Chemosphere 72 (2008) 1366–1372

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Chemosphere

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Effects of cold-dark storage on growth of Cylindrotheca closteriumand its sensitivity to copper

Cristiano V.M. Araújo *, Fernando R. Diz, Ignacio Moreno-Garrido, Luis M. Lubián, Julián BlascoInstituto de Ciencias Marinas de Andalucía (ICMAN), Campus Universitario Río San Pedro s/n, 11510, Puerto Real, Cádiz, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 January 2008Received in revised form 26 March 2008Accepted 10 April 2008Available online 3 June 2008

Keywords:DiatomsLong-term storageMarine ecotoxicologyMicrophytobenthos

0045-6535/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2008.04.022

* Corresponding author. Tel.: +34 956 832 612; faxE-mail address: cristiano.araujo@icman.csic.es (C.V

Cylindrotheca closterium cells were maintained at low temperature (4 ± 1 �C) and dark conditions up to 21weeks to assess the effect on survival and physiological status. From a control culture under standardconditions, three densities were prepared: (A) 2 � 104, (B) 10 � 104, and (C) 25 � 104 cells ml�1. Weekly,inoculums of each stored density were exposed to continuous light and at 20 ± 1 �C. Sensitivity to copperfor microalgal cultures was evaluated in order to assess possible changes in cells sensitivity due to stor-age. Concurrently, assays with a control culture were carried out in order to assess the sensitivity ofC. closterium to copper and to be able to generate a standard sensitivity control chart with a mean valueof EC50-72 h ± 2SD (standard deviation). Density-C presented higher cell yield values, between 40% and80% relative to control culture. Cell density showed to be important feature that may be taken intoaccount in cell storage experiments. There was an increase in sensitivity of cells submitted to storage;however results always kept in the range established as standard sensitivity with no statistically signif-icant difference with regards to control culture. EC50-72 h mean value for the control culture was29 ± 10 lg Cu l�1, while for densities-A, B and C were 22 ± 7; 23 ± 9 and 23 ± 8 lg Cu l�1, respectively.In spite of drastic changes in the environmental conditions due to storage, it is concluded that C. closte-rium cells stored during 5 months remained metabolically active and with no significant change in itssensitivity.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Search for ecotoxicological assays with ecologically relevantspecies is an important task for environmental researchers. Ecolog-ical relevance of a single species is related to the key role in theecosystem, as well as representativeness of the species in the tro-phic level (Calow, 1989). Test organisms should be chosen accord-ing to the information intended to be obtained (Rojicková et al.,1998). Thus, environmental risk analysis of sediments should takeinto account the benthic community (Moreno-Garrido et al.,2003a), while terrestrial impacts should be assessed through soilorganisms (DelValls et al., 2004). On the other hand, when wateris the main route of uptake for chemical compounds, then toxicitytest with planktonic organisms should be employed (Walker et al.,2001). In spite of the efforts employed to develop algal assay pro-cedures, the majority of the internationally standardized assays arefocused on planktonic freshwater algae (ISO, 1989; EnvironmentalCanada, 1992; OECD, 1998; USEPA, 2002; ABNT, 2005), with quiteless attention to species of the microphytobenthos (Moreno-Garr-ido et al., 2003a). Thus, tests should be developed following thespecific environmental conditions using representative species of

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the ecosystems. Benthic diatoms are a major component of themicrophytobenthos in aquatic systems (MacIntyre et al. 1996; deBower et al., 2003) and can therefore be used as a measure of waterquality (Kelly et al., 2001). Diatoms are the main source of food formeiofauna and they are very important in material and energytransfer through the benthic food webs (Miller et al., 1996; Blan-chard et al., 2000). Moreover, the excreted polysaccharides play arelevant role in the stability of the marine surface sediment layer(Miller et al., 1996), being key factor of the structure and functionof benthic intertidal zones (Scala and Bowler, 2001). Althoughthere is an unquestionable ecological importance of benthic dia-toms there are very few studies that have adopted these organismsin toxicity assays. In general, among microphytobenthos, Cylindrot-heca closterium is the main species used in ecotoxicology (Stauberand Florence, 1985, 1989; Moreno-Garrido et al., 2003a,b, 2007;Hogan et al., 2005).

Microalgae cultures, such as diatoms, are widely used to feedorganisms in aquaculture (Cañavate and Lubián, 1995) especiallyin shellfish hatcheries (Redekar and Wagh, 2000), and they havealso been used in ecotoxicological assays (Nörnström, 1990). How-ever, sometimes ecotoxicology laboratories need to maintain amicroalgae culture only exclusively to perform a determined assaywhose frequency may be spaced in time. The maintenance of livingmicroalgae cultures is an expensive task (Sánchez-Saavedra, 2006)

C.V.M. Araújo et al. / Chemosphere 72 (2008) 1366–1372 1367

mainly if toxicity assays are not routinely carried out. Mitbavkarand Anil (2006) suggested cryopreservation as a very useful tech-nique for the maintenance of phytoplankton culture collections.However, this technique results to be very aggressive at an ultra-structural level during freezing and thawing and once cells survivepost-thaw severe damages can occur (Tzovenis et al., 2004). Addi-tionally, the maintenance of cryopreserved algae is expensive andlaborious. Nevertheless, many factors may be taken into accountfor the success of cryopreservation such as state and density ofthe culture, the type of cryoprotectant and rate of cooling andthawing (Cañavate and Lubián, 1995; Poncet and Véron, 2003).According to Admiraal (1977) benthic diatoms cultures can bestored in dark, at 4 �C and subsequently inoculated in fresh med-ium in order to preserve cells for long time. Maintenance of cul-tures in cold and darkness is less expensive and more realisticand viable (Sánchez-Saavedra, 2006). Studies about physiologicaleffects due to cold and darkness exposition in diatoms are per-formed, in general, with polar species naturally adapted to changesin light and temperature cycle (Gleitz and Thomas, 1992). Manypolar diatom species can survive under total darkness for periodsup to 10 months (Peters and Thomas, 1996). Studies on cell viabil-ity (growth rate) of microalgae (not exclusively polar) exposed tolow temperature and darkness can be found in literature (Peters,1996; Murphy and Cowles, 1997; Chen, 2007). Not only algal sur-vival and growth need to be considered, but also changes in sensi-tivity to contaminants due to other stressors such as storageconditions, low temperature, darkness and nutrient depletion.

The aim of this paper was to assess if the storage of C. closteriumcells under low temperature (4 �C) and darkness could affect itsgrowth and verify if it caused variations in its sensitivity in toxicitytests. Information with regards to sensitivity changes after cold-dark storage for a further ecotoxicological application becomesvery useful for sediment toxicity tests including C. closterium cells.This study was undertaken for about 5 months because it was con-sidered that 5 months was enough time in view of a commercialapplication and to avoid significant decreases cell viability.

2. Materials and methods

2.1. Organism and culture medium

A strain of C. closterium (Ehrenberg) Lewin & Reimann (formerlyNitzschia closterium (Ehrenberg) W. Smith) was isolated in May2000 from a salt pond near the Marine Sciences Institute fromAndalusia (ICMAN) in Puerto Real (SW of Spain). The strain (C. clos-terium #010301) is currently included in the Culture Collection ofMarine Microalgae of the ICMAN (CCMM-ICMAN, BIOCISE). Forroutine cultures, seawater is filtered through a GF/C Whatman fil-ter, autoclaved, and later enriched with Guillard’s f/2 medium(Guillard and Ryther, 1962) plus 500 lg l�1 SiO2. Artificial substi-tute ocean water (ASTM, 1975) enriched with SiO2 (500 lg l�1),NO�3 (150 lg l�1), and PO3�

4 (10 lg l�1) was used in toxicity assayswith copper, in order to avoid the chelator effect of EDTA, includedin the f/2 medium. Routine culture and assays were performed at20 ± 1 �C under continuous white light (35.2 ± 1.1 lmol m�2 s�1)in a controlled culture chamber (Ibercex).

2.2. Preparation of cell densities and storage at low temperature anddarkness conditions

Three-day algal cultures (at exponential growth phase) wereused to prepare the three densities, named A, B, and C, which con-tained: 2 � 104, 10 � 104 and 25 � 104 cells ml�1, respectively. Alldensities were prepared with the same culture medium. Two mil-liliters of aliquots of densities-A, B and C were collected in sterile

plastic Pasteur pipettes, and then they were hermetically sealedby heating the pipettes’ tips. To avoid heating the algae the pip-ettes were placed upside-down and the cells were concentratedin the no heated part of the pipette during sealing. All procedureswere carried out in laminar flow chamber. Once the pipettes weresealed, they were stored at 4 ± 1 �C and darkness conditions up to21 weeks.

2.3. Sampling and growth assay (re-activation)

Stored pipettes submitted to low temperature and darknessconditions were sampled weekly for each density, in triplicate.The cells were re-activated using culture medium prepared as de-scribed in Section 2.1. Initially sampled volume was of 2 ml (re-ferred to one Pasteur pipette) introduced in 48 ml of medium.However, throughout the time assayed the growth velocity was de-creased (increase in lag-phase) in all densities studied, thus a big-ger volume of cells was needed to be inoculated. Stored inoculumvolume plus the culture medium volume resulted to be always of50 ml. When the inoculated volume was of 2 ml (one Pasteur pip-ette), cell densities in Erlenmeyer flask were 800, 4000 and10000 cells ml�1 for densities-A, B and C, respectively. After inocu-lation four growth periods for cell re-activation were established:4, 11, 18 and 25 days, this way when there was not enough numberof cells (around 1 � 105 cells ml�1) to carry out a toxicity assay in 4days, the culture was maintained for one more week and succes-sively. At the end of the re-activation assay cell yield (a) valueswere calculated using the following formula, for the three storeddensities studied:

a ¼ N � N0

N0ð1Þ

where N and N0 are the number of cells ml�1 at final time (t, in days)and initial time (t0, in days), respectively. Cell yield values werecompared with the control culture – kept under controlled condi-tions of temperature (20 ± 1 �C) and light (continuous white light35.2 ± 1.1 lmol m�2 s�1) – considered as 100% cell yield and wereexpressed as relative values (%). In order to compare cell yield frominoculated volume, initial cell density and growth days were takeninto account. Sensitivity of the re-activated culture was also evalu-ated with a toxicity assay using copper as reference compound.Samples from week 21 were re-inoculated for one more week andthen a toxicity assay was carried out. These samples, named re-inoculation, were only followed for densities-B and C because therewas no growth in density-A at 21st week.

2.4. Toxicity tests

Algal cultures of the three re-activated densities were centri-fuged (MES, Micro Centaur, SANYO) for 4 min at 4000 rpm andre-suspended in clean medium (artificial substitute ocean water –ASTM, 1975). This procedure was followed three times before usein the assay in order to guarantee the elimination of f/2 mediumand with the aim of avoiding the effect of the chelator EDTA in-cluded in the f/2 medium. Initial cellular concentration forgrowth-inhibition assay of 104 cells ml�1 (OECD, 1998) wasadopted. Bioassays were carried out in 96-well microplates (Grein-er 96-well flat bottom white, 12 � 8, 400 ll capacity) based onBlaise et al. (1998). Each well was filled with 300 ll of sample. Dur-ing incubation, microplates were covered with their transparentlids and maintained without agitation. Assays were carried out atfive replicates for each concentration, control (with no copper)and blank (only culture medium). To avoid possible edge effects,resulting from the circumferential wells, only centrally locatedwells were used. To reduce evaporation, wells along the microplatemargin were filled with Milli-Q water (Lukavsky, 1992). Samples

Table 1Data of density-A (2 � 104 cells ml�1) including sampling week (storage time),volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) ofgrowth until the carrying out of the assay and final cell density after growth

Storageweek

Volumeinoculated(ml)a

Cell density initial(104 cells ml�1)

Growthperiod(days)

Final cell density(104 cells ml�1)

1 2 0.08 11 39.16 (±3.40)2 2 0.08 11 38.55 (±8.29)3 2 0.08 18 126.66 (±9.86)4 2 0.08 18 174.00 (±31.57)5 4 0.16 18 150.75 (±37.12)7 4 0.16 18 120.75b

9 4 0.16 18 57.50b

11 4 0.16 18 90.58 (±29.95)13 4 0.16 18 46.50b

15 4 0.16 25 213.25b

17 8 0.32 – –c

19 8 0.32 25 161.75 (±16.97)21 8 0.32 – –c

Growth data result from the mean of three replicates. ±: standard deviation.a Each 2 ml are equivalent to 1 pipette (total volume of inoculum plus culture

medium was always of 50 ml).b Growth registered only in a replicate.c There was no growth.

Table 2Data of density-B (10 � 104 cells ml�1) including sampling week (storage time),volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) ofgrowth until the carrying out of the assay and final cell density after growth

Storageweek

Volumeinoculated(ml)a

Cell density initial(�104 cells ml�1)

Growthperiod(days)

Final cell density(�104 cells ml�1)

1 2 0.40 4 21.44 (±1.17)2 2 0.40 4 7.88 (±2.60)3 2 0.40 11 151.00 (±12.93)4 2 0.40 11 140.83 (±26.83)5 2 0.40 11 139.00 (±16.71)6 2 0.40 11 124.66 (±33.85)7 2 0.40 11 76.16 (±16.32)8 2 0.40 11 65.91 (±24.98)9 2 0.40 11 63.08 (±11.54)

10 2 0.40 11 50.41 (±3.81)11 2 0.40 11 42.41 (±30.88)12 2 0.40 11 38.16 (±6.45)13 4 0.80 11 31.41 (±2.32)14 4 0.80 11 30.33 (±11.00)15 4 0.80 11 30.25 (±20.07)17 4 0.80 11 33.91 (±10.27)19 8 1.60 18 127.00 (±18.77)21 8 1.60 18 203.50 (±10.40)

Growth data result from the mean of three replicates. ±: standard deviation.a Each 2 ml are equivalent to 1 pipette (total volume of inoculum plus culture

medium was always of 50 ml).

1368 C.V.M. Araújo et al. / Chemosphere 72 (2008) 1366–1372

were randomly distributed to avoid pseudoreplication effects dueto possible spatial differences in illumination and temperature.For all bioassays, the pH drift was less than 0.5 pH unit over 72 hin accordance with established by OECD (1998). Measurement ofthe initial and final pH values in all samples were registered (Cri-son, MicropH 2001, electrode Hamilton, Slimtrode). Fluorescencevalues were compared with the mean value of fluorescence activitybelonging to algae with no toxic (control), considered as 100%activity. Fluorometric data were obtained with a spectrophotome-ter (TECAN Multiplate Reader, GENios) at 0, 24, 48, and 72 h withthe following settings: excitation wavelength: 485 nm and emis-sion wavelength: 680 nm. Concurrently, toxicity assays (n = 12,during the weeks 1, 3, 4, 8, 9, 11, 13, 15, 17, 19, 21 and concomi-tantly the re-inoculation assay) with a control culture (kept undertemperature and light standard conditions) were carried out to as-sess sensitivity to copper, and possible variations of the sensitivityof control cultures.

2.5. Reference substance

Copper was used as copper sulphate pentahydrate (Cu-SO4 � 5H2O), reagent grade (99% purity), provided by Merck. Allglassware was cleaned with diluted nitric acid and rinsed severaltimes with Milli-Q ultrapure water. Test concentrations were ob-tained from a 100 mg Cu l�1 solution. Copper concentrations usedin the assays were 1, 5, 10, 15, 20, 30, 40 and 50 lg l�1.

2.6. Statistical analysis

For each assay, biomass measurement values such as chloro-phyll fluorescence were used to calculate inhibition percentages.Mean inhibition percentages for each concentration were calcu-lated by comparison of area under the control population (100%)and copper treatment curves (Hampel et al., 2001) obtained foreach 24 h throughout the 72 h duration of the assay. From theseinhibition, percentages EC50-72 h (concentration of copper thatcaused a 50% reduction in chlorophyll fluorescence) values wereestablished by Trimmed Spearman-Karber – TSK (Hamilton et al.,1977). Inhibition values exceeding 100% were expressed as a100% and negative inhibition values were expressed as 0%. Fromthe toxicity assays data (EC50-72 h) obtained from the control cul-ture a graph of control culture sensitivity (mean and ±2SD, stan-dard deviation) was generated. Control culture bioassays wereused to establish a fitness baseline in terms of sensitivity (EC50)values to calculate a mean and variations for a population consid-ered as reference (Lee, 1980). Thus, by plotting toxicity assay re-sults for each stored cell density in this sensitivity control chartwe can evaluate possible changes in cells sensitivity due to cold-dark storage. Therefore values obtained in the toxicity assays ofthe stored cells included in the range of ±2SD (warning limits) inthe sensitivity control chart were considered acceptable (ABNT,2005; Blaise and Vasseur, 2005). Additionally, EC50 values of thedensities-A, B and C were statistically compared with control cul-ture EC50 values by one-was ANOVA followed by Dunnett’s test,with significance level at p < 0.05 (Zar, 1996).

3. Results and discussion

Data about inoculum volume used each week for re-activation,cell density relative to the inoculated volume, final cell density, aswell as growth time in re-activation assays, are summarized in Ta-bles 1–3 for densities-A, B and C, respectively.

For density-A (Table. 1) the inoculum volume of 2 ml (repre-senting 0.08 � 104 cells ml�1) was very low. Thus, 11 days werenecessary to reach a satisfactory cell growth to perform the toxicity

assays. Increasing storage time, made necessary a higher inoculumvolume, using 4 ml from the 5th up to 15th week. From the 17thweek up to 21st 8 ml of inoculum was used. There was no growthfor samples referred to weeks 17 and 21. For density-B (Table. 2)after the 2nd week of storage it was possible to obtain cell growthafter 4 days in re-activation. From the third up to the 17th weekthe growth period necessary to perform toxicity assays was of 11days. 8 ml of inoculum with 18 days of incubation were necessaryfor the last 2 weeks. In the case of density-C (Table. 3) lowergrowth time was necessary with regards to the other densities.Samples belonging to the first 7 weeks of storage grew in only 4days, and up to the 12th week of storage 2 ml was enough forthe inoculation. The maximum growth time resulted in 11 days,including the cells stored during weeks 19 and 21, however in thiscase an inoculum of 8 ml was necessary.

Table 3Data of density-C (25 � 104 cells ml�1) including sampling week (storage time),volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) ofgrowth until the carrying out of the assay and final cell density after growth

Storageweek

Volumeinoculated(ml)a

Cell density initial(�104 cells ml�1)

Growthperiod(days)

Final cell density(�104 cells ml�1)

1 2 1.00 4 33.16 (±3.75)2 2 1.00 4 27.61 (±3.75)3 2 1.00 4 30.33 (±3.49)4 2 1.00 4 23.54 (±3.86)5 2 1.00 4 15.37 (±3.73)6 2 1.00 4 13.29 (±4.63)7 2 1.00 4 14.95 (±3.79)8 2 1.00 11 142.75 (±24.96)9 2 1.00 11 143.83 (±31.45)

10 2 1.00 11 137.91 (±3.35)11 2 1.00 11 106.16 (±20.53)12 2 1.00 11 98.91 (±17.88)13 4 2.00 11 96.25 (±18.22)14 4 2.00 11 104.91 (±19.09)15 4 2.00 11 109.50 (±3.27)17 4 2.00 11 84.58 (±15.36)19 8 4.00 11 167.50 (±2.61)21 8 4.00 11 83.91 (±8.66)

Growth data result from the mean of three replicates. ±: standard deviation.a Each 2 ml are equivalent to 1 pipette (total volume of inoculum plus culture

medium was always of 50 ml).

Density-A

Weeks of incubation in dark and cold1 2 3 4 5 7 9 11 13 15 19

Cel

l yie

ld (%

rela

tive

to c

ontro

l)

0

20

40

60

80After 11 days (2 ml)After 18 days (2 ml)After 18 days (4 ml)After 25 days (4 ml)After 25 days (8 ml)

Density-C

Weeks of incubation in dark and cold1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21

Cel

l yie

ld (%

rela

tive

to c

ontro

l)

0

20

40

60

80

100

After 4 days (2 ml)After 11 days (2ml)After 11 days (4 ml)After 11 days (8 ml)

Density-B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21

Cel

l yie

ld (%

rela

tive

to c

ontro

l)

0

25

50

75

150

175After 4 days (2 ml)After 11 days (2 ml)After 11 days (4 ml)After 18 days (8 ml)

Fig. 1. Mean cell yield (% relative to control) with respective standard deviation(n = 3) of cold-dark storage Cylindrotheca closterium populations after re-activation.Data belong to the three cell densities assessed: A (2 � 104 cells ml�1); B (10 �104 cells ml�1); and C (25 � 104 cells ml�1).

C.V.M. Araújo et al. / Chemosphere 72 (2008) 1366–1372 1369

After storage in cold and darkness conditions, when cells wereexposed to optimal conditions of light and temperature they pre-sented a delay in growth, indicating an extended lag phase. Thesame phenomenon was also found in marine diatom cells exposedto dark and re-illumination (Smayda and Mitchell-Innes, 1974). Inexperiments with marine Antarctic diatoms, Peters and Thomas(1996) found an increase of lag phase with the increase of storagetime in dark. After 21 days of dark storage exponential growth ofthe diatoms was registered immediately, while in 127 days in darkconditions the lag phase was extended up to 13 days; however dia-toms belonging to temperate regions lost growth capability after49 days in dark (Peters, 1996). Maximum period of darkness sur-vival of Cylindrotheca fusiformis at 2 and 20 �C was of 52 weeks,but other two diatom species, Navicula incerta and Nitzschia angu-laris, were able to remain viable after 3 years in darkness (Antia,1976). Benthic diatoms immobilized in alginate beads without li-quid medium and at 4 �C in dark after more than 1 year were capa-ble of growing and initiating new culture when seeded to freshmedium and adequate growth conditions; however 4 weeks werenecessary to reach similar cell densities from those obtained in acontrol culture (Chen, 2007).

In general, there was an increase in cell yield when re-activationperiod and inoculum volume increased. Higher values of cell yieldwere observed for density-C, between 40% and 80% relative to con-trol culture cell yield. For both the same inoculated volume andgrowth period (re-activation) a decrease in cell yield values in alldensities can be noticed with increasing cold-dark storage time.On the other hand, when increasing the growth period (re-activa-tion) but maintaining the same inoculum volume the cell yield in-creased. These data confirm that the delay in cell growth is due toan increase in storage time. A significant cell yield relative to con-trol culture can be obtained increasing the inoculated volume and/or growth time (re-activation). Data about cell yield for each den-sity assessed is represented in Fig. 1.

According to Dhargalkar (2004), some species of macroalgaeacclimated to a given temperature alter the growth rate withincreasing exposure time to cold. Jochen (1999) showed that Chrys-ochromulina hirta (Prymnesiophyte) cells strongly decrease theirabundance after 10 days of exposition to darkness; however for

Brachiomonas submarina (Chlorophyta) there were no changes inabundance. Furthermore, it was demonstrated that recoverycapacity of cells exposed to darkness when re-illuminated, mea-sured as metabolic activity by the use of FDA (fluorescein diace-tate) or cell growth, resulted in to be species-specific. For twopennate diatoms species (Prymnesium parvum and Bacteriastrumsp.) the metabolic activity did not change, and almost all cells re-mained metabolically active. Moreover, these species were capableto sustain a constant cell density after 12 days of darkness and pre-sented a rapid growth after re-illumination (Jochen, 1999). Some

Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21 R.I.

EC50

- 72

h (

ug C

ul-1

)

10

20

30

40

50

Density-A Density-B Density-C Control culture

Mean EC50 - Control Culture

upper SD

lower SD

+2SD

-2SD

Fig. 2. Sensitivity checking (EC50-72 h) from 12 assays with a control culture ofCylindrotheca closterium with the ±2SD (standard deviation) respective upper andlower interval values, and results of EC50-72 h copper values from assays carried outwith the three densities (A: 2 � 104 cells ml�1; B: 10 � 104 cells ml�1; C: 25 �104 cells ml�1) exposed to storage in cold and dark after re-activation. RI (Re-ino-culation) reefers to sample of week 21 after the second seeding.

1370 C.V.M. Araújo et al. / Chemosphere 72 (2008) 1366–1372

marine Antarctic diatom species after 3 months of darkness periodbegan to grow at similar rates as those established before the expo-sition to darkness when returned to light regime (Peters and Tho-mas, 1996). Thalassiosira weissflogii (marine diatom) wasmaintained alive during 2 months in dark, and it was capable toexponentially grow when illuminated (Murphy and Cowles, 1997).

Probably, the higher number of cells in density-C was suitablefor a faster growth, reaching the exponential phase much faster,likely due to a higher number of viable cells. Cell density plays animportant role in the viability of microalgae storage under low tem-perature (Sánchez-Saavedra, 2006). Two problems could arise ifhigher cell densities are used for storage: oxygen depletion by res-piration (once there is no photosynthetic activity) and nutrientdepletion. Due to a decrease in metabolism during storage in dark-ness and cold, there is low oxygen consumption (Antia, 1976), thisis why 2 ml of medium in 3 ml pipettes seem to be adequate tomaintain enough oxygen quantity for cells during storage. Duringre-activation of cells exposed to darkness, in the lag phase of accli-mation to new conditions, oxygen consumption and production arenot appreciable, indicating that the photosynthesis is compensatedwith respiration (Anderson, 1975a). Thus, the lag period in photo-synthesis becomes progressively longer as cold-dark storage timeincreases (Smayda and Mitchell-Innes, 1974; Anderson, 1975a).As cell storage was carried out with high nutrient concentration(f/2 medium), due to decreasing cell metabolic rate, cells shouldnot suffer a nutritional limitation during the storage time. More-over, this is supported by the higher growth observed in the caseof density-C (higher cell density). Heterotrophic activity (capacityto use glucose as nutrient) when exposed to darkness conditions(30 days) was observed in polar pennate diatoms (Palmisano andSullivan, 1982). Changes in heterotrophic mode of nutrition andreduction in metabolic activity are behaviours expected for micro-algae exposed to long periods of darkness, mostly when they do notcontain enough nutrient reserves (Jochen, 1999; Sánchez-Saavedra,2006). Other important factor is the formation of resting spores andcells when microalgae cells are submitted to unfavourable condi-tions (Anderson, 1975a; Peters and Thomas, 1996) such as low tem-peratures and darkness. Morphological, physiological andbiochemical changes are expected in dark-stressed cells (Smaydaand Mitchell-Innes, 1974). During the storage time no spore re-mains or morphological changes were observed by microscopicalanalysis. Similar results were obtained by Peters and Thomas(1996) in diatom species exposed to darkness. With regards to rest-ing cells it is difficult to confirm its presence due to morphologicalsimilarity to vegetative cells (Anderson, 1975b), once resting cellsposses an unmodified frustule (Anderson, 1975a). Palmisano andSullivan (1982) observed that diatoms size also remained constantafter 30 days expose to dark. This was also observed in this work.The diatom Fragilariopsis cylindrus exposed to �1.8 �C and low lightintensity showed decrease in photochemical efficiency, increase incell concentration of chlorophyll a and c, and changes in expressionof photosynthesis related genes (Mock and Valentin, 2004).

Cell growth observed in sensitivity toxicity assays with coppercarried out with re-activated cells and control culture cells wasof 22–32 times (after 72 h) with regards to initial concentration(for treatment without copper). It was possible to prove that afteracclimation under controlled conditions of light and temperature,cells presented a tendency to keep a similar growth rate to theone obtained for the control culture under optimum conditions,corroborating that a lower growth was resulted in of an acclima-tion lag to standard culture conditions. Short-term physiologicaladjustments in response to altered environmental conditions, asshown here, are very common in photosynthetic organisms (Mor-gan-Kiss et al., 2006). The final cell density of controls in assayswith cadmium, copper, chromium, and atrazine to Selenastrum cap-ricornutum cells submitted to cryopreservation over a 3-month

period at �80 �C was always higher than 16-fold after 72-h (Ben-hra et al., 1997).

A more reliable and certain procedure to measure status orhealth of the test organism is carrying out a bioassay using a refer-ence toxicant (Lee, 1980). In the present study successive bioassayswere carried out with a control culture to establish a standard va-lue of sensitivity using copper as reference substance. Sensitivitydata for the three stored densities assayed were plotted under astandard sensitivity chart to verify changes in sensitivity duringthe 21 weeks of storage (Fig. 2). EC50-72 h mean value for the con-trol culture was 29 ± 10 lg Cu l�1. These data were obtained from12 assays during the weeks 1, 3, 4, 8, 9, 11, 13, 15, 17, 19, 21 andin the reference week to re-inoculation (data plotted in Fig. 2 asdiamond). A standard sensitivity chart containing EC50-72 h meanvalue and lower and upper standard deviation multiplied by two(±2SD) was plotted, and the obtained values in toxicity assays forthe stored density cells included in this range of sensitivity controlgraph were considered acceptable (ABNT, 2005; Blaise and Vas-seur, 2005). There was an increase in sensitivity of all cell densitiesstudied; however the results always maintained inside the estab-lished range for standard sensitivity (Fig. 2). EC50-72 h mean valuesfor densities-A, B and C were 22 ± 7; 23 ± 9 and 23 ± 8 lg Cu l�1,respectively. EC50-72 h values for re-inoculation samples (samplesfrom the 21st week that were re-inoculated for one more week)were 25.55 and 18.79 lg Cu l�1 for densities-B and C showing thatthe sensitivity remained unmodified. EC50-72 h values were inde-pendent of the cell density stored. In general, sensitivity to copperof C. closterium measured as EC50-72 h was not significantly modi-fied with regards to control culture (p > 0.05; F3;57 = 1.994) during21 weeks of cold-dark storage for the three cell densities studied.Cryopreservation (3-month period at �80 �C) significantly in-creased the sensitivity of Selenastrum capricornutum cells when ex-posed to cadmium, copper, chromium, and atrazine; moreoversensitivity increased during long-term storage period (Benhraet al., 1997). In this study, Cu toxicity EC50-72 h values were of28.5 ± 2.8 lg l�1 for the algae under standard conditions and of21.7 ± 0.8 lg l�1 for the cryopreserved algae.

Sensitivity found for C. closterium was similar to informationfound for other microalgae species widely used in ecotoxicology.Hogan et al. (2005) found IC50-48 h (inhibition concentration) val-

C.V.M. Araújo et al. / Chemosphere 72 (2008) 1366–1372 1371

ues for copper of 18 (16–20) lg l�1 and 21 (18–24) lg l�1 usingflasks (50 ml) and minivials (6 ml) respectively. EC50-96 h valuesobtained by Stauber and Florence (1989) for Nitzschia closterium(=C. closterium) were of 10 lg Cu l�1 in assays employing Erlen-meyer flasks and using cell division rate as endpoint. Satoh et al.(2005), in bioassays using a Cylindrotheca species found a medianinhibitory concentration value (IC50-72 h) for Cu of 7.7 mg l�1,based on chlorophyll fluorescence. However, the high value ob-served by Satoh et al. (2005) could be due to the presence of highEDTA content in the culture medium. Using cell biomass as end-point, EC50-72 h values for copper established by Moreno-Garridoet al. (2000) using Erlenmeyer flasks were of 38.3 lg l�1 for Chlo-rella autotrophyca, 46.2 lg l�1 for Nannochloris atomus, 35.0 lg l�1

for Phaeodactylum tricornutum and of 4.4 lg l�1 for Isochrysis aff.galbana. For Rhodomonas salina the EC50-72 h value (cell biomassas endpoint) for copper was of 30 lg l�1 (Moreno-Garrido et al.,1999). Using esterase activity as endpoint, Franklin et al. (2001)found EC50-24 h values of 51 (38–70) lg Cu l�1 for Selenastrum cap-ricornutum and when cell division rate (after 72 h) was used asendpoint values of 7.5 (6.8–8.2) lg Cu l�1. In the same study, car-ried out on Entomoneis cf. punctulata the EC50-24 h value usingesterase activity was of 9.1 (7.6–11) lg Cu l�1, and the EC50-72 husing cell division rate as endpoint was of 18 (17–20) lg Cu l�1.Thus, sensitivity depends on the species and it is referred to theendpoint employed in the bioassays. Although many studies dem-onstrate that miniaturized bioassays are less sensitive than classi-cal bioassays, they present some advantages such as lessoccupation of space in laboratory, less time of preparation andevaluation, and require less volume of test sample (Rojickováet al., 1998). According to Pavlic et al. (2006), microplate algal as-says are advantageous because they reduce laboratory necessitiesof resources. Additionally, the use of the 96-well microplate makesable the possibility of more parallel assays and tests with a highernumber of concentrations. Undoubtedly, miniaturized assays withalgae are simple, practical and reproducible. Due to simplicity ofthe miniaturized bioassay and to the reduced interference of theoperator with algae its implementation in environmental assess-ment is recommended. In the present study, miniaturized bioas-says employed to assess sensitivity of C. closterium to copperwere found as appropriate.

4. Conclusions

Results from this work confirm that in spite of drastic changesin the environmental conditions due to storage in dark and lowtemperature, C. closterium cells remained metabolically active withno statistically significant changes in sensitivity to copper. In spiteof stored cells showed an extended lag phase, cell growth was nor-malized after re-activation under standard conditions of light andtemperature. Density-C, containing 25 � 104 cells ml�1, was themost appropriate for storage of C. closterium cells due to the highercell yield presented. Due to the lack of available references in liter-ature it is not possible to compare the data here obtained withother similar studies. This study represents a first approach in stor-age of benthic diatom cells for latter application in kits for toxicitymeasurements based in the response of marine microphytoben-thos, susceptible to be used by any person without previoustraining.

Acknowledgements

This work has been funded by the Spanish Ministry of Scienceand Education (project MECASEC: CTM2006-01437/MAR). C.V.M.Araújo is grateful to Brazilian Coordination of Improvement of Per-sonnel of Superior Level (CAPES) by PhD scholarships conceded.

The authors are thankful to Bibiana Debelius for revision of theEnglish text and to two reviewers for their contributions.

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