quantum yield of PSII (Schreiber et al., 1993;

Parkhill et al., 2001; Ralph et al., 2007), hereafter referred to as

Fv/Fm:

Fmax ¼ Fv=Fm ¼ ðFm � F0Þ=Fm (1)

The percent suppression of Fv/Fm over time in response to

toxins was calculated as:

% suppression ¼ ðFv=Fm0� Fv=FmtÞ=Fv=Fm0 � 100 (2)

where Fv/Fm0 is the initial Fv/Fm at time 0, prior to toxin

exposure, and Fv/Fmt is the Fv/Fm after exposure time t

(2e80 min) to the toxin. For each culture subsample tested,

cell density and PAM settings were adjusted to give similar

initial F0 output across trials.

Measurements were made just prior to toxin addition and

then at 2, 5, 10, 20, 40, and 80 min after toxin addition. During

exposure to toxins, samples were incubated in the dark in

a shaking incubator at 18e22 �C, and subsamples were

removed for each time-point measurement in a darkened

room. The exception was for Diuron� which required low

irradiance (<5 mmol photons m�2 s�1) during the 80 min

exposure to be effective. However, the initial Fv/Fm, prior to

Diuron� addition, was determined as for other toxins,

following 30 min dark incubation.

In order to examine more detailed doseeresponse rela-

tionships of the Fv/Fm suppression in response to 6 of the

toxins, a sub-set of the toxin-species combinations examined

were tested with a finer resolution of toxin concentrations.

When significant inhibitory effects of toxin were detected

after 80 min, cell viability was assessed in 1 mL of culture by

adding 20 mL of 1% (w/v) Evans Blue and incubating for 5 min

before observing and enumerating cell staining using an

Olympus BX-41microscope. Evans Blue is impermeable to live

cells but freely penetrates the membrane of dead cells and

stains blue (Crippen and Perrier, 1974)), thus blue-stained cells

were identified as dead, with heat-killed cultures (80 �C for

10 min) serving as positive controls.

2.4. Statistical analysis of toxin effects

For each species and toxin concentration, the Fv/Fm measured

at time zero (immediately before toxin addition), and at 80min

were compared for significant differences using analysis of

variance (1-way ANOVA of Fv/Fm variables with time as the

factor, for each toxinespecies combination). When significant

declines were observed, the inhibitory effects of different

toxins in the six algal species were examined by plotting Fv/Fmagainst time over 80 min. Most plots showed an exponential

decline in Fv/Fm over time so the time axis was log-

transformed to linearize the plots. A linear model was fitted

to the decline and the slope of line tested for significant

differences from zero. Toxin sensitivity was evaluated as

diverse toxic water pollutants on chlorophyll a fluorescence:h (2012), doi:10.1016/j.watres.2012.02.027



Table 1 e The toxins tested and solvents used, their chemical names, classes, modes of action and the summary toxicity determined in other studies.

Toxin(solvent)

IUPAC chemical name Chemical class, use, CAS number,EPA toxin classa

Mode of action LC50b aquaticvertebratesmg mL�1

EC50 greenalgaeb

mg mL�1

Conc. rangeused

mg mL�1

Diuron�

(ethanol)

3-(3,4-dichlorophenyl)-1,

1-dimethylurea (DCMU)

Phenyl-urea herbicide 330-54-1, EPA III PS II inhibitor 6.7e47 0.005 0e58

Glyphosate

(water)

N-(phosphonomethyl) glycine Phosphonoglycine herbicide 1071-83-6,

EPA II

Aromatic amino acid

synthesis inhibitor

5e240 7e400 0e750

KCN (water) Potassium cyanide Inorganic rodenticide 151-50-8, EPA I Oxidative metabolism

inhibitor (mitochond.)

0.065e1.4 0.025e0.045 0e1950

Atrazine

(acetone)

6-chloro-N2-ethyl-N4-isopropyl-1,

3,5-triazine-2,4-diamine

Triazine herbicide 1912-24-9, EPA III PS II inhibitor 5e50 0.04e1.5 0e10

Metribuzin

(methanol)

4-amino-6-tert-butyl-3-methylthio-1,

2,4-triazin-5(4H)-one

Triazinone herbicide 21087-64-9, EPA III PS II inhibitor 0.030e0.15 0.043 0e5

Terbuthylazine

(methanol)

N2etert-butyl-6-chloro-N4-ethyl-1,3,

5-triazine-2,4-diamine

Triazine herbicide algaecide, microbiocide

5915-41-3, EPA III

PS II inhibitor 7e8 0.016 0e50

Methyl parathion

(methanol)

O,O-dimethyl O-4-nitrophenyl-

phosphorothioate

Organothiophosphate insecticide 298-00-0,

EPA I

Cholinesterase inhibitor 0.8e25 2.9e100 0e10

Carbofuran

(methanol)

2,3-dihydro-2,2-dimethylbenzo-furan7-

yl-methylcarbamate

Carbamate insecticide nematicide 1563-66-2,

EPA I/II

Cholinesterase inhibitor 0.1e5 270 0e10

Malathion

(methanol)

Diethyl (dimethoxyphos-phinothioylthio)

succinate

Organothiophosphate insecticide 121-75-5,

EPA III

Cholinesterase inhibitor 0.3e15 0.01 0e10

a EPA Toxin classes: I (highly toxic), II (moderately toxic), III (slightly toxic).

b Summary LC50 (median lethal dose) and EC50 (half maximum effective concentration) values from EcotoxNet (2010) and PAN (2010).

water

research

xxx

(2012)1e12

4Please

citeth

isarticle

inpress

as:C

hoi,C

.J.,etal.,R

apid

effe

ctsofdiverse

toxic

waterpollu

tants

onch

loro

phyllafluoresce

nce:

Varia

ble

resp

onse

sam

ongfre

shwaterm

icroalga

e,W

aterRese

arch

(2012),doi:1

0.1016/j.w

atres.2

012.02.027




percent suppression of Fv/Fm at 80min relative to Fv/Fm values

just prior to toxin addition. To examine toxin sensitivity over

a finer range of toxin concentrations, the doseeresponse

relationships of selected species to 6 of the toxins were

analyzed as % suppression of Fv/Fm, and the relationships

were modeled with non-linear regression, using a rectangular

hyperbola (for MichaeliseMenten kinetics), or, in one case

where the data clearly did not follow saturation kinetics, an

exponential model. All statistical analyses used Sigmastat (v.

3.1, Systat Software Inc, Chicago, IL).

Fig. 1 e A range responses of Fv/Fm to exposure to toxins in

six microalgal species. Suppression of Fv/Fm is shown over

80 min following addition at 0 min of A) glyphosate

(440 mM), B) KCN (1 mM), C) atrazine (460 nM) and D)

metribuzin (40 nM) Species symbols shown in panel D are

the same for all plots. Points are means ± standard error of

measurements in three replicate trials.

3. Results and discussion

3.1. Rapid toxin detection using Fv/Fm

The chl a fluorescence parameter Fv/Fm of algal cells provided

an extremely rapid method to detect responses to several

aquatic toxins in the EPAmodel test species P. subcapitata, and

a range of common freshwater algal taxa. In each case, for the

controls with solvent-only additions, the slope of regression

lines for Fv/Fm over time was not significantly different from

zero (P> 0.05 ANOVA). However, following exposure to 6 of the

9 toxins tested, Fv/Fm declined significantly over < 80 min

( p < 0.05 ANOVA) (Fig. 1, Table 2). In response to atrazine,

metribuzin (Fig. 1) and terbuthylazine, Fv/Fm declined rapidly

overw10 min but thereafter remained stable. Fv/Fm decline in

S. petersenii, N. pelliculosa, A. formosa and C. erosa in response

to KCN or glyphosate continued almost throughout the full

80 min (Fig. 1). Most of the suppression of Fv/Fm was observed

within 20 min following toxin addition (Fig. 1), and 80 min

certainly could serve as an appropriate duration for a bioassay

based on algal Fv/Fm suppression. Fv/Fm was not significantly

suppressed in most algal species in response to the cholin-

esterase inhibitors methyl parathion, carbofuran and mala-

thion (Table 2) demonstrating that these pesticides, which

inhibit nerve function in insects, will not readily induce rapid

responses in Fv/Fm in microalgae.

Despite strong toxin effects on Fv/Fm, there was little

evidence of cell mortality within the 80 min exposure for any

species. Only in cultures exposed to very high glyphosate

concentrations (400 mM) did Evans Blue staining indicate

compromised cell membranes in approximately 30e50% of

cells of each species. The rapid responses of Fv/Fm over 80min

therefore represent sub-lethal effects on algal cells.

3.2. Algal species differences

To address our objective of determining algal species sensi-

tivity to toxins, we found that there clearly were different

sensitivities between algal species, but no single algal taxon

was themost sensitive to all nine toxins (Fig. 1; criteria defined

in Table 2). For example, based on percent suppression of Fv/

Fm in response to the toxins glyphosate, KCN, atrazine, met-

ribuzin and terbuthylazine, the EPA model species P. sub-

capitatawas not themost sensitive algal taxon (Fig. 1, Table 2).

Therefore, we predict that use of data on toxin suppression of

Fv/Fm from the model species P. subcapitata will result in

underestimating the sensitivities of photosynthesis in

commonly found freshwater species or natural assemblages.

Please cite this article in press as: Choi, C.J., et al., Rapid effects of diverse toxic water pollutants on chlorophyll a fluorescence:Variable responses among freshwater microalgae, Water Research (2012), doi:10.1016/j.watres.2012.02.027



Table

2eSum

mary

ofeffectsoftoxin

sonFv/F

mwithm

inim

um

effect

conce

ntrationsforth

e6algals

peciesand9toxin

stested.T

oxin

effectsare

reportedwhendeclin

esin

Fv/F

msh

owedlinearregress

ionslopesover80m

insignifica

ntlydifferentfrom

zero

(P<

0.05).

Toxin

Diuro

n�

Glyphosa

teKCN

Atrazine

Metribuzin

Terb

uth

ylazine

Meth

ylparath

ion

Carb

ofu

ran

Malath

ion

Maxim

um

conc.

tested

58mgm

L�1

250mM

75mgm

L�1

440mM

195

0mgm

L�1

30m

M10mgm

L�1

46mM

10mgm

L�1

47mM

46mgm

L�1

200

mM

10mgmL�1

38mM

10mgm

L�1

45mM

10mgm

L�1

30mM

Division

Species

Chloro

phyta

P.su

bcapitata

Very

stro

ng

<43nM

Slight

<440mM

Strong

<1mM

Slight

<460nM

Slight

<40nM

Moderate

<400nM

ns

Slight

<45mM

ns

Chloro

phyta

C.reinhardtii

Very

stro

ng

<43nM

Strong

<440mM

Slight

<2mM

Strong

<460nM

Moderate

<40nM

Moderate

<400nM

ns

ns

ns

Bacillariophyta

N.pellicu

losa

Very

stro

ng

<43nM

Strong

<220mM

Moderate

<0.5

mM

Slight

<460nM

ns

ns

ns

ns

ns

Bacillariophyta

A.form

osa

Very

stro

ng

<43nM

Strong

<90mM

Strong

<1mM

ns

ns

ns

Slight

<40mM

ns

ns

Hetero

kontophyta

S.petersenii

Very

stro

ng

<43nM

Strong

<440mM

Strong

<1mM

Slight

<460nM

Moderate

<40nM

Slight

<400nM

ns

ns

ns

Cryptophyta

C.erosa

Very

stro

ng

<43nM

Strong

<220mM

Strong

<1mM

ns

ns

ns

ns

ns

ns

Statisticalresp

onse

criteriafrom

linearregressionanalysis:

ns¼

noeffect

where

P>

0.05(linearregression)forslopeofFv/F

mover80min

athighest

conce

ntrationtested.1

.Slighteffect

¼significa

nt

decline(P

<0.05)in

Fv/F

mover80min,finalFv/F

m>0.4

forallco

nce

ntrationstested.2.Moderate

effect

¼significa

ntdecline(P

<0.05)in

Fv/F


m>0.3

and<0.4.3.Strong

effect

¼significa

ntdecline(P

<0.05)in

Fv/F


m<0.3.4.Very

stro

ngeffect

¼significa

ntandalm

ost

immediate

decline(P

<0.05),very

stro

ngsu

ppressionofFv/F

m.


Please cite this article in press as: Choi, C.J., et al., Rapid effects of dVariable responses among freshwater microalgae, Water Research

Differences in algal sensitivity to a range of toxins have been

reported (Juneau et al., 2002; Fairchild et al., 1998) and may

relate to genetically-determined mechanisms for assimila-

tion and detoxification or to variable toxin uptake kinetics

and capacity between algae (Weiner et al., 2004) which in

turn depend on the presence of other ions such as Cl� (Lee

et al., 2005). Rapid screening methods for toxin detection

using algal Fv/Fm responses could readily incorporate a wider

range of themost sensitive or ecologically important species.

Broad taxonomic group was not a good indicator of toxin

sensitivity (Fig. 1, Table 2). The two chlorophytes, P. sub-

capitata and C. reinhardtii showed divergent responses of Fv/

Fm to toxin exposure. P. subcapitata was highly sensitive to

KCN, relatively insensitive to glyphosate and atrazine while

C. reinhardtii was relatively insensitive to KCN but highly

sensitive to glyphosate and atrazine (Fig. 1, Table 2). The two

diatoms, A. formosa and N. pelliculosa also showed different

sensitivity to toxins. Both species were highly sensitive to

glyphosate but while A. formosa was less sensitive to atra-

zine, it was more sensitive to methyl parathion (Table 2). In

conventional multi-day algal growth bioassays, N. pelliculosa

growth was more sensitive to atrazine than growth of

a marine green alga Dunaliella tertiolecta (Hughes et al., 1988)

but for Fv/Fm, N. pelliculosa was less sensitive than C. rein-

hardtii (Table 2). The differences in responses to toxins

between groups suggest that no one species is likely to

provide the ideal, most sensitive test organism for all the

toxin types tested. However, the recommended ideal, to

include a suite of test species (Fairchild et al., 1998), becomes

more practical with such rapid screening methods based on

Fv/Fm, while this would be more expensive and less practical

for standard 3e4 day growth inhibition assays.

Taxa differences in Fv/Fm sensitivities to toxins suggests

that, in mixed algal species assemblages, suppression of Fv/

Fm in more sensitive species could potentially be masked by

chl a emission from less sensitive species (see discussion in

Franklin et al., 2009). This, along with effects of nutrient

limitation, temperature variability and photoacclimation or

photoinhibition processes in natural conditions (Holmes

et al., 1989; Franklin and Forster, 1997; Chalifour and

Juneau, 2011), may complicate application of this method

to natural phytoplankton assemblages (e.g. Rodriguez and

Greenbaum, 2009). We initially screened three cyanobacte-

rial taxa in our experiments, but optimizing Fv/Fm in cya-

nobacteria using PAM is problematic (Campbell et al., 1998).

We tried different filters (620e630 nm band-pass filters) to

optimize the excitation and emission wavelengths to

increase Fv/Fm values from cyanobacterial cultures, but were

not satisfied with the results so we restricted our measure-

ments to eukaryotes. Lower and more variable Fv/Fm values

in cyanobacteria could also complicate interpretation of net

Fv/Fm responses to toxins in natural populations which

include cyanobacteria.

One ecological consequence of variable toxin sensitivity

is that sub-lethal effects of toxin contamination of natural

waters may influence competition and stimulate changes in

species composition of natural phytoplankton assemblages

(Seguin et al., 2002; Zananski et al., 2010). In contaminated

waters where species composition of natural populations

has acclimated to chronic toxin exposure, Fv/Fm may not

iverse toxic water pollutants on chlorophyll a fluorescence:(2012), doi:10.1016/j.watres.2012.02.027




change in response to experimental toxin exposure as

dramatically as in individual test taxa with no previous

exposure history.

3.3. Toxin effects on F0, and Fm

As Fv/Fm ratios are derived from measured parameters F0 and

Fm (eq. (1)), changes in Fv/Fm can involve changes in one or

both parameters (Table 3). In response to DCMU, both F0 and

Fm increased in all species. This is the basis for Diuron�

(DCMU) use in measuring maximum chl a fluorescence

emission (Parkhill et al., 2001), but responses of F0 and Fm to

other toxins have been lesswell documented. Changes in F0 vs

Fm varied between algal species (Table 3). For example, in

response to KCN and glyphosate, in all species, Fv/Fm declines

involved decreases in Fm. In contrast, in response to glyph-

osate, F0 declined in all species except A. formosa, but the F0increased, decreased or was unchanged in response to KCN in

different species (Table 3). Between closely related Chlor-

ophyte species, in response to atrazine, F0 and Fm both

increased in P. subcapitata, but F0 was almost stable and Fmdeclined in C. reinhardtii (Fig. 2). In responses tometribuzin, Fv/

Fm declines involved increases in F0 in all species, but no

consistent changes in Fm between species including the two

green algae.

Differences in contribution of F0 vs Fm to Fv/Fm suppression

reflect different toxin modes of action. Differences between

even closely related taxa may indicate that responses to

specific toxin action on photosynthesis vary. Short-term

changes in chl a fluorescence emission over <80 min (i.e.

responses not dependent on de novo protein synthesis), reflect

changes in energy flux through chlorophyll harvesting

antennae (LHCII), specific inhibition of photosynthetic elec-

tron transport, or changes in use of ATP and NADPH products

of electron transport. Increases in F0 could relate to specific

inhibition of electron transport (PSII, plastoquinone pool),

induced by the herbicides. These expected F0 increases were

observed with all sensitive species with Diuron�, terbuthyla-

zine andmetribuzin but only with P. subcapitata and C. erosa in

response to atrazine.With glyphosate, F0 declines could relate

to impaired light absorption or uncoupling of electron trans-

port, while decreases in Fm observed consistently with both

glyphosate and KCN, could relate to uncoupling of LCHII from

PSII via energy-dependent non-photochemical quenching

(qN) (Holmes et al., 1989; Young and Beardall, 2003). However,

Table 3 e Summary of toxins effects on F0 and Fm for each speduring 80 min exposure (see Table 2). D [ increase, 0/D [ slidecrease. Blank spaces indicate non-significant responses of F

Algal species Diuron� Glyphosate KCN Atrazine Met

F0 Fm F0 Fm F0 Fm F0 Fm F0

P. subcapitata þ þ e e þ e þ þ þC. reinhardtii þ þ e e þ e 0/þ e þN. pelliculosa þ þ e e 0 e 0 e þA. formosa þ þ 0/þ e þ e

S. petersenii þ þ e e e e þ e þC. erosa þ þ e e 0/� e


as incubations were in the dark (except Diuron�), any qN

changes will not relate to actinic light-dependent energy

responses, but may reflect direct chemical effects on trans-

thylakoid pH gradient dissipation, possibly via perturbations

of ATP and NADPH use (Holmes et al., 1989; Young and

Beardall, 2003). Quenching parameters have been specifically

examined to investigate toxin modes of action (Brack and

Frank, 1998; Juneau and Popovic, 1999; Juneau et al., 2002,

2005; Fai et al., 2007). Our data suggest that as both F0 and

Fm can change, suggesting that steady state F output also

varies during incubation, quenching parameters may be

complicated to interpret between taxa (see discussion in

Juneau and Popovic, 1999; Juneau et al., 2005). Therefore, for

rapid toxicological assessment using a range of species,

maximum quantum yield may be a more straightforward

parameter than any of the quenching parameters.

3.4. Toxin sensitivity - dose-responses

There were clear differences in the dose-responses of

suppression of Fv/Fm among different toxins tested (Fig. 3) and

minimum toxin concentrations required for a significant

decline in Fv/Fm over 80 min in each species (Table 2; selected

species in Fig. 3). Although concentrations tested were not

always comparable between toxins (due to different detection

ranges, modes of action and solubility limitations), generally,

Fv/Fm in the test algae was less sensitive to suppression by

organothiophosphate or carbamate pesticides than to the

triazine herbicides and phenyl-urea herbicides, that specifi-

cally inhibit PSII and electron transport. Some of the effects of

specific PSII toxins (atrazine, metribuzin, terbuthylazine,

Table 1) on chl a fluorescence parameters in microalgae, have

been reported in some algal taxa (Brack and Frank, 1998; Fai

et al., 2007; Vallotton et al., 2008; Dorigo and Leboulanger,

2001). However, this study also shows significant dose-

dependent suppression of Fv/Fm by toxins KCN and glyph-

osate, which are not known to directly inhibit PSII. To address

the objective of determining the concentration ranges over

which different toxins affect Fv/Fm, each toxin is discussed

below.

3.4.1. Diuron�

The specific inhibition of electron transport from PSII to

plastoquinone by Diuron� (DCMU) resulted in extremely high

sensitivities of Fv/Fm in all species to Diuron� (minimumeffect

cies for which there were significant suppression of Fv/Fmght increase, 0 [ no change, e [ decrease, 0/L [ slight

v/Fm (see Table 2).

ribuzin Terbuthylazine Methyl parathion Carbofuran

Fm F0 Fm F0 Fm F0 Fm

0 þ 0 þ 0

0/� þ 0

0

0/þ e

e þ e




Fig. 2 e Contrasts in changes in F0 vs Fm as components of

Fv/Fm suppression in response to atrazine in P. subcapitata

and C. reinhardtii. Fm symbols are filled and F0 points are

open symbols. Points are means ± standard error of 2e3

replicate measurements.


concentration <0.01 mg mL�1 or <43 nM), a MichaeliseMenten

half-saturation constant (Km) of 140 nM, and saturation at

<5 mM in P. subcapitata (Table 2, Fig. 3A). Despite this extreme

sensitivity, therewas only amaximumsuppression of Fv/Fm of

w80% over the range tested (0e250 mM).

3.4.2. AtrazineFv/Fmwas not sensitive to atrazine in all species over the range

of concentrations tested (Table 2), but for the sensitive species

S. petersenii, the effect on Fv/Fm saturated at a low concentra-

tions (<10 mM) with a Km of 0.18 mM. Maximum suppression

was onlyw32% (Figs. 1and 3D), suggesting partial inhibition or

limitation by cell uptake. The threshold concentration of

atrazine required for detection of changes in Fv/Fm over 80min

in the three most sensitive species (<0.1 mg mL�1 for C. rein-

hardtii, N. pelliculosa and P. subcapitata) was similar to atrazine

half maximal inhibitory concentration (IC50) concentrations

for effects on chl a fluorescence parameters and growth in P.

subcapitata (0.07e0.08 mg mL�1) (Fai et al., 2007; Vallotton et al.,

2008) and half maximal effective concentration (EC50) for 5

day growth of other algae (0.17 mg mL�1) (Hughes et al., 1988).

Research by Walsh (1972) suggested that inhibition photo-

synthetic oxygen evolution over 90 min and 10 day growth

assays showed similar sensitivity to atrazine in marine

microalgae. Therefore, high sensitivity (low Km) of algal Fv/Fmresponses to atrazine suggest that short-term changes in Fv/

Fm could be used for toxicological studies of atrazine, although

freshwater species will need to selected carefully, as two

species (A. formosa and C. erosa) were not sensitive to 46 mM

atrazine (Table 2).

3.4.3. Metribuzin and terbuthylazineThe Km for Fv/Fm suppression was 0.14 mM for metribuzin in P.

subcapitata, and 6.57 mM for terbuthylazine in C. reinhardtii

(Fig. 3E,F). The low Km and saturation concentration may

relate to specific binding to plastoquinone components and

suggests that binding sites are saturated at relatively low toxin


concentrations. Maximum suppression in response to metri-

buzin and terbuthylazine was only 35e42% (Figs. 1 and 3E,F) in

contrast to suppression in response to Diuron� which

approached 80% of initial values. This might suggest that

binding of these herbicides may be less effective than for

Diuron� or that toxic effect is limited by cell uptake capacity

(Weiner et al., 2004).

3.4.4. GlyphosateGlyphosate had strong effects on Fv/Fm in all species except P.

subcapitata, resulting in almost 100% suppression in N. pelli-

culosa (Fig. 1). Glyphosate inhibits aromatic amino acid

synthesis derived from the shikimate pathway (Funke et al.,

2006) rather than PSII directly, so the rapid and dramatic

inhibitory effect of glyphosate on Fv/Fm (Fig. 1, Table 1) may

involve disruption of integrated carbon and nitrogen assimi-

lation which serves as a sink for photochemically derived

energy (Holmes et al., 1989; Young and Beardall, 2003). The

consistent effects of glyphosate on F0 (declined in all but A.

formosa) and Fm (declined in all species) (Table 3) also suggests

some consistent mechanism leading to suppression of Fv/Fmin all algal species by glyphosate. Fv/Fm in the EPA model

species P. subcapitata was the least sensitive to glyphosate

while Fv/Fm inA. formosawas themost sensitive in response to

glyphosate (threshold <90 mM (15 mg mL�1)). In A. formosa,

suppression of Fv/Fm by glyphosate was not saturated over

0e440 mM but increased exponentially (Fig. 3B), the only toxin

to show this doseeresponse relationship. In comparison to

the specific PSII inhibitors (Diuron�, atrazine, metribuzin,

terbuthylazine), which saturated afterw10min (Fig. 1C,D), the

decline in Fv/Fm with glyphosate continued over a longer time

period in the most sensitive species (Fig. 1A), suggesting that

glyphosate effects on PSII might result from cumulative toxic

effects on other cellular processes. The threshold concentra-

tions for effects of glyphosate on Fv/Fm in the algae (90e440 mM

(15e75 mg mL�1)) were similar to typical median lethal doses

(LC50) over 96 h for fish (Fig. 3B, Tables 1and 2) so Fv/Fmresponses in microalgae may be suitable to detect rapid and

sensitive responses to glyphosate. Glyphosate-based herbi-

cides (e.g. Roundup�) are the most widely used herbicides

globally for agricultural and urban weed control and are

readily transported into aquatic systems via surface runoff

(Byer et al., 2008). The variable sensitivity observed among

algal taxa and known insensitivity to glyphosate in some

cyanobacteria (Powell et al., 1991) suggests that glyphosate

contamination in freshwater ecosystems may promote

changes in species dominance.

3.4.5. KCNSuppression of Fv/Fm by KCN in P subcapitata was virtually

100%, and shown to saturate at w2 mM in P. subcapitata with

a Km of 330 mM (Fig. 3C). Rapid declines in Fv/Fm in natural algal

assemblages have been shown in response to 2 mM

(130 mgmL�1) KCN (Rodriguez et al., 2002) but in this study, five

algal species showed threshold effect concentrations of less

than 1 mM (65 mg mL�1) (Table 2). Cyanide directly inhibits

cytochrome oxidase in mitochondrial electron transport so

KCN may affect PSII and thus chl a fluorescence indirectly via

a disruption of cellular energy metabolism which links mito-

chondrial and chloroplast processes (Holmes et al., 1989).




Fig. 3 e The maximum suppression of Fv/Fm after 80 min, relative to initial values prior to exposure to a range of

concentrations for toxins A) Diuron�, B) glyphosate, C) KCN, D) atrazine, E) metribuzin and (F) terbuthylazine. Plots are

shown for species for which the widest range of doses was examined. Each point is a single determination. Lines were fitted

by non-linear regression, using a rectangular hyperbola (MichaeliseMenten) (A, C-F) or an exponential (B), model. For the

MichaeliseMenten models, estimates of maximum suppression (Max) and half-saturation constants (Km) are shown on the

graphs. AeC are on the same y axis scale, and DeF on the same y axis scale. r2 values for non-linear models are asymptotic

values.


Compared to the specific PSII inhibitors which had fast effects

on Fv/Fm (mostly within 10 min of addition, Fig. 1C,D)

suppression by KCN continued over the full 80 min in most

sensitive species (Fig. 1B), which may relate to delayed effects

via inhibition of non-photosynthetic processes, resulting in

escalating toxic effects on cell function over time.It is possible

that in some algal species, cyanide could have more direct

effects on chl a fluorescence via inhibition of photosynthesis


enzymes Rubisco (observed in spinach by Wishnick and Lane

(1969)) and a chlororespiration oxidase (observed in a xan-

thophyte alga by Buchel and Garab (1995), but if so, KCN

should have induced an increase in Fm, but it declined in all

species tested (Table 3). Possible toxic effects in chloroplasts

may remain throughout the experiment, but partial recovery

of Fv/Fm observed in N. pelliculosa, C. reinhardtii and P. sub-

capitata (Figs. 1B and 4) suggests induction of a mitochondrial




Fig. 4 e Changes in Fv/Fm in P. subcapitata in response to

KCN illustrating partial recovery of Fv/Fm following rapid

initial suppression. Each point represents measurements

in subsamples of three replicate cultures exposed to

KCN ± standard error. Dose of KCN (0e30 mM) shown next

to each response plot.


alternative oxidase pathway, which is widespread in algal

taxa (Eriksen and Lewitus, 1999). The relatively lower sensi-

tivity of C. reinhardtii to KCN (Table 2) may relate to more

efficient induction of this alternative pathway. Despite these

clear doseeresponse relationships, the algae were relatively

insensitive to KCN, requiring much higher concentrations of

KCN to examine effects on Fv/Fm than for the other toxins,

especially the PSII inhibitors (Fig. 3). For comparison, model

fish species showed aminimum toxic dose during 2e4 days of

cyanide exposure three orders of magnitude lower

(0.15e0.3 mM, 0.01e0.02 mg mL�1) than for multi-day exposure

in algae (USEPA Acquire database, 2011), suggesting that algae

are not sensitive test organisms for studies of cyanide.

4. Conclusions and significance

� Diverse freshwater algal taxa showed variable sensitivity of

Fv/Fm suppression in response to nine toxins; there was no

one taxon which was the most sensitive, and lower sensi-

tivity of the laboratory model organism P. subcapitata than

other taxa suggest that for studies on these toxins, using

a suite of algal species will give more ecologically repre-

sentative responses.

� Rapid suppression in Fv/Fm offers considerable time-saving

advantages over traditional 3e4 day growth bioassays,

allowing a larger number of algal species, toxins and toxin

concentrations to be tested in the laboratory, with fewer

resources.

� While PSII inhibitor toxins were expected to have strong

effects on chl a fluorescence parameters, rapid Fv/Fmsuppression in response to glyphosate and KCN, which do


not directly target PSII, suggest that Fv/Fm changes can

reflect whole cell physiological stress.

� Bioassays based on rapid Fv/Fm changes offer most promise

in laboratory testing of toxins, where multiple species

selection can be customized to the ecosystem or taxa of

interest; field-portable fluorometers will facilitate field

testing, but distinct responses between species will

complicate interpretation of net Fv/Fm signals from

a community.

Acknowledgments

This study was supported by a grant from the Defense

Advanced Research Projects Agency (DARPA) through the

UWM Center for Water Security to E.B.Y and J.A.B. C-J.C. was

also supported by a UWM Graduate School Fellowship. Zac

Driscoll assisted with some measurements. Elias Greenbaum

and Miguel Rodriguez, Oak Ridge National Laboratory,

provided perspectives which were valuable in shaping this

research. Comments from two anonymous reviewers greatly

helped improve the clarity of the manuscript.

r e f e r e n c e s

Andersen, R.A., 2005. Algal Culturing Techniques. Elsevier/Academic Press, Burlington, Mass, 578 p.

Bozeman, J., Koopman, B., Bitton, G., 1989. Toxicity testing usingimmobilized algae. Aquatic Toxicology 14, 345e352.

Brack, W., Frank, H., 1998. Chlorophyll a fluorescence: a tool forthe investigation of the toxic effects in the photosyntheticapparatus. Ecotoxicology and Environmental Safety 40, 34e41.

Buchel, C., Garab, G., 1995. Evidence for the operation ofa cyanide-sensitive oxidase in chlororespiration in thethylakoids of the chlorophyll-c-containing alga Pleurochloris-meiringensis (Xanthophyceae). Planta 197, 69e75.

Buonasera, K., Lambreva, M., Rea, G., Touloupakis, E., Giardi, M.T.,2011. Technological applications of chlorophyll a fluorescencefor the assessment of environmental pollutants. Analyticaland Bioanalytical Chemistry 401, 1139e1151.

Byer, J., Struger, J., Klawunn, P., Todd, A., Sverko, E., 2008. Lowcost monitoring of glyphosate in surface waters using theELISA method: an evaluation. Environmental Science &Technology 42, 6052e6057.

Campbell, D., Hurry, V., Clarke, A.K., Gustafsson, P., Oquist, G.,1998. Chlorophyll fluorescence analysis of cyanobacterialphotosynthesis and acclimation. Microbiology and MolecularBiology Reviews 62, 667e683.

Chalifour, A., Juneau, P., 2011. Temperature-dependentsensitivity of growth and photosynthesis of Scenedesmusobliquus, Navicula pelliculosa and two strains of Microcystisaeruginosa to the herbicide atrazine. Aquatic Toxicology 103,9e17.

Crippen, R.W., Perrier, J.L., 1974. Use of neutral red and Evans bluefor live-dead determinations of marine plankton (withcomments on use of rotenone for inhibition of grazing). StainTechnology 49, 97.

Dorigo, U., Leboulanger, C., 2001. A pulse-amplitude modulatedfluorescence-based method for assessing the effects ofphotosystem II herbicides on freshwater periphyton. Journalof Applied Phycology 13, 509e515.





Drabkova, M., Admiraal, W., Mar�salek, B., 2007. Combinedexposure to hydrogen peroxide and light - Selective effects oncyanobacteria, green algae, and diatoms. EnvironmentalScience & Technology 41, 309e314.

EcotoxNet, 2010. The Extension Toxicology Network. http://extoxnet.orst.edu/.

Eriksen, N.T., Lewitus, A.J., 1999. Cyanide-resistant respiration indiverse marine phytoplankton. Evidence for the widespreadoccurrence of the alternative oxidase. Aquatic MicrobialEcology 17, 145e152.

Fai, P.B., Grant, A., Reid, B., 2007. Chlorophyll a fluorescence asa biomarker for rapid toxicity assessment. EnvironmentalToxicology and Chemistry 26, 1520e1531.

Fairchild, J.F., Ruessler, D.S., Carlson, A.R., 1998. Comparativesensitivity of five species of macrophytes and six speciesof algae to atrazine, metribuzin, alachlor, andmetolachlor. Environmental Toxicology and Chemistry 17,1830e1834.

Franklin, L.A., Forster, R.M., 1997. The changing irradianceenvironment: consequences for marine macrophytephysiology, productivity and ecology. European Journal ofPhycology 32, 207e232.

Franklin, D.J., Choi, C.J., Hughes, C., Malin, G., Berges, J.A., 2009.Large proportions of dead cells have little effect on variablechlorophyll fluorescence (Fv:Fm): implications for field surveys.Marine Ecology Progress Series 382, 35e40.

Funke, T., Han, H., Healy-Fried, M.L., Fischer, M., Schonbrunn, E.,2006. Molecular basis for the herbicide resistance of roundup-ready crops. Proceedings of the National Academy of SciencesUSA 103, 13010e13015.

Galloway, T.S., Brown, R.J., Browne, M.A., Dissanayake, A.,Lowe, D., Jones, M.B., Depledge, M.H., 2004. A multibiomarkerapproach to environmental assessment. EnvironmentalScience & Technology 38, 1723e1731.

Gilliom, R.J., Barbash, J.E., Kolpin, D.W., Larson, S.J., 1999. Testingwater quality for pesticide pollution. Environmental Science &Technology 33, 164e169.

Holmes, J.J., Weger, H.G., Turpin, D.H., 1989. Chlorophylla fluorescence predicts total photosynthetic electron flow toCO2 or NO3

�/NO2� under transient conditions. Plant Physiology

91, 331e337.Hughes, J.S., Alexander, M.M., Balu, K., 1988. In: Adams, W.J.,

Chapman, G.A., Landis, W.G. (Eds.), 1988. An Evaluation ofAppropriate Expressions of Toxicity in Aquatic Plant Bioassaysas Demonstrated by the Effects of Atrazine on Algae andDuckweed. Aquatic Toxicology and Hazard Assessment, vol.10. American Soc. for Testing and Materials, Philadelphia,pp. 531e547. ASTM STP 971.

Juneau, P., Popovic, R., 1999. Evidence for the rapid phytotoxicityand environmental stress evaluation using the PAMfluorometric method: importance and future application.Ecotoxicology 8, 449e455.

Juneau, P., El Berdey, A., Popovic, R., 2002. PAM fluorometry in thedetermination of the sensitivity of Chlorella vulgaris,Selenastrum capricornutum, and Chlamydomonas reinhardtii tocopper. Archives of Environmental Contamination andToxicology 42, 155e164.

Juneau, P., Green, B.R., Harrison, P.J., 2005. Simulation of Pulse-Amplitude-Modulated (PAM) fluorescence: limitations of somePAM-parameters in studying environmental stress effects.Photosynthesis 43, 75e83.

Kviderova, J., 2009. Rapid algal toxicity assay using variablechlorophyll fluorescence for Chlorella kessleri (Chlorophyta).Environmental Toxicology. 25, 554e563.

Lee, D.Y., Fortin, C., Campbell, P.G.C., 2005. Contrasting effects ofchloride on the toxicity of silver to two green algae,Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii.Aquatic Toxicology 75, 127e135.


Lukavsky, J., 1992. The evaluation of algal growth-potential (Agp)and toxicity of water by miniaturized growth bioassay. WaterResearch 26, 1409e1413.

Macedo, R.S., Lombardi, A.T., Omachi, C.Y., Rorig, L.R., 2008.Effects of the herbicide bentazon on growth and photosystemII maximum quantum yield of the marine diatomSkeletonema costatum. Toxicology In Vitro 22, 716e722.

Muller, R., Schreiber, U., Escher, B.I., Quayle, P., Nash, S.M.B.,Mueller, J.F., 2008. Rapid exposure assessment of PSIIherbicides in surface water using a novel chlorophylla fluorescence imaging assay. Science of The TotalEnvironment 401, 51e59.

OECD (Organisation for Economic Co-operation andDevelopment), 1984. Guidelines for Testing of Chemicals,Number 201: Alga, Growth Inhibition Test. Publication Service,Paris, France, pp. 246e59.

PAN, 2010. Pesticide Action Network Pesticides Database. http://www.pesticideinfo.org/ (accessed July 2010).

Parkhill, J.P., Maillet, G., Cullen, J.J., 2001. Fluorescence-basedmaximal quantum yield for PSII as a diagnostic of nutrientstress. Journal of Phycology 37, 517e529.

Podola, B., Melkonian, M., 2005. Selective real-time herbicidemonitoring by an array chip biosensor employing diversemicroalgae. Journal of Applied Phycology 17, 261e271.

Powell, H.A., Kerby, N.W., Rowell, P., 1991. Natural tolerance ofcyanobacteria to the herbicide glyphosate. New Phytology 119,421e426.

Ralph, P.J., Smith, R.A., Macinnis-Ng, C.M.O., Seery, C.R., 2007. Useof fluorescence-based ecotoxicological bioassays inmonitoring toxicants and pollution in aquatic systems:review. Toxicological and Environmental Chemistry. 89,589e607.

Rodriguez, M., Greenbaum, E., 2009. Detection limits for real-timesource water monitoring using indigenous freshwatermicroalgae. Water Environment Research 81, 2363e2371.

Rodriguez, M., Sanders, C.A., Greenbaum, E., 2002. Biosensors forrapid monitoring of primary-source drinking water usingnaturally occurring photosynthesis. Biosensors &Bioelectronics 17, 843e849.

Schreiber, U., Neubauer, C., Schliwa, U., 1993. PAM fluorometerbased on medium-frequency pulsed Xe-flash measuring light- a highly sensitive new tool in basic and appliedphotosynthesis research. Photosynthesis Research 36, 65e72.

Schreiber, U., Muller, J.F., Haugg, A., Gademann, R., 2002. Newtype of dual-channel PAM chlorophyll fluorometer for highlysensitive water toxicity biotests. Photosynthesis Research. 74,317e330.

Seguin, F., LeBihan, F., Leboulanger, C., Berard, A., 2002.Assessment of pollution: induction of atrazine tolerance inphytoplankton communities in freshwater outdoormesocosms using chlorophyll fluorescence as an endpoint.Water Research 36, 3227e3236.

USEPA, 2002. Pseudokirchneriella Subcapitata Chronic ToxicityTest Method, Based on Short Term Methods for Measuring theChronic Toxicity of Effluents and Receiving Waters toFreshwater Organisms, fourth ed.. EPA-821-R-02e013 TestMethod 1003.0.

USEPA Acquire database, 2011. http://cfpub.epa.gov/ecotox/(accessed Oct 2011).

Vallotton, N., Eggen, R.I.L., Escher, B.I., Krayenbuhl, J., Chevre, N.,2008. Effect of pulse herbicide exposure on Scenedesmusvacuolatus: a comparison of two photosystem II inhibitors.Environmental Toxicology and Chemistry 27, 1399e1407.

Walsh, G.E., 1972. Effects of herbicides on photosynthesis andgrowth of marine unicellular algae. Hyacinth Control Journal10, 45e48.

Wehr, J.D., Sheath, R.G., 2003. Freshwater Algae of North America:Ecology and Classification. Academic Press.


http://extoxnet.orst.edu/

http://extoxnet.orst.edu/

http://www.pesticideinfo.org/

http://www.pesticideinfo.org/

http://cfpub.epa.gov/ecotox/




Weiner, J.A., DeLorenzo, M.E., Fulton, M.H., 2004. Relationshipbetween uptake capacity and differential toxicity of theherbicide atrazine in selected microalgal species. AquaticToxicology 68, 121e128.

Wishnick, M., Lane, M.D., 1969. Inhibition of ribulose diphosphatecarboxylase by cyanide. Journal of Biological Chemistry 244,55e59.


Young, E., Beardall, J., 2003. Transient perturbations inchlorophyll a fluorescence elicited by nitrogen re-supply tonitrogen-stressed microalgae: distinct responses to NO3

�

versus NH4þ. Journal of Phycology 39, 332e342.

Zananski, T.J., Twiss, M.R., Mihuc, T.B., 2010. Use of fluorimetry toevaluate atrazine toxicity to phytoplankton communities.Aquatic Ecosystem Health & Management 13, 56e65.




Documents

Rapid effects of diverse toxic water pollutants on ... › ... › files › 2016 › 07 › Choi_etal_201… · on chlorophyll a ﬂuorescence: Variable responses among freshwater