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Effects of cold-dark storage on growth of Cylindrotheca closterium and its sensitivity to copper
Chemosphere 72 (2008) 1366–1372
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Effects of cold-dark storage on growth of Cylindrotheca closterium and its sensitivity to copper Cristiano V.M. Araújo *, Fernando R. Diz, Ignacio Moreno-Garrido, Luis M. Lubián, Julián Blasco Instituto 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
Article history: Received 8 January 2008 Received in revised form 26 March 2008 Accepted 10 April 2008 Available online 3 June 2008 Keywords: Diatoms Long-term storage Marine ecotoxicology Microphytobenthos
a b s t r a c t Cylindrotheca closterium cells were maintained at low temperature (4 ± 1 °C) and dark conditions up to 21 weeks to assess the effect on survival and physiological status. From a control culture under standard conditions, three densities were prepared: (A) 2 104, (B) 10 104, and (C) 25 104 cells ml1. Weekly, inoculums of each stored density were exposed to continuous light and at 20 ± 1 °C. Sensitivity to copper for microalgal cultures was evaluated in order to assess possible changes in cells sensitivity due to storage. Concurrently, assays with a control culture were carried out in order to assess the sensitivity of C. closterium to copper and to be able to generate a standard sensitivity control chart with a mean value of EC50-72 h ± 2SD (standard deviation). Density-C presented higher cell yield values, between 40% and 80% relative to control culture. Cell density showed to be important feature that may be taken into account 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 significant difference with regards to control culture. EC50-72 h mean value for the control culture was 29 ± 10 lg Cu l1, while for densities-A, B and C were 22 ± 7; 23 ± 9 and 23 ± 8 lg Cu l1, respectively. In spite of drastic changes in the environmental conditions due to storage, it is concluded that C. closterium cells stored during 5 months remained metabolically active and with no significant change in its sensitivity. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Search for ecotoxicological assays with ecologically relevant species is an important task for environmental researchers. Ecological relevance of a single species is related to the key role in the ecosystem, as well as representativeness of the species in the trophic level (Calow, 1989). Test organisms should be chosen according to the information intended to be obtained (Rojicˇková et al., 1998). Thus, environmental risk analysis of sediments should take into account the benthic community (Moreno-Garrido et al., 2003a), while terrestrial impacts should be assessed through soil organisms (DelValls et al., 2004). On the other hand, when water is the main route of uptake for chemical compounds, then toxicity test with planktonic organisms should be employed (Walker et al., 2001). In spite of the efforts employed to develop algal assay procedures, the majority of the internationally standardized assays are focused on planktonic freshwater algae (ISO, 1989; Environmental Canada, 1992; OECD, 1998; USEPA, 2002; ABNT, 2005), with quite less attention to species of the microphytobenthos (Moreno-Garrido et al., 2003a). Thus, tests should be developed following the specific environmental conditions using representative species of * Corresponding author. Tel.: +34 956 832 612; fax: +34 956 834 701. E-mail address: [email protected] (C.V.M. Araújo). 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.04.022
the ecosystems. Benthic diatoms are a major component of the microphytobenthos in aquatic systems (MacIntyre et al. 1996; de Bower et al., 2003) and can therefore be used as a measure of water quality (Kelly et al., 2001). Diatoms are the main source of food for meiofauna and they are very important in material and energy transfer through the benthic food webs (Miller et al., 1996; Blanchard et al., 2000). Moreover, the excreted polysaccharides play a relevant role in the stability of the marine surface sediment layer (Miller et al., 1996), being key factor of the structure and function of benthic intertidal zones (Scala and Bowler, 2001). Although there is an unquestionable ecological importance of benthic diatoms there are very few studies that have adopted these organisms in toxicity assays. In general, among microphytobenthos, Cylindrotheca closterium is the main species used in ecotoxicology (Stauber and Florence, 1985, 1989; Moreno-Garrido et al., 2003a,b, 2007; Hogan et al., 2005). Microalgae cultures, such as diatoms, are widely used to feed organisms in aquaculture (Cañavate and Lubián, 1995) especially in shellfish hatcheries (Redekar and Wagh, 2000), and they have also been used in ecotoxicological assays (Nörnström, 1990). However, sometimes ecotoxicology laboratories need to maintain a microalgae culture only exclusively to perform a determined assay whose frequency may be spaced in time. The maintenance of living microalgae cultures is an expensive task (Sánchez-Saavedra, 2006)
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mainly if toxicity assays are not routinely carried out. Mitbavkar and Anil (2006) suggested cryopreservation as a very useful technique for the maintenance of phytoplankton culture collections. However, this technique results to be very aggressive at an ultrastructural level during freezing and thawing and once cells survive post-thaw severe damages can occur (Tzovenis et al., 2004). Additionally, the maintenance of cryopreserved algae is expensive and laborious. Nevertheless, many factors may be taken into account for the success of cryopreservation such as state and density of the culture, the type of cryoprotectant and rate of cooling and thawing (Cañavate and Lubián, 1995; Poncet and Véron, 2003). According to Admiraal (1977) benthic diatoms cultures can be stored in dark, at 4 °C and subsequently inoculated in fresh medium in order to preserve cells for long time. Maintenance of cultures in cold and darkness is less expensive and more realistic and viable (Sánchez-Saavedra, 2006). Studies about physiological effects due to cold and darkness exposition in diatoms are performed, in general, with polar species naturally adapted to changes in light and temperature cycle (Gleitz and Thomas, 1992). Many polar diatom species can survive under total darkness for periods up to 10 months (Peters and Thomas, 1996). Studies on cell viability (growth rate) of microalgae (not exclusively polar) exposed to low temperature and darkness can be found in literature (Peters, 1996; Murphy and Cowles, 1997; Chen, 2007). Not only algal survival and growth need to be considered, but also changes in sensitivity to contaminants due to other stressors such as storage conditions, low temperature, darkness and nutrient depletion. The aim of this paper was to assess if the storage of C. closterium cells under low temperature (4 °C) and darkness could affect its growth and verify if it caused variations in its sensitivity in toxicity tests. Information with regards to sensitivity changes after colddark storage for a further ecotoxicological application becomes very useful for sediment toxicity tests including C. closterium cells. This study was undertaken for about 5 months because it was considered that 5 months was enough time in view of a commercial application 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 (formerly Nitzschia closterium (Ehrenberg) W. Smith) was isolated in May 2000 from a salt pond near the Marine Sciences Institute from Andalusia (ICMAN) in Puerto Real (SW of Spain). The strain (C. closterium #010301) is currently included in the Culture Collection of Marine Microalgae of the ICMAN (CCMM-ICMAN, BIOCISE). For routine cultures, seawater is filtered through a GF/C Whatman filter, autoclaved, and later enriched with Guillard’s f/2 medium (Guillard and Ryther, 1962) plus 500 lg l1 SiO2. Artificial substitute ocean water (ASTM, 1975) enriched with SiO2 (500 lg l1), 1 1 NO ), and PO3 ) was used in toxicity assays 3 (150 lg l 4 (10 lg l with copper, in order to avoid the chelator effect of EDTA, included in the f/2 medium. Routine culture and assays were performed at 20 ± 1 °C under continuous white light (35.2 ± 1.1 lmol m2 s1) in a controlled culture chamber (Ibercex). 2.2. Preparation of cell densities and storage at low temperature and darkness conditions Three-day algal cultures (at exponential growth phase) were used to prepare the three densities, named A, B, and C, which contained: 2 104, 10 104 and 25 104 cells ml1, respectively. All densities were prepared with the same culture medium. Two milliliters of aliquots of densities-A, B and C were collected in sterile
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plastic Pasteur pipettes, and then they were hermetically sealed by heating the pipettes’ tips. To avoid heating the algae the pipettes were placed upside-down and the cells were concentrated in the no heated part of the pipette during sealing. All procedures were carried out in laminar flow chamber. Once the pipettes were sealed, they were stored at 4 ± 1 °C and darkness conditions up to 21 weeks. 2.3. Sampling and growth assay (re-activation) Stored pipettes submitted to low temperature and darkness conditions were sampled weekly for each density, in triplicate. The cells were re-activated using culture medium prepared as described in Section 2.1. Initially sampled volume was of 2 ml (referred to one Pasteur pipette) introduced in 48 ml of medium. However, throughout the time assayed the growth velocity was decreased (increase in lag-phase) in all densities studied, thus a bigger volume of cells was needed to be inoculated. Stored inoculum volume plus the culture medium volume resulted to be always of 50 ml. When the inoculated volume was of 2 ml (one Pasteur pipette), cell densities in Erlenmeyer flask were 800, 4000 and 10 000 cells ml1 for densities-A, B and C, respectively. After inoculation four growth periods for cell re-activation were established: 4, 11, 18 and 25 days, this way when there was not enough number of cells (around 1 105 cells ml1) to carry out a toxicity assay in 4 days, the culture was maintained for one more week and successively. At the end of the re-activation assay cell yield (a) values were calculated using the following formula, for the three stored densities studied: a¼
N N0 N0
ð1Þ
where N and N0 are the number of cells ml1 at final time (t, in days) and initial time (t0, in days), respectively. Cell yield values were compared with the control culture – kept under controlled conditions of temperature (20 ± 1 °C) and light (continuous white light 35.2 ± 1.1 lmol m2 s1) – considered as 100% cell yield and were expressed as relative values (%). In order to compare cell yield from inoculated volume, initial cell density and growth days were taken into account. Sensitivity of the re-activated culture was also evaluated with a toxicity assay using copper as reference compound. Samples from week 21 were re-inoculated for one more week and then a toxicity assay was carried out. These samples, named reinoculation, were only followed for densities-B and C because there was no growth in density-A at 21st week. 2.4. Toxicity tests Algal cultures of the three re-activated densities were centrifuged (MES, Micro Centaur, SANYO) for 4 min at 4000 rpm and re-suspended in clean medium (artificial substitute ocean water – ASTM, 1975). This procedure was followed three times before use in the assay in order to guarantee the elimination of f/2 medium and with the aim of avoiding the effect of the chelator EDTA included in the f/2 medium. Initial cellular concentration for growth-inhibition assay of 104 cells ml1 (OECD, 1998) was adopted. Bioassays were carried out in 96-well microplates (Greiner 96-well flat bottom white, 12 8, 400 ll capacity) based on Blaise et al. (1998). Each well was filled with 300 ll of sample. During incubation, microplates were covered with their transparent lids and maintained without agitation. Assays were carried out at five 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 located wells were used. To reduce evaporation, wells along the microplate margin were filled with Milli-Q water (Lukavsky´, 1992). Samples
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were randomly distributed to avoid pseudoreplication effects due to possible spatial differences in illumination and temperature. For all bioassays, the pH drift was less than 0.5 pH unit over 72 h in accordance with established by OECD (1998). Measurement of the initial and final pH values in all samples were registered (Crison, MicropH 2001, electrode Hamilton, Slimtrode). Fluorescence values were compared with the mean value of fluorescence activity belonging to algae with no toxic (control), considered as 100% activity. Fluorometric data were obtained with a spectrophotometer (TECAN Multiplate Reader, GENios) at 0, 24, 48, and 72 h with the following settings: excitation wavelength: 485 nm and emission wavelength: 680 nm. Concurrently, toxicity assays (n = 12, during the weeks 1, 3, 4, 8, 9, 11, 13, 15, 17, 19, 21 and concomitantly the re-inoculation assay) with a control culture (kept under temperature and light standard conditions) were carried out to assess sensitivity to copper, and possible variations of the sensitivity of control cultures.
Table 1 Data of density-A (2 104 cells ml1) including sampling week (storage time), volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) of growth until the carrying out of the assay and final cell density after growth
2.5. Reference substance
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.
Copper was used as copper sulphate pentahydrate (CuSO4 5H2O), reagent grade (99% purity), provided by Merck. All glassware was cleaned with diluted nitric acid and rinsed several times with Milli-Q ultrapure water. Test concentrations were obtained from a 100 mg Cu l1 solution. Copper concentrations used in the assays were 1, 5, 10, 15, 20, 30, 40 and 50 lg l1. 2.6. Statistical analysis For each assay, biomass measurement values such as chlorophyll fluorescence were used to calculate inhibition percentages. Mean inhibition percentages for each concentration were calculated by comparison of area under the control population (100%) and copper treatment curves (Hampel et al., 2001) obtained for each 24 h throughout the 72 h duration of the assay. From these inhibition, percentages EC50-72 h (concentration of copper that caused a 50% reduction in chlorophyll fluorescence) values were established by Trimmed Spearman-Karber – TSK (Hamilton et al., 1977). Inhibition values exceeding 100% were expressed as a 100% and negative inhibition values were expressed as 0%. From the toxicity assays data (EC50-72 h) obtained from the control culture a graph of control culture sensitivity (mean and ±2SD, standard deviation) was generated. Control culture bioassays were used to establish a fitness baseline in terms of sensitivity (EC50) values to calculate a mean and variations for a population considered as reference (Lee, 1980). Thus, by plotting toxicity assay results for each stored cell density in this sensitivity control chart we can evaluate possible changes in cells sensitivity due to colddark storage. Therefore values obtained in the toxicity assays of the stored cells included in the range of ±2SD (warning limits) in the sensitivity control chart were considered acceptable (ABNT, 2005; Blaise and Vasseur, 2005). Additionally, EC50 values of the densities-A, B and C were statistically compared with control culture 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, as well as growth time in re-activation assays, are summarized in Tables 1–3 for densities-A, B and C, respectively. For density-A (Table. 1) the inoculum volume of 2 ml (representing 0.08 104 cells ml1) was very low. Thus, 11 days were necessary to reach a satisfactory cell growth to perform the toxicity
Storage week
Volume inoculated (ml)a
Cell density initial (104 cells ml1)
Growth period (days)
Final cell density (104 cells ml1)
1 2 3 4 5 7 9 11 13 15 17 19 21
2 2 2 2 4 4 4 4 4 4 8 8 8
0.08 0.08 0.08 0.08 0.16 0.16 0.16 0.16 0.16 0.16 0.32 0.32 0.32
11 11 18 18 18 18 18 18 18 25 – 25 –
39.16 (±3.40) 38.55 (±8.29) 126.66 (±9.86) 174.00 (±31.57) 150.75 (±37.12) 120.75b 57.50b 90.58 (±29.95) 46.50b 213.25b –c 161.75 (±16.97) –c
Table 2 Data of density-B (10 104 cells ml1) including sampling week (storage time), volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) of growth until the carrying out of the assay and final cell density after growth Storage week
Volume inoculated (ml)a
Cell density initial (104 cells ml1)
Growth period (days)
Final cell density (104 cells ml1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21
2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 4 8 8
0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.80 0.80 0.80 0.80 1.60 1.60
4 4 11 11 11 11 11 11 11 11 11 11 11 11 11 11 18 18
21.44 (±1.17) 7.88 (±2.60) 151.00 (±12.93) 140.83 (±26.83) 139.00 (±16.71) 124.66 (±33.85) 76.16 (±16.32) 65.91 (±24.98) 63.08 (±11.54) 50.41 (±3.81) 42.41 (±30.88) 38.16 (±6.45) 31.41 (±2.32) 30.33 (±11.00) 30.25 (±20.07) 33.91 (±10.27) 127.00 (±18.77) 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).
assays. Increasing storage time, made necessary a higher inoculum volume, using 4 ml from the 5th up to 15th week. From the 17th week up to 21st 8 ml of inoculum was used. There was no growth for samples referred to weeks 17 and 21. For density-B (Table. 2) after the 2nd week of storage it was possible to obtain cell growth after 4 days in re-activation. From the third up to the 17th week the growth period necessary to perform toxicity assays was of 11 days. 8 ml of inoculum with 18 days of incubation were necessary for the last 2 weeks. In the case of density-C (Table. 3) lower growth time was necessary with regards to the other densities. Samples belonging to the first 7 weeks of storage grew in only 4 days, and up to the 12th week of storage 2 ml was enough for the inoculation. The maximum growth time resulted in 11 days, including the cells stored during weeks 19 and 21, however in this case an inoculum of 8 ml was necessary.
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Cell density initial (104 cells ml1)
Growth period (days)
Final cell density (104 cells ml1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21
2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 4 8 8
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00 2.00 2.00 2.00 4.00 4.00
4 4 4 4 4 4 4 11 11 11 11 11 11 11 11 11 11 11
33.16 (±3.75) 27.61 (±3.75) 30.33 (±3.49) 23.54 (±3.86) 15.37 (±3.73) 13.29 (±4.63) 14.95 (±3.79) 142.75 (±24.96) 143.83 (±31.45) 137.91 (±3.35) 106.16 (±20.53) 98.91 (±17.88) 96.25 (±18.22) 104.91 (±19.09) 109.50 (±3.27) 84.58 (±15.36) 167.50 (±2.61) 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).
After storage in cold and darkness conditions, when cells were exposed to optimal conditions of light and temperature they presented a delay in growth, indicating an extended lag phase. The same phenomenon was also found in marine diatom cells exposed to dark and re-illumination (Smayda and Mitchell-Innes, 1974). In experiments with marine Antarctic diatoms, Peters and Thomas (1996) found an increase of lag phase with the increase of storage time in dark. After 21 days of dark storage exponential growth of the diatoms was registered immediately, while in 127 days in dark conditions the lag phase was extended up to 13 days; however diatoms belonging to temperate regions lost growth capability after 49 days in dark (Peters, 1996). Maximum period of darkness survival of Cylindrotheca fusiformis at 2 and 20 °C was of 52 weeks, but other two diatom species, Navicula incerta and Nitzschia angularis, were able to remain viable after 3 years in darkness (Antia, 1976). Benthic diatoms immobilized in alginate beads without liquid medium and at 4 °C in dark after more than 1 year were capable of growing and initiating new culture when seeded to fresh medium and adequate growth conditions; however 4 weeks were necessary to reach similar cell densities from those obtained in a control culture (Chen, 2007). In general, there was an increase in cell yield when re-activation period and inoculum volume increased. Higher values of cell yield were observed for density-C, between 40% and 80% relative to control culture cell yield. For both the same inoculated volume and growth period (re-activation) a decrease in cell yield values in all densities can be noticed with increasing cold-dark storage time. On the other hand, when increasing the growth period (re-activation) but maintaining the same inoculum volume the cell yield increased. These data confirm that the delay in cell growth is due to an increase in storage time. A significant cell yield relative to control culture can be obtained increasing the inoculated volume and/ or growth time (re-activation). Data about cell yield for each density assessed is represented in Fig. 1. According to Dhargalkar (2004), some species of macroalgae acclimated to a given temperature alter the growth rate with increasing exposure time to cold. Jochen (1999) showed that Chrysochromulina hirta (Prymnesiophyte) cells strongly decrease their abundance after 10 days of exposition to darkness; however for
Cell yield (% relative to control)
Volume inoculated (ml)a
After 11 days (2 ml) After 18 days (2 ml) After 18 days (4 ml) After 25 days (4 ml) After 25 days (8 ml)
80
60
40
20
0 1 2 3 4 5
Cell yield (% relative to control)
Storage week
Density-A
175
7
9
11
13
15
19
Weeks of incubation in dark and cold Density-B After 4 days (2 ml) After 11 days (2 ml) After 11 days (4 ml) After 18 days (8 ml)
150
75 50 25 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Density-C
Cell yield (% relative to control)
Table 3 Data of density-C (25 104 cells ml1) including sampling week (storage time), volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) of growth until the carrying out of the assay and final cell density after growth
100
17
19
21
After 4 days (2 ml) After 11 days (2ml) After 11 days (4 ml) After 11 days (8 ml)
80
60
40
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
17
19
21
Weeks of incubation in dark and cold 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 ml1); B (10 104 cells ml1); and C (25 104 cells ml1).
Brachiomonas submarina (Chlorophyta) there were no changes in abundance. Furthermore, it was demonstrated that recovery capacity of cells exposed to darkness when re-illuminated, measured as metabolic activity by the use of FDA (fluorescein diacetate) or cell growth, resulted in to be species-specific. For two pennate diatoms species (Prymnesium parvum and Bacteriastrum sp.) the metabolic activity did not change, and almost all cells remained metabolically active. Moreover, these species were capable to sustain a constant cell density after 12 days of darkness and presented a rapid growth after re-illumination (Jochen, 1999). Some
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period at 80 °C was always higher than 16-fold after 72-h (Benhra et al., 1997). A more reliable and certain procedure to measure status or health of the test organism is carrying out a bioassay using a reference toxicant (Lee, 1980). In the present study successive bioassays were carried out with a control culture to establish a standard value of sensitivity using copper as reference substance. Sensitivity data for the three stored densities assayed were plotted under a standard sensitivity chart to verify changes in sensitivity during the 21 weeks of storage (Fig. 2). EC50-72 h mean value for the control culture was 29 ± 10 lg Cu l1. These data were obtained from 12 assays during the weeks 1, 3, 4, 8, 9, 11, 13, 15, 17, 19, 21 and in the reference week to re-inoculation (data plotted in Fig. 2 as diamond). A standard sensitivity chart containing EC50-72 h mean value and lower and upper standard deviation multiplied by two (±2SD) was plotted, and the obtained values in toxicity assays for the stored density cells included in this range of sensitivity control graph were considered acceptable (ABNT, 2005; Blaise and Vasseur, 2005). There was an increase in sensitivity of all cell densities studied; however the results always maintained inside the established range for standard sensitivity (Fig. 2). EC50-72 h mean values for densities-A, B and C were 22 ± 7; 23 ± 9 and 23 ± 8 lg Cu l1, respectively. EC50-72 h values for re-inoculation samples (samples from the 21st week that were re-inoculated for one more week) were 25.55 and 18.79 lg Cu l1 for densities-B and C showing that the sensitivity remained unmodified. EC50-72 h values were independent of the cell density stored. In general, sensitivity to copper of C. closterium measured as EC50-72 h was not significantly modified with regards to control culture (p > 0.05; F3;57 = 1.994) during 21 weeks of cold-dark storage for the three cell densities studied. Cryopreservation (3-month period at 80 °C) significantly increased the sensitivity of Selenastrum capricornutum cells when exposed to cadmium, copper, chromium, and atrazine; moreover sensitivity increased during long-term storage period (Benhra et al., 1997). In this study, Cu toxicity EC50-72 h values were of 28.5 ± 2.8 lg l1 for the algae under standard conditions and of 21.7 ± 0.8 lg l1 for the cryopreserved algae. Sensitivity found for C. closterium was similar to information found for other microalgae species widely used in ecotoxicology. Hogan et al. (2005) found IC50-48 h (inhibition concentration) val-
Density-A Density-B Density-C Control culture
50
+2SD
19 21 R .I.
14 15 17
12 13
9 10 11
-2SD
8
10
3
lower SD
2
20
7
Mean EC50 - Control Culture
6
30
5
upper SD
4
40
1
marine Antarctic diatom species after 3 months of darkness period began to grow at similar rates as those established before the exposition to darkness when returned to light regime (Peters and Thomas, 1996). Thalassiosira weissflogii (marine diatom) was maintained alive during 2 months in dark, and it was capable to exponentially grow when illuminated (Murphy and Cowles, 1997). Probably, the higher number of cells in density-C was suitable for a faster growth, reaching the exponential phase much faster, likely due to a higher number of viable cells. Cell density plays an important role in the viability of microalgae storage under low temperature (Sánchez-Saavedra, 2006). Two problems could arise if higher cell densities are used for storage: oxygen depletion by respiration (once there is no photosynthetic activity) and nutrient depletion. Due to a decrease in metabolism during storage in darkness and cold, there is low oxygen consumption (Antia, 1976), this is why 2 ml of medium in 3 ml pipettes seem to be adequate to maintain enough oxygen quantity for cells during storage. During re-activation of cells exposed to darkness, in the lag phase of acclimation to new conditions, oxygen consumption and production are not appreciable, indicating that the photosynthesis is compensated with respiration (Anderson, 1975a). Thus, the lag period in photosynthesis becomes progressively longer as cold-dark storage time increases (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 should not suffer a nutritional limitation during the storage time. Moreover, this is supported by the higher growth observed in the case of density-C (higher cell density). Heterotrophic activity (capacity to use glucose as nutrient) when exposed to darkness conditions (30 days) was observed in polar pennate diatoms (Palmisano and Sullivan, 1982). Changes in heterotrophic mode of nutrition and reduction in metabolic activity are behaviours expected for microalgae exposed to long periods of darkness, mostly when they do not contain enough nutrient reserves (Jochen, 1999; Sánchez-Saavedra, 2006). Other important factor is the formation of resting spores and cells when microalgae cells are submitted to unfavourable conditions (Anderson, 1975a; Peters and Thomas, 1996) such as low temperatures and darkness. Morphological, physiological and biochemical changes are expected in dark-stressed cells (Smayda and Mitchell-Innes, 1974). During the storage time no spore remains or morphological changes were observed by microscopical analysis. Similar results were obtained by Peters and Thomas (1996) in diatom species exposed to darkness. With regards to resting cells it is difficult to confirm its presence due to morphological similarity to vegetative cells (Anderson, 1975b), once resting cells posses an unmodified frustule (Anderson, 1975a). Palmisano and Sullivan (1982) observed that diatoms size also remained constant after 30 days expose to dark. This was also observed in this work. The diatom Fragilariopsis cylindrus exposed to 1.8 °C and low light intensity showed decrease in photochemical efficiency, increase in cell concentration of chlorophyll a and c, and changes in expression of photosynthesis related genes (Mock and Valentin, 2004). Cell growth observed in sensitivity toxicity assays with copper carried out with re-activated cells and control culture cells was of 22–32 times (after 72 h) with regards to initial concentration (for treatment without copper). It was possible to prove that after acclimation under controlled conditions of light and temperature, cells presented a tendency to keep a similar growth rate to the one obtained for the control culture under optimum conditions, corroborating that a lower growth was resulted in of an acclimation lag to standard culture conditions. Short-term physiological adjustments in response to altered environmental conditions, as shown here, are very common in photosynthetic organisms (Morgan-Kiss et al., 2006). The final cell density of controls in assays with cadmium, copper, chromium, and atrazine to Selenastrum capricornutum cells submitted to cryopreservation over a 3-month
EC50 - 72 h (ug Cu l -1)
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Weeks Fig. 2. Sensitivity checking (EC50-72 h) from 12 assays with a control culture of Cylindrotheca closterium with the ±2SD (standard deviation) respective upper and lower interval values, and results of EC50-72 h copper values from assays carried out with the three densities (A: 2 104 cells ml1; B: 10 104 cells ml1; C: 25 104 cells ml1) exposed to storage in cold and dark after re-activation. RI (Re-inoculation) reefers to sample of week 21 after the second seeding.
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ues for copper of 18 (16–20) lg l1 and 21 (18–24) lg l1 using flasks (50 ml) and minivials (6 ml) respectively. EC50-96 h values obtained by Stauber and Florence (1989) for Nitzschia closterium (=C. closterium) were of 10 lg Cu l1 in assays employing Erlenmeyer flasks and using cell division rate as endpoint. Satoh et al. (2005), in bioassays using a Cylindrotheca species found a median inhibitory concentration value (IC50-72 h) for Cu of 7.7 mg l1, based on chlorophyll fluorescence. However, the high value observed by Satoh et al. (2005) could be due to the presence of high EDTA content in the culture medium. Using cell biomass as endpoint, EC50-72 h values for copper established by Moreno-Garrido et al. (2000) using Erlenmeyer flasks were of 38.3 lg l1 for Chlorella autotrophyca, 46.2 lg l1 for Nannochloris atomus, 35.0 lg l1 for Phaeodactylum tricornutum and of 4.4 lg l1 for Isochrysis aff. galbana. For Rhodomonas salina the EC50-72 h value (cell biomass as endpoint) for copper was of 30 lg l1 (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 l1 for Selenastrum capricornutum and when cell division rate (after 72 h) was used as endpoint values of 7.5 (6.8–8.2) lg Cu l1. In the same study, carried out on Entomoneis cf. punctulata the EC50-24 h value using esterase activity was of 9.1 (7.6–11) lg Cu l1, and the EC50-72 h using cell division rate as endpoint was of 18 (17–20) lg Cu l1. Thus, sensitivity depends on the species and it is referred to the endpoint employed in the bioassays. Although many studies demonstrate that miniaturized bioassays are less sensitive than classical bioassays, they present some advantages such as less occupation of space in laboratory, less time of preparation and evaluation, and require less volume of test sample (Rojicˇková et al., 1998). According to Pavlic´ et al. (2006), microplate algal assays are advantageous because they reduce laboratory necessities of resources. Additionally, the use of the 96-well microplate makes able the possibility of more parallel assays and tests with a higher number of concentrations. Undoubtedly, miniaturized assays with algae are simple, practical and reproducible. Due to simplicity of the miniaturized bioassay and to the reduced interference of the operator with algae its implementation in environmental assessment is recommended. In the present study, miniaturized bioassays employed to assess sensitivity of C. closterium to copper were found as appropriate. 4. Conclusions Results from this work confirm that in spite of drastic changes in the environmental conditions due to storage in dark and low temperature, C. closterium cells remained metabolically active with no statistically significant changes in sensitivity to copper. In spite of stored cells showed an extended lag phase, cell growth was normalized after re-activation under standard conditions of light and temperature. Density-C, containing 25 104 cells ml1, was the most appropriate for storage of C. closterium cells due to the higher cell yield presented. Due to the lack of available references in literature it is not possible to compare the data here obtained with other similar studies. This study represents a first approach in storage of benthic diatom cells for latter application in kits for toxicity measurements based in the response of marine microphytobenthos, susceptible to be used by any person without previous training. Acknowledgements This work has been funded by the Spanish Ministry of Science and Education (project MECASEC: CTM2006-01437/MAR). C.V.M. Araújo is grateful to Brazilian Coordination of Improvement of Personnel of Superior Level (CAPES) by PhD scholarships conceded.
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microplate growth-inhibition assays. Bull. Environ. Contam. Toxicol. 76, 883– 890. Peters, E., 1996. Prolonged darkness and diatom mortality. II. Marine temperate species. J. Ex. Mar. Biol. Ecol. 207, 43–58. Peters, E., Thomas, D.N., 1996. Prolonged darkness and diatom mortality. I. Marine Antarctic species. J. Ex. Mar. Biol. Ecol. 207, 25–41. Poncet, J.-M., Véron, B., 2003. Cryopreservation of the unicellular marine alga, Nannochloropsis oculata. Biotechnol. Lett. 25, 2017–2022. Redekar, P.D., Wagh, A.B., 2000. Cryopreservation studies on the marine diatom Navicula subinflata Grun. Seaweed Res. Utiln. 22, 183–187. Rojicˇková, R., Dvorˇákova, D., Maršálek, B., 1998. The use of miniaturized algal bioassays in comparison to the standard flask assay. Environ. Toxicol. Water Qual. 13, 235–241. Sánchez-Saavedra, M.P., 2006. The effect of cold storage on cell viability and composition of two benthic diatoms. Aquacultur. Eng. 34, 131–136. Satoh, A., Vudikaria, L.Q., Kurano, N., Miyachi, S., 2005. Evaluation of the sensitivity of marine microalgal strains to the heavy metals, Cu, As, Sb, Pb and Cd. Environ. Int. 31, 713–722. Scala, S., Bowler, C., 2001. Molecular insights into the novel aspects of diatom biology. Cell. Mol. Life Sci. 58, 1666–1673. Smayda, T.J., Mitchell-Innes, B., 1974. Dark survival of autotrophic, planktonic marine diatoms. Mar. Biol. 25, 195–202. Stauber, J.L., Florence, T.M., 1985. The influence of iron on copper toxicity to the marine diatom, Nitzschia closterium (Ehrenberg) W. Smith. Aquat. Toxicol. 6, 297–305. Stauber, J.L., Florence, T.M., 1989. The effect of culture medium on metal toxicity to the marine diatom Nitzschia closterium and the freshwater green alga Chlorella pyrenoidosa. Wat. Res. 23 (7), 907–911. Tzovenis, I., Triantaphyllidis, G., Naihong, X., Chatzinikolaou, E., Papadopoulou, K., Xouri, G., Tafas, T., 2004. Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain. Aquaculture 230, 457–473. USEPA, 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. U.S. Environmental Protection Agency, EPA-821-R-02-013. Walker, C.H., Hopkin, S.P., Sibly, R.M., Peakall, D.B., 2001. Principles of Ecotoxicology, second ed. Taylor and Francis, London. Zar, J.H., 1996. Biostatistical Analysis, third ed. Prentice-Hall Int., Upper Saddle River, NJ, USA.
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Effects of cold-dark storage on growth of Cylindrotheca closterium and its sensitivity to copper Cristiano V.M. Araújo *, Fernando R. Diz, Ignacio Moreno-Garrido, Luis M. Lubián, Julián Blasco Instituto 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
Article history: Received 8 January 2008 Received in revised form 26 March 2008 Accepted 10 April 2008 Available online 3 June 2008 Keywords: Diatoms Long-term storage Marine ecotoxicology Microphytobenthos
a b s t r a c t Cylindrotheca closterium cells were maintained at low temperature (4 ± 1 °C) and dark conditions up to 21 weeks to assess the effect on survival and physiological status. From a control culture under standard conditions, three densities were prepared: (A) 2 104, (B) 10 104, and (C) 25 104 cells ml1. Weekly, inoculums of each stored density were exposed to continuous light and at 20 ± 1 °C. Sensitivity to copper for microalgal cultures was evaluated in order to assess possible changes in cells sensitivity due to storage. Concurrently, assays with a control culture were carried out in order to assess the sensitivity of C. closterium to copper and to be able to generate a standard sensitivity control chart with a mean value of EC50-72 h ± 2SD (standard deviation). Density-C presented higher cell yield values, between 40% and 80% relative to control culture. Cell density showed to be important feature that may be taken into account 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 significant difference with regards to control culture. EC50-72 h mean value for the control culture was 29 ± 10 lg Cu l1, while for densities-A, B and C were 22 ± 7; 23 ± 9 and 23 ± 8 lg Cu l1, respectively. In spite of drastic changes in the environmental conditions due to storage, it is concluded that C. closterium cells stored during 5 months remained metabolically active and with no significant change in its sensitivity. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Search for ecotoxicological assays with ecologically relevant species is an important task for environmental researchers. Ecological relevance of a single species is related to the key role in the ecosystem, as well as representativeness of the species in the trophic level (Calow, 1989). Test organisms should be chosen according to the information intended to be obtained (Rojicˇková et al., 1998). Thus, environmental risk analysis of sediments should take into account the benthic community (Moreno-Garrido et al., 2003a), while terrestrial impacts should be assessed through soil organisms (DelValls et al., 2004). On the other hand, when water is the main route of uptake for chemical compounds, then toxicity test with planktonic organisms should be employed (Walker et al., 2001). In spite of the efforts employed to develop algal assay procedures, the majority of the internationally standardized assays are focused on planktonic freshwater algae (ISO, 1989; Environmental Canada, 1992; OECD, 1998; USEPA, 2002; ABNT, 2005), with quite less attention to species of the microphytobenthos (Moreno-Garrido et al., 2003a). Thus, tests should be developed following the specific environmental conditions using representative species of * Corresponding author. Tel.: +34 956 832 612; fax: +34 956 834 701. E-mail address: [email protected] (C.V.M. Araújo). 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.04.022
the ecosystems. Benthic diatoms are a major component of the microphytobenthos in aquatic systems (MacIntyre et al. 1996; de Bower et al., 2003) and can therefore be used as a measure of water quality (Kelly et al., 2001). Diatoms are the main source of food for meiofauna and they are very important in material and energy transfer through the benthic food webs (Miller et al., 1996; Blanchard et al., 2000). Moreover, the excreted polysaccharides play a relevant role in the stability of the marine surface sediment layer (Miller et al., 1996), being key factor of the structure and function of benthic intertidal zones (Scala and Bowler, 2001). Although there is an unquestionable ecological importance of benthic diatoms there are very few studies that have adopted these organisms in toxicity assays. In general, among microphytobenthos, Cylindrotheca closterium is the main species used in ecotoxicology (Stauber and Florence, 1985, 1989; Moreno-Garrido et al., 2003a,b, 2007; Hogan et al., 2005). Microalgae cultures, such as diatoms, are widely used to feed organisms in aquaculture (Cañavate and Lubián, 1995) especially in shellfish hatcheries (Redekar and Wagh, 2000), and they have also been used in ecotoxicological assays (Nörnström, 1990). However, sometimes ecotoxicology laboratories need to maintain a microalgae culture only exclusively to perform a determined assay whose frequency may be spaced in time. The maintenance of living microalgae cultures is an expensive task (Sánchez-Saavedra, 2006)
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mainly if toxicity assays are not routinely carried out. Mitbavkar and Anil (2006) suggested cryopreservation as a very useful technique for the maintenance of phytoplankton culture collections. However, this technique results to be very aggressive at an ultrastructural level during freezing and thawing and once cells survive post-thaw severe damages can occur (Tzovenis et al., 2004). Additionally, the maintenance of cryopreserved algae is expensive and laborious. Nevertheless, many factors may be taken into account for the success of cryopreservation such as state and density of the culture, the type of cryoprotectant and rate of cooling and thawing (Cañavate and Lubián, 1995; Poncet and Véron, 2003). According to Admiraal (1977) benthic diatoms cultures can be stored in dark, at 4 °C and subsequently inoculated in fresh medium in order to preserve cells for long time. Maintenance of cultures in cold and darkness is less expensive and more realistic and viable (Sánchez-Saavedra, 2006). Studies about physiological effects due to cold and darkness exposition in diatoms are performed, in general, with polar species naturally adapted to changes in light and temperature cycle (Gleitz and Thomas, 1992). Many polar diatom species can survive under total darkness for periods up to 10 months (Peters and Thomas, 1996). Studies on cell viability (growth rate) of microalgae (not exclusively polar) exposed to low temperature and darkness can be found in literature (Peters, 1996; Murphy and Cowles, 1997; Chen, 2007). Not only algal survival and growth need to be considered, but also changes in sensitivity to contaminants due to other stressors such as storage conditions, low temperature, darkness and nutrient depletion. The aim of this paper was to assess if the storage of C. closterium cells under low temperature (4 °C) and darkness could affect its growth and verify if it caused variations in its sensitivity in toxicity tests. Information with regards to sensitivity changes after colddark storage for a further ecotoxicological application becomes very useful for sediment toxicity tests including C. closterium cells. This study was undertaken for about 5 months because it was considered that 5 months was enough time in view of a commercial application 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 (formerly Nitzschia closterium (Ehrenberg) W. Smith) was isolated in May 2000 from a salt pond near the Marine Sciences Institute from Andalusia (ICMAN) in Puerto Real (SW of Spain). The strain (C. closterium #010301) is currently included in the Culture Collection of Marine Microalgae of the ICMAN (CCMM-ICMAN, BIOCISE). For routine cultures, seawater is filtered through a GF/C Whatman filter, autoclaved, and later enriched with Guillard’s f/2 medium (Guillard and Ryther, 1962) plus 500 lg l1 SiO2. Artificial substitute ocean water (ASTM, 1975) enriched with SiO2 (500 lg l1), 1 1 NO ), and PO3 ) was used in toxicity assays 3 (150 lg l 4 (10 lg l with copper, in order to avoid the chelator effect of EDTA, included in the f/2 medium. Routine culture and assays were performed at 20 ± 1 °C under continuous white light (35.2 ± 1.1 lmol m2 s1) in a controlled culture chamber (Ibercex). 2.2. Preparation of cell densities and storage at low temperature and darkness conditions Three-day algal cultures (at exponential growth phase) were used to prepare the three densities, named A, B, and C, which contained: 2 104, 10 104 and 25 104 cells ml1, respectively. All densities were prepared with the same culture medium. Two milliliters of aliquots of densities-A, B and C were collected in sterile
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plastic Pasteur pipettes, and then they were hermetically sealed by heating the pipettes’ tips. To avoid heating the algae the pipettes were placed upside-down and the cells were concentrated in the no heated part of the pipette during sealing. All procedures were carried out in laminar flow chamber. Once the pipettes were sealed, they were stored at 4 ± 1 °C and darkness conditions up to 21 weeks. 2.3. Sampling and growth assay (re-activation) Stored pipettes submitted to low temperature and darkness conditions were sampled weekly for each density, in triplicate. The cells were re-activated using culture medium prepared as described in Section 2.1. Initially sampled volume was of 2 ml (referred to one Pasteur pipette) introduced in 48 ml of medium. However, throughout the time assayed the growth velocity was decreased (increase in lag-phase) in all densities studied, thus a bigger volume of cells was needed to be inoculated. Stored inoculum volume plus the culture medium volume resulted to be always of 50 ml. When the inoculated volume was of 2 ml (one Pasteur pipette), cell densities in Erlenmeyer flask were 800, 4000 and 10 000 cells ml1 for densities-A, B and C, respectively. After inoculation four growth periods for cell re-activation were established: 4, 11, 18 and 25 days, this way when there was not enough number of cells (around 1 105 cells ml1) to carry out a toxicity assay in 4 days, the culture was maintained for one more week and successively. At the end of the re-activation assay cell yield (a) values were calculated using the following formula, for the three stored densities studied: a¼
N N0 N0
ð1Þ
where N and N0 are the number of cells ml1 at final time (t, in days) and initial time (t0, in days), respectively. Cell yield values were compared with the control culture – kept under controlled conditions of temperature (20 ± 1 °C) and light (continuous white light 35.2 ± 1.1 lmol m2 s1) – considered as 100% cell yield and were expressed as relative values (%). In order to compare cell yield from inoculated volume, initial cell density and growth days were taken into account. Sensitivity of the re-activated culture was also evaluated with a toxicity assay using copper as reference compound. Samples from week 21 were re-inoculated for one more week and then a toxicity assay was carried out. These samples, named reinoculation, were only followed for densities-B and C because there was no growth in density-A at 21st week. 2.4. Toxicity tests Algal cultures of the three re-activated densities were centrifuged (MES, Micro Centaur, SANYO) for 4 min at 4000 rpm and re-suspended in clean medium (artificial substitute ocean water – ASTM, 1975). This procedure was followed three times before use in the assay in order to guarantee the elimination of f/2 medium and with the aim of avoiding the effect of the chelator EDTA included in the f/2 medium. Initial cellular concentration for growth-inhibition assay of 104 cells ml1 (OECD, 1998) was adopted. Bioassays were carried out in 96-well microplates (Greiner 96-well flat bottom white, 12 8, 400 ll capacity) based on Blaise et al. (1998). Each well was filled with 300 ll of sample. During incubation, microplates were covered with their transparent lids and maintained without agitation. Assays were carried out at five 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 located wells were used. To reduce evaporation, wells along the microplate margin were filled with Milli-Q water (Lukavsky´, 1992). Samples
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were randomly distributed to avoid pseudoreplication effects due to possible spatial differences in illumination and temperature. For all bioassays, the pH drift was less than 0.5 pH unit over 72 h in accordance with established by OECD (1998). Measurement of the initial and final pH values in all samples were registered (Crison, MicropH 2001, electrode Hamilton, Slimtrode). Fluorescence values were compared with the mean value of fluorescence activity belonging to algae with no toxic (control), considered as 100% activity. Fluorometric data were obtained with a spectrophotometer (TECAN Multiplate Reader, GENios) at 0, 24, 48, and 72 h with the following settings: excitation wavelength: 485 nm and emission wavelength: 680 nm. Concurrently, toxicity assays (n = 12, during the weeks 1, 3, 4, 8, 9, 11, 13, 15, 17, 19, 21 and concomitantly the re-inoculation assay) with a control culture (kept under temperature and light standard conditions) were carried out to assess sensitivity to copper, and possible variations of the sensitivity of control cultures.
Table 1 Data of density-A (2 104 cells ml1) including sampling week (storage time), volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) of growth until the carrying out of the assay and final cell density after growth
2.5. Reference substance
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.
Copper was used as copper sulphate pentahydrate (CuSO4 5H2O), reagent grade (99% purity), provided by Merck. All glassware was cleaned with diluted nitric acid and rinsed several times with Milli-Q ultrapure water. Test concentrations were obtained from a 100 mg Cu l1 solution. Copper concentrations used in the assays were 1, 5, 10, 15, 20, 30, 40 and 50 lg l1. 2.6. Statistical analysis For each assay, biomass measurement values such as chlorophyll fluorescence were used to calculate inhibition percentages. Mean inhibition percentages for each concentration were calculated by comparison of area under the control population (100%) and copper treatment curves (Hampel et al., 2001) obtained for each 24 h throughout the 72 h duration of the assay. From these inhibition, percentages EC50-72 h (concentration of copper that caused a 50% reduction in chlorophyll fluorescence) values were established by Trimmed Spearman-Karber – TSK (Hamilton et al., 1977). Inhibition values exceeding 100% were expressed as a 100% and negative inhibition values were expressed as 0%. From the toxicity assays data (EC50-72 h) obtained from the control culture a graph of control culture sensitivity (mean and ±2SD, standard deviation) was generated. Control culture bioassays were used to establish a fitness baseline in terms of sensitivity (EC50) values to calculate a mean and variations for a population considered as reference (Lee, 1980). Thus, by plotting toxicity assay results for each stored cell density in this sensitivity control chart we can evaluate possible changes in cells sensitivity due to colddark storage. Therefore values obtained in the toxicity assays of the stored cells included in the range of ±2SD (warning limits) in the sensitivity control chart were considered acceptable (ABNT, 2005; Blaise and Vasseur, 2005). Additionally, EC50 values of the densities-A, B and C were statistically compared with control culture 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, as well as growth time in re-activation assays, are summarized in Tables 1–3 for densities-A, B and C, respectively. For density-A (Table. 1) the inoculum volume of 2 ml (representing 0.08 104 cells ml1) was very low. Thus, 11 days were necessary to reach a satisfactory cell growth to perform the toxicity
Storage week
Volume inoculated (ml)a
Cell density initial (104 cells ml1)
Growth period (days)
Final cell density (104 cells ml1)
1 2 3 4 5 7 9 11 13 15 17 19 21
2 2 2 2 4 4 4 4 4 4 8 8 8
0.08 0.08 0.08 0.08 0.16 0.16 0.16 0.16 0.16 0.16 0.32 0.32 0.32
11 11 18 18 18 18 18 18 18 25 – 25 –
39.16 (±3.40) 38.55 (±8.29) 126.66 (±9.86) 174.00 (±31.57) 150.75 (±37.12) 120.75b 57.50b 90.58 (±29.95) 46.50b 213.25b –c 161.75 (±16.97) –c
Table 2 Data of density-B (10 104 cells ml1) including sampling week (storage time), volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) of growth until the carrying out of the assay and final cell density after growth Storage week
Volume inoculated (ml)a
Cell density initial (104 cells ml1)
Growth period (days)
Final cell density (104 cells ml1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21
2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 4 8 8
0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.80 0.80 0.80 0.80 1.60 1.60
4 4 11 11 11 11 11 11 11 11 11 11 11 11 11 11 18 18
21.44 (±1.17) 7.88 (±2.60) 151.00 (±12.93) 140.83 (±26.83) 139.00 (±16.71) 124.66 (±33.85) 76.16 (±16.32) 65.91 (±24.98) 63.08 (±11.54) 50.41 (±3.81) 42.41 (±30.88) 38.16 (±6.45) 31.41 (±2.32) 30.33 (±11.00) 30.25 (±20.07) 33.91 (±10.27) 127.00 (±18.77) 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).
assays. Increasing storage time, made necessary a higher inoculum volume, using 4 ml from the 5th up to 15th week. From the 17th week up to 21st 8 ml of inoculum was used. There was no growth for samples referred to weeks 17 and 21. For density-B (Table. 2) after the 2nd week of storage it was possible to obtain cell growth after 4 days in re-activation. From the third up to the 17th week the growth period necessary to perform toxicity assays was of 11 days. 8 ml of inoculum with 18 days of incubation were necessary for the last 2 weeks. In the case of density-C (Table. 3) lower growth time was necessary with regards to the other densities. Samples belonging to the first 7 weeks of storage grew in only 4 days, and up to the 12th week of storage 2 ml was enough for the inoculation. The maximum growth time resulted in 11 days, including the cells stored during weeks 19 and 21, however in this case an inoculum of 8 ml was necessary.
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Cell density initial (104 cells ml1)
Growth period (days)
Final cell density (104 cells ml1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 21
2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 4 8 8
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.00 2.00 2.00 2.00 4.00 4.00
4 4 4 4 4 4 4 11 11 11 11 11 11 11 11 11 11 11
33.16 (±3.75) 27.61 (±3.75) 30.33 (±3.49) 23.54 (±3.86) 15.37 (±3.73) 13.29 (±4.63) 14.95 (±3.79) 142.75 (±24.96) 143.83 (±31.45) 137.91 (±3.35) 106.16 (±20.53) 98.91 (±17.88) 96.25 (±18.22) 104.91 (±19.09) 109.50 (±3.27) 84.58 (±15.36) 167.50 (±2.61) 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).
After storage in cold and darkness conditions, when cells were exposed to optimal conditions of light and temperature they presented a delay in growth, indicating an extended lag phase. The same phenomenon was also found in marine diatom cells exposed to dark and re-illumination (Smayda and Mitchell-Innes, 1974). In experiments with marine Antarctic diatoms, Peters and Thomas (1996) found an increase of lag phase with the increase of storage time in dark. After 21 days of dark storage exponential growth of the diatoms was registered immediately, while in 127 days in dark conditions the lag phase was extended up to 13 days; however diatoms belonging to temperate regions lost growth capability after 49 days in dark (Peters, 1996). Maximum period of darkness survival of Cylindrotheca fusiformis at 2 and 20 °C was of 52 weeks, but other two diatom species, Navicula incerta and Nitzschia angularis, were able to remain viable after 3 years in darkness (Antia, 1976). Benthic diatoms immobilized in alginate beads without liquid medium and at 4 °C in dark after more than 1 year were capable of growing and initiating new culture when seeded to fresh medium and adequate growth conditions; however 4 weeks were necessary to reach similar cell densities from those obtained in a control culture (Chen, 2007). In general, there was an increase in cell yield when re-activation period and inoculum volume increased. Higher values of cell yield were observed for density-C, between 40% and 80% relative to control culture cell yield. For both the same inoculated volume and growth period (re-activation) a decrease in cell yield values in all densities can be noticed with increasing cold-dark storage time. On the other hand, when increasing the growth period (re-activation) but maintaining the same inoculum volume the cell yield increased. These data confirm that the delay in cell growth is due to an increase in storage time. A significant cell yield relative to control culture can be obtained increasing the inoculated volume and/ or growth time (re-activation). Data about cell yield for each density assessed is represented in Fig. 1. According to Dhargalkar (2004), some species of macroalgae acclimated to a given temperature alter the growth rate with increasing exposure time to cold. Jochen (1999) showed that Chrysochromulina hirta (Prymnesiophyte) cells strongly decrease their abundance after 10 days of exposition to darkness; however for
Cell yield (% relative to control)
Volume inoculated (ml)a
After 11 days (2 ml) After 18 days (2 ml) After 18 days (4 ml) After 25 days (4 ml) After 25 days (8 ml)
80
60
40
20
0 1 2 3 4 5
Cell yield (% relative to control)
Storage week
Density-A
175
7
9
11
13
15
19
Weeks of incubation in dark and cold Density-B After 4 days (2 ml) After 11 days (2 ml) After 11 days (4 ml) After 18 days (8 ml)
150
75 50 25 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Density-C
Cell yield (% relative to control)
Table 3 Data of density-C (25 104 cells ml1) including sampling week (storage time), volume of inoculum seeded in Erlenmeyer flask, initial cell density, period (days) of growth until the carrying out of the assay and final cell density after growth
100
17
19
21
After 4 days (2 ml) After 11 days (2ml) After 11 days (4 ml) After 11 days (8 ml)
80
60
40
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
17
19
21
Weeks of incubation in dark and cold 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 ml1); B (10 104 cells ml1); and C (25 104 cells ml1).
Brachiomonas submarina (Chlorophyta) there were no changes in abundance. Furthermore, it was demonstrated that recovery capacity of cells exposed to darkness when re-illuminated, measured as metabolic activity by the use of FDA (fluorescein diacetate) or cell growth, resulted in to be species-specific. For two pennate diatoms species (Prymnesium parvum and Bacteriastrum sp.) the metabolic activity did not change, and almost all cells remained metabolically active. Moreover, these species were capable to sustain a constant cell density after 12 days of darkness and presented a rapid growth after re-illumination (Jochen, 1999). Some
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period at 80 °C was always higher than 16-fold after 72-h (Benhra et al., 1997). A more reliable and certain procedure to measure status or health of the test organism is carrying out a bioassay using a reference toxicant (Lee, 1980). In the present study successive bioassays were carried out with a control culture to establish a standard value of sensitivity using copper as reference substance. Sensitivity data for the three stored densities assayed were plotted under a standard sensitivity chart to verify changes in sensitivity during the 21 weeks of storage (Fig. 2). EC50-72 h mean value for the control culture was 29 ± 10 lg Cu l1. These data were obtained from 12 assays during the weeks 1, 3, 4, 8, 9, 11, 13, 15, 17, 19, 21 and in the reference week to re-inoculation (data plotted in Fig. 2 as diamond). A standard sensitivity chart containing EC50-72 h mean value and lower and upper standard deviation multiplied by two (±2SD) was plotted, and the obtained values in toxicity assays for the stored density cells included in this range of sensitivity control graph were considered acceptable (ABNT, 2005; Blaise and Vasseur, 2005). There was an increase in sensitivity of all cell densities studied; however the results always maintained inside the established range for standard sensitivity (Fig. 2). EC50-72 h mean values for densities-A, B and C were 22 ± 7; 23 ± 9 and 23 ± 8 lg Cu l1, respectively. EC50-72 h values for re-inoculation samples (samples from the 21st week that were re-inoculated for one more week) were 25.55 and 18.79 lg Cu l1 for densities-B and C showing that the sensitivity remained unmodified. EC50-72 h values were independent of the cell density stored. In general, sensitivity to copper of C. closterium measured as EC50-72 h was not significantly modified with regards to control culture (p > 0.05; F3;57 = 1.994) during 21 weeks of cold-dark storage for the three cell densities studied. Cryopreservation (3-month period at 80 °C) significantly increased the sensitivity of Selenastrum capricornutum cells when exposed to cadmium, copper, chromium, and atrazine; moreover sensitivity increased during long-term storage period (Benhra et al., 1997). In this study, Cu toxicity EC50-72 h values were of 28.5 ± 2.8 lg l1 for the algae under standard conditions and of 21.7 ± 0.8 lg l1 for the cryopreserved algae. Sensitivity found for C. closterium was similar to information found for other microalgae species widely used in ecotoxicology. Hogan et al. (2005) found IC50-48 h (inhibition concentration) val-
Density-A Density-B Density-C Control culture
50
+2SD
19 21 R .I.
14 15 17
12 13
9 10 11
-2SD
8
10
3
lower SD
2
20
7
Mean EC50 - Control Culture
6
30
5
upper SD
4
40
1
marine Antarctic diatom species after 3 months of darkness period began to grow at similar rates as those established before the exposition to darkness when returned to light regime (Peters and Thomas, 1996). Thalassiosira weissflogii (marine diatom) was maintained alive during 2 months in dark, and it was capable to exponentially grow when illuminated (Murphy and Cowles, 1997). Probably, the higher number of cells in density-C was suitable for a faster growth, reaching the exponential phase much faster, likely due to a higher number of viable cells. Cell density plays an important role in the viability of microalgae storage under low temperature (Sánchez-Saavedra, 2006). Two problems could arise if higher cell densities are used for storage: oxygen depletion by respiration (once there is no photosynthetic activity) and nutrient depletion. Due to a decrease in metabolism during storage in darkness and cold, there is low oxygen consumption (Antia, 1976), this is why 2 ml of medium in 3 ml pipettes seem to be adequate to maintain enough oxygen quantity for cells during storage. During re-activation of cells exposed to darkness, in the lag phase of acclimation to new conditions, oxygen consumption and production are not appreciable, indicating that the photosynthesis is compensated with respiration (Anderson, 1975a). Thus, the lag period in photosynthesis becomes progressively longer as cold-dark storage time increases (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 should not suffer a nutritional limitation during the storage time. Moreover, this is supported by the higher growth observed in the case of density-C (higher cell density). Heterotrophic activity (capacity to use glucose as nutrient) when exposed to darkness conditions (30 days) was observed in polar pennate diatoms (Palmisano and Sullivan, 1982). Changes in heterotrophic mode of nutrition and reduction in metabolic activity are behaviours expected for microalgae exposed to long periods of darkness, mostly when they do not contain enough nutrient reserves (Jochen, 1999; Sánchez-Saavedra, 2006). Other important factor is the formation of resting spores and cells when microalgae cells are submitted to unfavourable conditions (Anderson, 1975a; Peters and Thomas, 1996) such as low temperatures and darkness. Morphological, physiological and biochemical changes are expected in dark-stressed cells (Smayda and Mitchell-Innes, 1974). During the storage time no spore remains or morphological changes were observed by microscopical analysis. Similar results were obtained by Peters and Thomas (1996) in diatom species exposed to darkness. With regards to resting cells it is difficult to confirm its presence due to morphological similarity to vegetative cells (Anderson, 1975b), once resting cells posses an unmodified frustule (Anderson, 1975a). Palmisano and Sullivan (1982) observed that diatoms size also remained constant after 30 days expose to dark. This was also observed in this work. The diatom Fragilariopsis cylindrus exposed to 1.8 °C and low light intensity showed decrease in photochemical efficiency, increase in cell concentration of chlorophyll a and c, and changes in expression of photosynthesis related genes (Mock and Valentin, 2004). Cell growth observed in sensitivity toxicity assays with copper carried out with re-activated cells and control culture cells was of 22–32 times (after 72 h) with regards to initial concentration (for treatment without copper). It was possible to prove that after acclimation under controlled conditions of light and temperature, cells presented a tendency to keep a similar growth rate to the one obtained for the control culture under optimum conditions, corroborating that a lower growth was resulted in of an acclimation lag to standard culture conditions. Short-term physiological adjustments in response to altered environmental conditions, as shown here, are very common in photosynthetic organisms (Morgan-Kiss et al., 2006). The final cell density of controls in assays with cadmium, copper, chromium, and atrazine to Selenastrum capricornutum cells submitted to cryopreservation over a 3-month
EC50 - 72 h (ug Cu l -1)
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Weeks Fig. 2. Sensitivity checking (EC50-72 h) from 12 assays with a control culture of Cylindrotheca closterium with the ±2SD (standard deviation) respective upper and lower interval values, and results of EC50-72 h copper values from assays carried out with the three densities (A: 2 104 cells ml1; B: 10 104 cells ml1; C: 25 104 cells ml1) exposed to storage in cold and dark after re-activation. RI (Re-inoculation) reefers to sample of week 21 after the second seeding.
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ues for copper of 18 (16–20) lg l1 and 21 (18–24) lg l1 using flasks (50 ml) and minivials (6 ml) respectively. EC50-96 h values obtained by Stauber and Florence (1989) for Nitzschia closterium (=C. closterium) were of 10 lg Cu l1 in assays employing Erlenmeyer flasks and using cell division rate as endpoint. Satoh et al. (2005), in bioassays using a Cylindrotheca species found a median inhibitory concentration value (IC50-72 h) for Cu of 7.7 mg l1, based on chlorophyll fluorescence. However, the high value observed by Satoh et al. (2005) could be due to the presence of high EDTA content in the culture medium. Using cell biomass as endpoint, EC50-72 h values for copper established by Moreno-Garrido et al. (2000) using Erlenmeyer flasks were of 38.3 lg l1 for Chlorella autotrophyca, 46.2 lg l1 for Nannochloris atomus, 35.0 lg l1 for Phaeodactylum tricornutum and of 4.4 lg l1 for Isochrysis aff. galbana. For Rhodomonas salina the EC50-72 h value (cell biomass as endpoint) for copper was of 30 lg l1 (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 l1 for Selenastrum capricornutum and when cell division rate (after 72 h) was used as endpoint values of 7.5 (6.8–8.2) lg Cu l1. In the same study, carried out on Entomoneis cf. punctulata the EC50-24 h value using esterase activity was of 9.1 (7.6–11) lg Cu l1, and the EC50-72 h using cell division rate as endpoint was of 18 (17–20) lg Cu l1. Thus, sensitivity depends on the species and it is referred to the endpoint employed in the bioassays. Although many studies demonstrate that miniaturized bioassays are less sensitive than classical bioassays, they present some advantages such as less occupation of space in laboratory, less time of preparation and evaluation, and require less volume of test sample (Rojicˇková et al., 1998). According to Pavlic´ et al. (2006), microplate algal assays are advantageous because they reduce laboratory necessities of resources. Additionally, the use of the 96-well microplate makes able the possibility of more parallel assays and tests with a higher number of concentrations. Undoubtedly, miniaturized assays with algae are simple, practical and reproducible. Due to simplicity of the miniaturized bioassay and to the reduced interference of the operator with algae its implementation in environmental assessment is recommended. In the present study, miniaturized bioassays employed to assess sensitivity of C. closterium to copper were found as appropriate. 4. Conclusions Results from this work confirm that in spite of drastic changes in the environmental conditions due to storage in dark and low temperature, C. closterium cells remained metabolically active with no statistically significant changes in sensitivity to copper. In spite of stored cells showed an extended lag phase, cell growth was normalized after re-activation under standard conditions of light and temperature. Density-C, containing 25 104 cells ml1, was the most appropriate for storage of C. closterium cells due to the higher cell yield presented. Due to the lack of available references in literature it is not possible to compare the data here obtained with other similar studies. This study represents a first approach in storage of benthic diatom cells for latter application in kits for toxicity measurements based in the response of marine microphytobenthos, susceptible to be used by any person without previous training. Acknowledgements This work has been funded by the Spanish Ministry of Science and Education (project MECASEC: CTM2006-01437/MAR). C.V.M. Araújo is grateful to Brazilian Coordination of Improvement of Personnel of Superior Level (CAPES) by PhD scholarships conceded.
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