Ecotoxicology and Environmental Safety 94 (2013) 37–44

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Ecotoxicology and Environmental Safety

0147-65http://d

n CorrE-m

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

Application of a fluorometric microplate algal toxicity assay for riverineperiphytic algal species

Takashi Nagai a,n, Kiyoshi Taya a, Hirochica Annoh b, Satoru Ishihara c

a National Institute for Agro-Environmental Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki 305-8604, Japanb ESCO Cooperation, Tomitake 173-2, Nagano, Nagano 381-0006, Japanc Food and Agricultural Materials Inspection Center, Agricultural Chemicals Inspection Station, Suzukicho 2-772, Kodaira, Tokyo 187-0011, Japan

a r t i c l e i n f o

Article history:Received 12 December 2012Received in revised form28 March 2013Accepted 23 April 2013Available online 21 May 2013

Keywords:Attached algaeIn-vivo fluorescencePesticidesSpecies sensitivity distribution

13/$ - see front matter & 2013 Elsevier Inc. Alx.doi.org/10.1016/j.ecoenv.2013.04.020

esponding author. Fax: +81 29 838 8199.ail address: nagait@affrc.go.jp (T. Nagai).

a b s t r a c t

Although riverine periphytic algae attached to riverbed gravel are dominant species in flowing rivers,there is limited toxicity data on them because of the difficulty in cell culture and assays. Moreover, it iswell known that sensitivity to pesticides differ markedly among species, and therefore the toxicity datafor multiple species need to be efficiently obtained. In this study, we investigated the use of fluorometricmicroplate toxicity assay for testing periphytic algal species. We selected five candidate test algal speciesDesmodesmus subspicatus, Achnanthidium minutissimum, Navicula pelliculosa, Nitzschia palea, andPseudanabaena galeata. The selected species are dominant in the river, include a wide range of taxon,and represent actual species composition. Other additional species were also used to compare thesensitivity and suitability of the microplate assay. A 96-well microplate was used as a test chamber andalgal growth was measured by in-vivo fluorescence. Assay conditions using microplate and fluorometricmeasurement were established, and sensitivities of 3,5-dichlorophenol as a reference substance wereassayed. The 50 percent effect concentrations (EC50s) obtained by fluorometric microplate assay andthose obtained by conventional Erlenmeyer flask assay conducted in this study were consistent.Moreover, the EC50 values of 3,5-dichlorophenol were within the reported confidence intervals inliterature. These results supported the validity of our microplate assay. Species sensitivity distribution(SSD) analysis was conducted using the EC50s of five species. The SSD was found to be similar to the SSDobtained using additional tested species, suggesting that SSD using the five species largely representsalgal sensitivity. Our results provide a useful and efficient method for high-tier probabilistic ecologicalrisk assessment of pesticides.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Paddy fields occupy more than half of the total agricultural landin Japan, and various herbicides are used for weed prevention inthese fields. Up to 50 percent of the applied herbicides areremoved as run-off, and they directly flow into rivers throughdrainage channels (Watanabe et al., 2008). Generally, algae is asensitive taxonomic group to herbicides (van den Brink et al.,2006). Moreover, herbicide effects on the species composition andcommunity structure of benthic algal assemblage were found innatural aquatic ecosystems (Sabater et al., 2007; Ricart et al.,2010). Therefore, an important concern of the herbicidal effect onnon-target organisms is algae in the river ecosystems.

Riverine periphytic algae attached to riverbed gravel plays animportant role in ecological function as the primary producers and

l rights reserved.

as food for invertebrates and fish (Finlay et al., 2002). Especially,diatoms are the most dominant algal group in terms of speciesnumber and biomass (Round et al., 1990). For example, Naviculasp. is extremely common and occurs in almost all periphytonsamples containing diatoms (Biggs and Kilroy, 2000). Further,Achnanthidium minutissimum is common and widespread in arange of ecological conditions but does best in clean, low con-ductivity streams. Nitzschia palea is widespread, common, andwell known as a pollution tolerant species (Biggs and Kilroy,2000). Besides diatoms, green algae and cyanobacteria are alsocommon periphytons. For example, Desmodesmus (Scenedesmus)sp. can be extremely common in the periphyton of low tomoderately enriched streams and Pseudanabaena (Phormidium)sp. is often very abundant in high-conductivity streams (Biggs andKilroy, 2000; ESCO, 2006).

However, in the conventional ecological effect assessment, theeffects of pesticides on algae are mostly assessed by using a singlestandard species, the green alga Pseudokirchneriella subcapitata,which is a planktonic algae and not the dominant species in river

T. Nagai et al. / Ecotoxicology and Environmental Safety 94 (2013) 37–4438

ecosystems (Kasai, 2003). Thus, only a few data are available forthe pesticide toxicity on riverine periphytic algal species (forexample Larras et al., 2012). Moreover, it is well known that thesensitivity to pesticides differs markedly among species (van denBrink et al., 2006; Nagai et al., 2011), and a single specific indicatoralgal species cannot be representative of the whole algal assem-blage (Freemark et al., 1990; Swanson et al., 1991). Therefore,toxicity data for multiple species should be obtained efficiently.

On the other hand, species sensitivity distribution (SSD) analysishas been developed to statistically deal with multiple species toxicitydata in ecological risk assessment (Posthuma et al., 2002). The SSD isa statistical distribution (often a log-normal distribution) describingthe variance among a set of species in their sensitivity to toxicants,for example, the 50 percent effect concentrations (EC50s) or noobserved effect concentrations (NOECs) of a certain chemical. TheSSD has been used for high-tier ecological risk assessment ofpesticides as a probabilistic method. The fifth percentile of adistribution (called the hazardous concentration for five percent ofthe species, HC5) has been chosen as a safe concentration thatprotects most species in a community (Posthuma et al., 2002).However, more data are required for the analysis, and in particulartoxicity data from more than five algal species is desired for SSDanalysis (OECD, 1995; TenBrook et al., 2008).

The purpose of this study was to demonstrate the applicationof an efficient and economical high-throughput algal toxicity assayusing five riverine periphytic species. The effects of pesticides onalgae are generally analyzed by standard test methods (OECD,2006), which require considerable labor for testing multiplespecies and is not suitable for periphytic species because of algalattachment to the surface of Erlenmeyer flasks (Swanson et al.,1991). Instead, Ishiahara et al. (2006) showed the use of amicroplate assay in which periphytic algae are attached to thebottom of a microplate. Microplate algal toxicity assay have beenwidely utilized as rapid and economical screening assay (Blaiseand Vasseur, 2005). Moreover, this test method is employedofficially for testing algal toxicity in Canada (EnvironmentCanada, 2007). The advantages of microplate-based toxicity assayare summarized as follows: (1) small sample volume requirement;(2) incubator space economy; (3) disposable microplate andpipette tips; and (4) increased bioanalytical output (Blaise andVasseur, 2005). Moreover, Eisentraeger et al. (2003) showed thatfluorometric measurement of algal growth has high measurementsensitivity. Therefore, we optimized fluorometric microplate toxi-city assay for testing multiple periphytic species.

In this study, five candidate test algal species were selectedconsidering not only their suitability for microplate assay but also

Table 1Test species, strains, and their culture conditions, including growth medium, applicabililight intensity used for maintenance or toxicity assay, and temperature.

Species Strain Taxonomicgroup

Medium

Pseudokirchneriella subcapitata NIES-35 Green algae CnDesmodesmus subspicatus NIES-797 Green algae CMayamaea atomus NIAES K11-11 Diatom CSiNitzschia palea NIAES PD3 Diatom CSiNitzschia palea NIAES U3-3 Diatom CSinNitzschia palea NIES-487 Diatom CSinAchnanthidium minutissimum NIES-71 Diatom CSiAchnanthidium minutissimum NIES-414 Diatom CSinNavicula pelliculosa UTEX-B673 Diatom CSinPseudanabaena galeata NIES-512 Cyanobacteria CTAnabaena flos-aquae NIES-73 Cyanobacteria CB

n Candidate species.

ecological relevance. Assay conditions using microplate andfluorometric measurements were then established, and the sensi-tivities of 3,5-dichlorophenol (DCP) were assayed and comparedamong species and among assay methods. Moreover, the validityof candidate species selection was discussed based on the obtainedresults.

2. Materials and Methods

2.1. Test organisms and maintenance

We selected candidate algal species by considering not only the above ease inassay handling but also the following ecological relevance: (1) widely distributedand frequently observed species in river ecosystems in Japan; (2) including a widerange of taxonomic groups (green algae, diatoms, cyanobacteria), and reflecting theactual species composition in Japanese rivers (diatoms are dominant followed bygreen algal and cyanobacteria); and (3) selection from several environmentalconditions, including saproxenous species (adapted to clean water), saprophilicspecies (adapted to organically polluted water), and eurysaprobic species(Watanabe et al., 1986). From the results of previous investigations on riverineperiphytons in Japanese rivers (Watanabe, 2005; ESCO, 2006), we selected a greenalga (Desmodesmus sp.), three diatoms (Achnanthidium sp., Nitzschia sp., andNavicula sp.), and a cyanobacteria (Pseudanabaena sp.) as representatives of riverineperiphytic algal genera. Moreover, other additional species were used for toxicitytests to verify the selection.

The algal strains used for the toxicity assay (eight species with eleven strains)are listed in Table 1. NIES strains were obtained from the National Institute forEnvironmental Studies, Japan, Microbial Culture Collection (Kasai et al., 2004).UTEX strain was obtained from UTEX The Culture Collection of Algae at TheUniversity of Texas at Austin. Although Navicula pelliculosa was renamed asFistulifera pelliculosa (Lange-Bertalot, 1997), original registration name by UTEXwas used in this paper.Mayamaea atomus strain NIAES K11-11, N. palea strain NIAESPD3, and N. palea strain NIAES U3-3 were collected and isolated from the SakuraRiver, paddy fields, and Saka River, Tsukuba city, Japan, respectively.

Stock cultures were maintained in the conditions listed in Table 1. Culturemediums, C, CT, CB, and CSi (Kasai et al., 2004), were prepared using Milli-Q water(resistance 18.3 MΩ; Millipore, Billerica, MA, USA). Light irradiation was continuousby using a daylight fluorescent lamp (color temperature 6500 K). Routine culturemaintenance was by using both aqueous and solid agar medium, but using solid agarmedium was impossible with two of the cyanobacterial strains and the use of anaqueous medium resulted in unstable growth for the two Nitzschia strains (Table 1).

2.2. Algal toxicity assay using microplate

Fluorometric algal toxicity assay were conducted using 96-well microplates,according to the standardized algal growth inhibition test (OECD, 2006). DCP,which was purchased from Wako Pure Chemical Inc. Ltd. (Osaka, Japan), was usedfor the toxicity assay as a reference substance for investigating the validity of thetoxicity test and sensitivity of the tested species (OECD, 2006). Stock solutions ofDCP were prepared in dimethyl sulfoxide (DMSO; Wako, Osaka, Japan). The finalconcentration of DMSO was less than 0.1 percent (v/v;≈1 g l−1), a concentration atwhich no adverse effects have been observed (Jay, 1996). In addition, the effect of

ty of solid and aqueous medium (○ indicates suitable and � indicates unsuitable),

Solidmedium

Aqueousmedium

Light intensity(maintenance),(lux)

Light intensity(toxicity assay),(lux)

Temp.(1C)

○ ○ 1000 5000 23○ ○ 1000 2000 23○ ○ 1000 2000 20○ � 1000 2000 20○ � 1000 2000 20○ ○ 1000 2000 20○ ○ 1000 2000 20○ ○ 1000 2000 20○ ○ 1000 2000 20� ○ 500 2000 23� ○ 1000 2000 23

T. Nagai et al. / Ecotoxicology and Environmental Safety 94 (2013) 37–44 39

DMSO on growth of candidate species was preliminarily assayed using 0.1, 0.5, and1.0 percentof DMSO in the same method as that described below.

Preculturing was performed in 15 ml medium taken in 50 ml borosilicate glasstubes for five days under the same conditions as those for the assay. However, thegrowth of N. palea strains NIAES PD3 and NIAES U3-3 were unstable in the aqueousculture medium, and therefore CSi agar medium was used for the preculture.Precultures were inoculated into the medium to give an initial fluorescence intensityof five, which corresponds to an approximate cell density of 0.6–2.9�104 cells ml−1

(details in Section 3.1). Algae were grown for 96 h in 200 ml culture medium per wellin 96-well polystyrene transparent microplates (Falcon 35–1172). A black microplatewith clear bottom, which is suitable for fluorescence measurement, was not used,because algal growth was inhibited probably due to light masking. Cross-wellcontamination of the fluorescence signal was not observed in our experimentalcondition. Cells were exposed to a geometric sequence of nine concentrations of DCP(0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 mg l−1). Each culture experiment,including a control test, was conducted with six replicates. The medium andtemperature used in the test were the same as that for culture maintenance, exceptfor the light intensity, which was higher than that for culture maintenance (Table 1).Although optimum light intensity for the standard test species (P. subcapitata) was4400–8900 lx, other species were more sensitive to strong light intensity (OECD,2006). Therefore, low light intensity was used for the test.

Algal growth was measured by in-vivo fluorescence every 24 h using amicroplate reader (Gemini EM, Molecular Devices, Sunnyvale, CA, USA) withSoftMax Pro Software for analysis. Bottom reading mode with well scanning (ninemeasurements of each well) was used for the measurement. The horizontalvariation in algal attachment to the well bottomwas corrected using well scanning.The detection of in-vivo fluorescence of chlorophyll a for green algae and diatomswas optimized at excitation and emission wavelengths of 435 and 685 nm,respectively. Similarly, the detection of in-vivo fluorescence of phycocyanin forcyanobacteria was optimized according to their pigment composition at excitationand emission wavelengths of 600 and 650 nm, respectively (Watras and Baker,1988). The quantification limits of fluorescence intensity were 0.65 for green algaeand diatom, and 1.7 for cyanobacteria. To verify the measurement, the relationshipbetween fluorescent intensities and the cell densities was preliminarily obtainedusing a flow cytometer (PAS, Partec GmbH, Münster, Germany) equipped with anair-cooled argon laser (excitation 488 nm, 15 mW) and red fluorescent (filterRG630) and side scatter detectors. FloMax Software ver. 2.3 (Partec) was used toanalyze the flow cytometry results. Attached and aggregated diatom cells werepreviously dispersed by vortex mixing and sonication, and filamentous cyanobac-teria P. galeata were previously dispersed by sonication (OECD, 2006).

2.3. Algal toxicity assay using an Erlenmeyer flask

To verify the accuracy of the microplate toxicity assay, an algal toxicity assay ofDCP using conventional Erlenmeyer flasks was conducted according to thestandardized algal growth inhibition test (OECD, 2006). Strongly attached algalspecies are difficult to test using Erlenmeyer flasks. Therefore, only three algalstrains, Desmodesmus subspicatus NIES-797, A. minutissimum NIES-414, andP. galeata NIES-512, were used for the test because of their weak attachment.Cultures were inoculated into the medium to obtain initial densities of 1.0�104

cells ml−1. During exposure periods, cultures were then mixed at 100 rpm using areciprocal shaker (TB-25RLS, Takasaki Kagaku Kikai, Kawaguchi, Japan) inD. subspicatus, and mixed by hand twice per day in A. minutissimum and P. galeata.Continuous mixing by 100 rpm was not suitable for growth of A. minutissimum andP. galeata. The culture conditions (medium, light, duration, endpoint, DCP concen-tration) used in the tests were the same as those for the microplate toxicity assaydescribed above. Each culture experiment was conducted with three samplereplicates and six replicates of the control test. Cell counting was conducted usinga particle counter (Colter counter Z1, Beckman Colter) for D. subspicatus andA. minutissimum, and using hemacytometer (Thoma) for P. galeata.

At the start (0 h) and end (96 h) of the assay, the concentrations of DCP in culturemedium were analyzed using solid-phase extraction and high-performance liquidchromatography (HPLC, LC-20AT, Shimadzu Co.) equipped with an L-Column (ChemicalsEvaluation and Research Institute, Japan) and an ultraviolet–visible spectrophotometricdetector (SPD-20 A, Shimadzu Co.). Samples (5–25ml) were passed through a solid-phase extraction column (InertSep PLS-2, GL Science, Tokyo, Japan), and then theretained DCP was eluted using 5 ml of methanol. Eluates (20 ml) were injected into anHPLC (mobile phase, methanol:0.1 percent H3PO4¼75:25; flow rate, 1 ml min−l;detection wavelength, 280 nm), and the concentration of DCP was determined usingthe calibration curve method. Average recovery of added 0.1 mg l−l of DCP was 90.3percent (CV 2.2 percent), and the limit of quantification was 0.02 mg l−l.

2.4. Concentration-response analysis

During the growth experiments, growth rate (day) from day t′ to day t werecalculated as follows:

Growth rate¼ lnðxt Þ−lnðxt′Þt−t′

ð1Þ

where xt is the fluorescence intensity or the cell density at time t. Then, relativegrowth rates in each test concentration were calculated by dividing the averagegrowth rate of the control tests (without test substance). Concentration-responsefunctions were determined using statistical regression analysis, that is the relativegrowth rate and DCP concentrations were fitted to logit model using nonlinearleast squares regression. Logit model can simply be expressed as follows:

Relative growth rate¼ 11þ exp ðf a þ f blnðCDCPÞÞ

ð2Þ

where CDCP is the DCP concentration (mg l−1), and fa and fb are coefficient values.EC50 and EC10 are CDCP values when relative growth rates are equivalent to 0.5 and0.9, respectively. Therefore, EC50 and EC10 are expressed as follows:

EC50 ¼ expð−f a=f bÞ ð3Þ

EC10 ¼ expððlnð0:1=0:9Þ−f aÞ=f bÞ ¼ expðð−2:197−f aÞ=f bÞ ð4ÞCoefficient values (fa and fb) in Eq. (2) can be substituted using Eqs. (3) and (4)

as follows:

Relative growth rate¼ 11þ expð−2:197ðlnðCDCP−lnðEC50ÞÞ=ðlnðEC10Þ−lnðEC50ÞÞÞ

ð5Þ

The EC50 and EC10 values with the 95 percent confidence intervals (CI) based onthe relative growth rates were calculated from the regressed function of Eq. (5).Statistical analyses were conducted using software R ver. 2.14.0.

2.5. Optimization of a simultaneous assay for five species

The culture conditions were optimized so that five candidate algal speciescould be tested simultaneously under the same conditions, such as light, tempera-ture, and medium. Control growth experiments were conducted under a lightintensity of 1000, 2000, 3000, and 5000 lx, temperature of 2271 1C, and using C,CT, and CSi medium. Diatoms need Si for growth, and therefore only CSi mediumwas tested. The validity criteria for the OECD test guideline 201 (OECD, 2006),which are growth magnification, replicate variability in control experiments, andlinearity of growth, were tested under each condition.

2.6. Species sensitivity distribution analysis

The results of the five species toxicity assay were applied for SSD analysis.An example of SSD analysis was conducted using the obtained EC50s for DCP.Multiple data for the same species were reduced by using the geometric mean, andthen, the species geometric means of the EC50 values were fitted to a log-normaldistribution. The fifth percentile of SSD (five percent hazard concentration, HC5)and the 50th percentile of SSD (HC50) at confidence levels of 50 percent and 5–95percent were calculated according to Aldenberg and Jaworska (2000). In addition,the SSD using our additional full test data (eight species) was also analyzed for thepurpose of comparison. The two-sample Kolmogorov–Smirnov test was conductedusing software R ver. 2.14.0 to test whether the data of two SSDs were sampledfrom the same distribution.

Acute toxicity data (EC50) on herbicide atrazine for five genera selected in ourstudy (Desmodesmus, Achnanthidium, Navicula, Nitzschia, and Pseudanabaena) werecollected from the ECOTOX database (US Environmental Protection Agency).Similarly, acute toxicity data (EC50) on herbicide simetryn for the five genera werecollected from Nagai et al. (2008) and Kasai and Hanazato (1995). SSDs of the twoherbicides were also analyzed similar to DCP. Furthermore, the reported SSD ofatrazine using acute toxicity data on freshwater primary producers (n¼29) (vanden Brink et al., 2006) and the reported SSD of simetryn using acute toxicity dataon freshwater algae (n¼31) (Nagai et al., 2008) were compared to the SSDs usingfive candidate species. The difference of SSDs was also tested using the two-sampleKolmogorov–Smirnov test.

3. Results

3.1. Microplate toxicity assay

Fluorescent intensity and cell density counts were well corre-lated for each species (Fig. 1). Algal growth in the microplate couldbe monitored successfully by in-vivo fluorescence using ourmeasurement conditions.

The toxicity of the organic solvent DMSO is presented in Fig. 2.The 0.1 percent DMSO concentration did not affect the growth ofall candidate species, but 0.5 and 1.0 percent DMSO concentrationshad statistically significant effects on the growth of A. minutissimumand N. pelliculosa (Fig. 2). Although DMSO can be used as an organicsolvent for the preparation of stock solutions, final experimental

1 10 100 1000103

104

105

106

107

Fluorescence intensityC

ell d

ensi

ty (c

ells

ml-1

)

Cel

l den

sity

(cel

ls m

l-1)

D. subpicutus

lnY = 8.61 + 1.03lnXr = 0.99

1 10 100 1000103

104

105

106

107

Fluorescence intensity

P. galeata

lnY = 7.90 + 0.95lnXr = 0.99

1 10 100 1000103

104

105

106

107

Fluorescence intensity

N. palea

lnY = 7.09 + 1.02lnXr = 0.99

1 10 100 1000103

104

105

106

107

Fluorescence intensity

A. minutissimum

lnY = 7.82 + 1.02lnXr = 0.99

1 10 100 1000103

104

105

106

107

Fluorescence intensity

N. pelliculosa

lnY = 8.37 + 0.93lnXr = 0.99

Fig. 1. The relationship between fluorescence intensity and cell density counted by flow cytometry for each candidate species.

0

0.4

0.8

1.2

1.6

0 0.1 0.5 1.0

D. subspicatus P. galeata N. paleaA. minutissimum N. pelliculosa

DMSO concentration(%)

Gro

wth

rate

(day

−1)

* * * *

Fig. 2. The effect of DMSO on the growth of each algal species. The mean andstandard deviation (error bars) of six replicate experiments are shown. Asterisksindicate a statistically significant effect compared with the control (zero concen-tration) growth rate (Po0.05, Dunnett's multiple comparisons test).

T. Nagai et al. / Ecotoxicology and Environmental Safety 94 (2013) 37–4440

solutions should be prepared using less than 0.1 percent of DMSO.Therefore, DMSO concentration was limited to 0.1 percent in ourDCP toxicity assay.

The results of the toxicity assay of DCP for eight species ofeleven strains are summarized in Table 2. In toxicity assays ofP. subcapitata, D. subspicatus, and N. palea strain NIAES PD3,growth rates were calculated during 0–72 h, because more thansixteen-fold growth in the control experiment was observedwithin 72 h. In toxicity assays of the other species, growth rateswere calculated for the 24–96 h period, because a lag time for algalgrowth and delayed toxic effect of DCP were observed during thefirst 24 h. All tested species, except Anabaena flos-aquae, grewmore than sixteen-fold within the 96 h test duration in the controlexperiment. This indicates that the test duration of 96 h wasrequired to fulfill the validity criteria of growth (more thansixteen-fold growth in control) in the standard test guideline(OECD, 2006). The other validity criteria, namely the coefficientof variation of growth rates in replicates of the control experi-ments and mean coefficient of variation of each one day growthrates during the period of growth rate calculation are less thanseven percent and 35 percent, respectively, were found to fulfillour test conditions, except for those of A. flos-aquae and M. atomus(Table 2).

The values of EC50 ranged from 0.38 mg l−1 to 3.30 mg l−1, andsensitivity differences between species were less than ten fold.The values of EC10 ranged from 0.24 mg l−1 to 2.30 mg l−1 (Table 2).

The most sensitive species was A. minutissimum strain NIES-71,and the least sensitive species was A. flos-aquae strain NIES-73.

3.2. Erlenmeyer flask toxicity assay

Three species including green algae, diatoms, and cyanobac-teria were also tested using Erlenmeyer flasks as well as micro-plates. The durations of growth rate calculation were the same asthe microplate assay (Table 2). The measured DCP concentrationswere within 80–120 percent of nominal concentrations, andtherefore all concentration-response analysis was conducted usingnominal concentrations (OECD, 2006). The EC50 and EC10 forD. subspicatus were 2.10 (95 percent confidence intervals of1.98–2.22) and 1.15 (95 percent confidence intervals of 1.04–1.28),respectively. The EC50 and EC10 for A. minutissimum were 0.90 (95percent confidence intervals of 0.73–0.99) and 0.63 (95 percentconfidence intervals of 0.39–0.79), respectively. The EC50 and EC10for P. galeata were 1.00 (95 percent confidence intervals of0.76–1.12) and 0.58 (95 percent confidence intervals of 0.28–0.86),respectively. Thus, the EC50 and EC10 values obtained from themicroplate assay and those obtained from conventional Erlenmeyerflask assay were consistent (Fig. 3).

3.3. Optimization of simultaneous assay for five species

The growth magnification of five candidate species undervarious culture conditions are shown in Fig. 4. Growth magnifica-tion was a sensitive parameter to the medium and lightingcondition. The use of CT and CSi media enhanced growth ofD. subspicatus compared to C medium (Fig. 4). The validity criteriaof growth (more than sixteen-fold) could be fulfilled within 72 hunder 2000–5000 lx using CT and CSi media and 3000–5000 lxusing C medium. The use of CT medium was the most appropriatefor the growth of P. galeata among the three selected media(Fig. 4). The validity criteria of growth during 96 h was fulfilledunder 1000–5000 lx using CT and CSi media and 1000–3000 lxusing C medium. Three diatoms were tested using only CSimedium. The validity criteria of growth during 96 h were fulfilledunder 1000–5000 lx for N. palea and A. minutissimum, and fulfilledunder 1000–3000 lx for N. pelliculosa (Fig. 4). The other validitycriteria, replicate variability in control experiments and linearity ofgrowth, were found to be fulfilled when the validity criteria ofgrowth was fulfilled.

Table 2Periods for concentration-response analysis, growth rates in control experiment, coefficient of variations of growth rates in replicates of the control experiment (CV control),mean coefficient of variations of each one day growth rates during the period of growth rate calculation (CV slope), EC50s, and EC10s with 95% confidence intervals of eachtested species.

Species Strain Period for analysis (h) Growth rate (day−1) CV control (%) CV slope (%) EC50 (mg l−1) 95% CI EC10 (mg l−1) 95% CI

P. subcapitata NIES-35 0–72 0.99 3.5 23.9 2.36 2.19–2.57 1.67 1.47–2.00nD. subspicatus NIES-797 0–72 1.10 4.6 13.4 2.61 2.37–2.87 1.26 1.00–1.56M. atomus NIAES K11-11 24–96 0.77 4.2 38.4 1.58 1.56–1.60 1.46 1.14–1.52N. palea NIAES PD3 0–72 1.21 1.0 22.3 0.90 0.78–1.03 0.33 0.20–0.52N. palea NIAES U3-3 24–96 0.71 1.4 32.6 1.11 0.94–1.33 0.69 0.50–1.06nN. palea NIES-487 24–96 0.95 1.7 34.8 1.19 1.11–1.28 0.80 0.71–0.96nA. minutissimum NIES-71 24–96 0.74 2.2 11.6 0.38 0.32–0.44 0.24 0.16–0.32A. minutissimum NIES-414 24–96 0.83 3.6 19.5 0.76 0.71–0.83 0.57 0.45–0.63nN. pelliculosa UTEX-B673 24–96 0.80 0.9 8.3 1.04 1.01–1.06 0.80 0.79–0.81nP. galeata NIES-512 24–96 0.95 4.4 34.9 0.60 0.52–0.69 0.39 0.28–0.55A. flos-aquae NIES-73 24–96 0.42 24.4 37.2 3.30 3.03–3.89 2.30 1.58–2.56

n Candidate species.

0

1

2

3

0 1 2 3EC50 and EC10 in microplate assay (mg l−1)

EC

50 a

nd E

C10

in fl

ask

assa

y (m

g l−1

)

D. subspicatusA. minutissimumP. galeata

Fig. 3. Comparison of EC50s and EC10s between the microplate assay andErlenmeyer flask assay. Filled symbols indicate EC50s and open symbols indicateEC10s. The solid line shows the 1:1 line and the dashed lines show the 1:2 and 2:1lines. Error bars indicate 95 percent confidence intervals.

T. Nagai et al. / Ecotoxicology and Environmental Safety 94 (2013) 37–44 41

3.4. Species sensitivity distribution analysis

The SSDs for DCP based on EC50 using data from the fivecandidate species and using additional full test data (n¼8, Table 2)were analyzed. The logarithmic mean and standard deviation (lnMean and ln SD) of the five candidate species and full data areshown in Table 3. The median estimates of HC5 and HC50 of DCPusing five candidate species data were 0.26 and 0.94 mg l−1,respectively (Table 3). On the other hand, the median estimatesof HC5 and HC50 of SSD using full data of all tested species were0.42 and 1.4 mg l−1, respectively (Table 3). The SSD using fivespecies data was found to be similar to the SSD using additionalfull data (Fig. 5A) and the difference was not significant (D¼0.30,p¼0.94).

Acute toxicity data of atrazine for the five genera selected inour study were obtained from the ECOTOX database (US EPA).The genus geometric mean of EC50 values ranged from 13.0 mg l−1

to 260 mg l−1. The ln Mean and ln SD of these values are shown inTable 3. The median estimates of the HC5 and HC50 were 11 and86 mg l−1, respectively (Table 3). On the other hand, the SSD ofatrazine using full data analyzed by van den Brink et al. (2006)resulted in the median estimates of the HC5 and HC50 of 13 and137 mg l−1, respectively (Table 3). The SSD using five species datawas found to be similar to the SSD using full data (Fig. 5B) and thedifference was not significant (D¼0.41, p¼0.37).

Acute toxicity data of simetryn for the five genera selected inour study were obtained from Nagai et al. (2008) and Kasai andHanazato (1995). The genus geometric mean of EC50 values rangedfrom 16.9 mg l−1 to 136 mg l−1. The ln Mean and ln SD of thesevalues are shown in Table 3. The median estimates of the HC5 andHC50 were 14 and 52 mg l−1, respectively (Table 3). On the otherhand, the SSD of simetryn using full data analyzed by Nagai et al.(2008) resulted in the median estimates of the HC5 and HC50 of8.2 and 37 mg l−1, respectively (Table 3). The SSD using five speciesdata was found to be similar to the SSD using full data (Fig. 5C) andthe difference was not significant (D¼0.44, p¼0.36).

4. Discussion

4.1. Microplate toxicity assay

Our results of EC50 obtained from the microplate assay wereconsistent with those obtained from conventional Erlenmeyerflask assay (Fig. 3). These results suggest the validity of ourmicroplate assay using not only green algae but also diatomsand cyanobacteria. Moreover, DCP has been used as referencesubstance for interlaboratory toxicity tests (ISO, 2004). The meanEC50 values for P. subcapitata and D. subspicatus were reported tobe 3.38 and 6.42 mg l−1 with SDs of 1.30 and 2.38 mg l−1 (n¼9 and18), respectively (ISO, 2004). Therefore, the 95 percent confidenceintervals of EC50 of DCP for P. subcapitata and D. subspicatuswere calculated by mean7t-value� SD as 0.38–6.38 and 1.40–11.44 mg l−1, respectively. Our results remained within theconfidence intervals (Table 2), demonstrating the validity of ourmicroplate assay.

In the results of the growth experiment using several lightintensities and growth mediums, the unified test conditions of2000–3000 lx using CSi medium is appropriate to fulfill thevalidity criteria of all five species of algal growth. The growth ofN. pelliculosawas the weakest during the test period, and optimumgrowth rate was achieved under a light intensity of 3000 lx(Fig. 4). From the results, culture conditions were optimized sothat the five algal species could be tested simultaneously underthe same conditions: light intensity of 3000 lx, temperature of22 1C, and use of CSi medium. These culture conditions are notnecessarily optimum conditions for each single species. However,using the same test medium and culture condition is easier interms of manipulation and better for comparison of toxicity dataamong test species. Consequently, a high-throughput and multi-species algal toxicity assay would enable the use of common assayconditions.

Table 3Comparison of HC5 and HC50 (confidence levels of 50% and 5−95%) values for SSDusing 5 candidate species and full toxicity data for DCP, atrazine, and simetryn.

Chemicals Used data ln Mean ln SD n HC5 HC50

50% 5–95% 50% 5–95%

DCP 5 Species −0.06 0.73 5 0.26 0.043–0.52 0.94 0.47–1.9Full data 0.30 0.68 8 0.42 0.16–0.71 1.4 0.86–2.1

Atrazine 5 Species 4.45 1.14 5 11 0.70–34 86 29–256Full data 4.92 1.43 29 13 5.7–23 137 88–214

Simetryn 5 Species 3.96 0.75 5 14 2.3–28 52 26–107Full data 3.61 0.90 31 8.2 4.9–12 37 28–49

0 1000 2000 3000 4000 50000

10

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40

50

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row

th

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P.galeata

0 1000 2000 3000 4000 50000

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Light intensity (lux) Light intensity (lux) Light intensity (lux)

Gro

wth

N.palea

0 1000 2000 3000 4000 50000

10

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50A.minutissimum

0 1000 2000 3000 4000 50000

10

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50N.pelliculosa

Fig. 4. The effects of light intensity and medium on the growth magnification during 72 h for D. subspicatus and 96 h for other species. Open square, filled triangle, and opencircle represent C, CT, and CSi media, respectively. The mean and standard deviation (error bars) of six replicate experiments are shown.

T. Nagai et al. / Ecotoxicology and Environmental Safety 94 (2013) 37–4442

4.2. Species selection

The selection of algal species to be tested should be based ontaxonomic diversity, availability of cultures, good taxonomic char-acterization of strains, ease of culturing in artificial media suitablefor toxicity testing, sufficiently rapid growth to allow reliableestimation of growth rate, suitable morphology for counting, andsusceptibility to phytotoxicants (Freemark et al., 1990). Desmodesmusis a green algae observed as riverine periphyton as well asphytoplankton in lakes and ponds (Biggs and Kilroy, 2000; ESCO,2006). There are plenty of toxicity data for D. subspicatus, becauseD. subspicatus is a recommended species in OECD test guideline(OECD, 2006). Moreover, it was found to be suitable for fluorometricmicroplate assay. Consequently, the use of D. subspicatus is adequateas a test species of green algae.

Achnanthidium, Navicula, and Nitzschia are very common gen-era of riverine periphytic diatoms (Biggs and Kilroy, 2000; Leiraand Sabater, 2005; ESCO, 2006; Ricart et al., 2010). Among them,A. minutissimum belongs to a saproxenous species (Watanabe,2005; Biggs and Kilroy, 2000). A. minutissimum strain NIES-71 wasisolated from a metal contaminated river and A. minutissimumstrain NIES-414 was isolated from an unpolluted river (Kasai et al.,2004). The EC50 of Cu for A. minutissimum strain NIES-71 is morethan ten times higher than that for A. minutissimum strain NIES-414 (Takamura et al., 1989). However, EC50 of DCP for A. minutissimum

strain NIES-71 is lower than that for A. minutissimum strain NIES-414(Table 2). Strains NIES-71 and NIES-414 were both suitable forfluorometric microplate assay, and strain NIES-71 was consideredbetter for assaying of organic pollutants, such as pesticides, because ofits high sensitivity.

Navicula and Mayamaea are taxonomically close genera andpreviously regarded as the same genus, Navicula (Lange-Bertalot,1997). N. pelliculosa and M. atomus are both widely distributeddiatoms in flowing freshwater environments (Watanabe, 2005;Ishiahara et al., 2006), and are both suitable for fluorometricmicroplate assay. There are abundant toxicity data for N. pelliculosa,because this is a recommended species in the OECD test guideline(OECD, 2006). Therefore, N. pelliculosa is thought to be a better testorganism considering the comparison purpose.

N. palea is a representative saprophilic species (Watanabe,2005; Biggs and Kilroy, 2000). Three strains of N. palea weretested in our study. Strain NIES-487 was isolated from a metalcontaminated river (Kasai et al., 2004), strain NIAES PD3 wasisolated from a paddy field, and NIAES U3-3 was isolated from anunpolluted river water (Ishiahara et al., 2006). The EC50 of DCP didnot differ markedly among the three strains (Table 2), regardless ofthe environment from which they were isolated. The growth ofstrains NIAES PD3 and NIAES U3-3 were unstable in aqueousmedium (Table 1), and preculturing had to be conducted usingsolid agar medium. Therefore, the assay procedure using the twostrains was cumbersome and complicated. In contrast, NIES-487 iseasy to handle using aqueous medium and is suitable for fluoro-metric microplate assay.

Pseudanabaena and Anabaena are frequently observed cyano-bacteria in riverine periphyton as well as in lakes and ponds (Biggsand Kilroy, 2000; ESCO, 2006). P. galeata was suitable for fluoro-metric microplate assay, but A. flos-aquae did not grow well in themicroplate (four−five-fold for 96 h). Moreover, sensitivity ofP. galeata for DCP was higher than that of A. flos-aquae (Table 2).Therefore, the use of P. galeata is better for use as a test speciesamong cyanobacteria than A. flos-aquae. Consequently, our fivecandidate species, D. subspicatus, A. minutissimum, N. pelliculosa,N. palea, and P. galeata, are reasonable for use as test species fromthe point of view of not only ease in assay but also ecologicalrelevance.

0

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Desmodesmus

Nitzschia

Pseudanabaena

Navicula

Achnanthidium

A

B

C

Fig. 5. Species sensitivity distribution of DCP (A), atrazine (B), and simetryn(C) using the EC50s of five candidate species (filled square with solid line) andusing the full data (dashed line). Geometric mean of EC50s within the same genuswas used for the SSD analyses of atrazine and simetryn.

T. Nagai et al. / Ecotoxicology and Environmental Safety 94 (2013) 37–44 43

4.3. Application to species sensitivity distribution

Our study selected five candidate algal species for SSD analysisof herbicides. TenBrook et al. (2008) reviewed the requirednumber of samples for SSD analysis and concluded that a samplesize of five is the minimum needed for applying parametric SSDanalysis. Data requirements for SSD analysis range from n¼4 to 10depending on organizations. Among them, OECD (1995) suggestedminimum dataset of five different species. Okkerman et al. (1991)concluded that data from five species is adequate for the SSDanalysis, although data from seven species would be ideal.Aldenberg and Slob (1993) showed that underprotection risk ofthe median estimates of the HC5 decrease considerably when thesample size is increased from two to five, but not as much whenthe sample size is increased from five to ten and from ten totwenty. Moreover, five standard aquatic plant species (four micro-algae and a duckweed) used for pesticide registration in the UnitesStates were reasonably representative dataset for estimating HC5

of an aquatic plant community (Thursby et al., 2011). These

discussions support our approach of SSD analysis using data fromfive algal species. Moreover, our results indicate consistencybetween SSDs using five data and full data (Table 3, Fig. 5),suggesting that the SSD using the five species largely representedalgal sensitivity.

For further validation of our approach, HC5s for atrazine andsimetryn by five species SSD (Table 3) were compared with theeffect level on a community in a freshwater ecosystem obtainedfrom mesocosm studies. The median estimate of HC5 from an SSDbased on acute toxicity have been reported to be protective againstadverse ecological effects from single short-term exposure infreshwater semi-field (microcosm/mesocosm) experiments(Maltby et al., 2005, van den Brink et al., 2006). Van den Brinket al. (2006) reviewed the mesocosm studies of herbicides andreported that the no observed effect concentrations of singleatrazine application were between 5 mg l−1 and 20 mg l−1.The median estimate of atrazine HC5 using five species data(11 mg l−1, Table 3) was consistent with 5–20 mg l−1. Chang et al.(2011) conducted outdoor mesocosm study for twelve days using20 and 100 mg l−1 of simetryn. The phytoplankton community wasslightly affected after application of 20 mg l−1 of simetryn, but arecovery was found in a few days. This result was evaluated aslowest observed effect concentration in the ecosystem of 20 mg l−1.The median estimate of simetryn HC5 using five species data(14 mg l−1, Table 3) was lower than 20 mg l−1. The comparisonsbetween HC5 and the mesocosm study suggested that SSD analysisusing the five species data selected in our study would have theappropriate protective ability. Moreover, the most sensitive speciesamong the five species differed between DCP, atrazine, andsimetryn (Fig. 5), indicating the importance of multispeciestoxicity testing. Therefore, our multispecies fluorometric micro-plate algal toxicity assay provides a useful and efficient method forSSD analysis.

5. Conclusion

Previously, toxicity assay using riverine periphytic algal specieswas technically difficult to conduct by standardized protocol. Wefound that fluorometric microplate toxicity assay could be appliedto test such species. Five candidate riverine periphytic algal specieswere selected for SSD analysis. The selection of test species wasreasonable from the point of view of dominance in the river,applicability for fluorometric microplate assay, selection from ataxonomically wide range of species, and fraction of actual speciescomposition. Our assay procedure does not require considerablelabor, and a number of assays can be performed simultaneously.Culture conditions were optimized so that the five algal speciescould be tested simultaneously under the same conditions.Consequently, a high-throughput and multispecies algal toxicityassay was developed. Our approach is useful for high-tier prob-abilistic ecological risk assessment of pesticides using SSD.

Acknowledgment

This research was supported by the Environment Research andTechnology Development Fund (C-1102) of the Ministry of theEnvironment, Japan.

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