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Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae)
Ecotoxicology and Environmental Safety 54 (2003) 290–295
Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae) I. Moreno-Garrido, M. Hampel, L.M. Lubia´n, and J. Blasco Campus Rı´o San Pedro, Instituto de Ciencias Marinas de Andalucı´a (CSIC), s/n. Apdo. Oficial, 11510, Puerto Real, Ca´diz, Spain Received 31 December 2001; accepted 8 July 2002
Abstract A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC50 values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC50 values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg 1 for Cd, 26 mg kg 1 for Cu, and 29 mg kg 1 for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Sediment toxicity tests; Cylindrotheca closterium; cadmium; copper; lead, microphytobenthos
1. Introduction Benthic microalgae (microphytobenthos) are quantitatively very important components of aquatic environments, inhabiting diverse environments from intertidal mudflats to continental shelf systems (Light and Beardall, 2001; Blanchard et al., 2000). In certain situations, biomass of microphytobentos matches or even exceeds bacterial biomass in intertidal sediments (La Rosa et al., 2001). Epipelic species dominate in sheltered habitats, where they are not easily suspended (Absil and Van Scheppingen, 1996). The importance of microphytobenthos for sediment stability has been investigated recently (de Brower et al., 2000), finding twofold sediment stability in spring due to the increase of epipelic microalgae populations; which implies a larger amount of exopolymers released by these cells into the medium. Possibility of predicting critical erosion by measuring chlorophyll a levels (due to microphytoben
Corresponding author. Fax: 34-956-834-701. E-mail address: [email protected] (I. Moreno-Garrido).
tos) in the first millimeters of sediment has also been reported (Riethmu¨ller et al., 2000). Primary productivity and oxygen production at the upper level of intertidal sediments are, in great part, due to microphytobentos. Characterization of sediments by routine bulk chemical analysis provides no useful information about toxicity of sediment-bound contaminants (Munawar and Munawar, 1987). Sediment toxicity tests and bioassays are tools of increasing importance for regulators, scientists, and technologists involved in testing the toxicity and bioavailability of chemical compounds in the sediment on benthic organisms, but little or no effort in standardization has been made on microalgae (SETAC, 1993; Lamberson et al., 1992). In fact, sediment toxicity tests involving microalgae directly exposed to sediments have not been found in the literature: The small number of experiments involving sediment toxicity tests with microalgae were carried out on elutriates (Munawar and Munawar, 1987; Matthiesen et al., 1998; Wong and Couture, 1986). In planktonic environments, microalgae demonstrate higher sensitivity to different toxicants than other organisms
0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00077-5
I. Moreno-Garrido et al. / Ecotoxicology and Environmental Safety 54 (2003) 290–295
for which they should be considered in environmental safety assessment (Radix et al., 2000; Stauber and Florence, 1990; Servos, 1999). Microphytobentos (constituted mainly by diatoms) (Delgado, 1989) is the major food supply for numerous intertidal species. Thus, it is obvious that microflora might play an important role in accumulation of contaminants through the coastal food chains (Absil et al., 1996). Many studies revealed the high accumulation capacity of microalgae for heavy metals (MorenoGarrido, 1997; Ferna´ndez-Leborans and Novillo, 1996; Absil and Van Scheppingen, 1996; Fisher, 1985; Maeda and Sakaguchi, 1990; Ross, 1897). In this work, a novel approach to sediment toxicity tests is developed, taking growth inhibition as the endpoint. Populations of cultured benthic microalgae were directly exposed to laboratory-spiked sediment with three separate heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)]. 2. Materials and methods 2.1. Organisms A strain of Cylindrotheca closterium (Ehremberg) Lewin & Reimann [formerly Nitzschia closterium (Ehremberg) Wm. Smith] was isolated in May 2000 from a salt pond near the Instituto Ciencias Marinas Andalucı´ a (ICMAN) in Puerto Real (SW of Spain). Since its isolation, the strain was included in the Culture Collection of Marine Microalgae of the ICMAN (CCMM-ICMAN, BIOCISE). The strain was cultured in artificial substitute ocean water (ASTM, 1975) medium during months before its employment in the experiments in order to avoid possible adaptation problems that would increase the lag phase in experimental cultures. Organisms used for toxicity tests were always in exponential growth phase (3-day-old cultures).
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2.3. Sediment Natural marine sand obtained from a nonpolluted area (Sancti Petri, SW of Spain) was washed in laboratory with 0.1 N HNO3 and rinsed with ultrapure water (Milli Q) several times. Aliquots of this material were moulted ground in a mill (Fritsch, model Pulverisette 6) for 12 min at 380 cpm. The fraction o63 mm was not significant in the initial sediment. Initial content of silt-sized particles (o63 mm) was not significant in sand sediment. After grinding, more than 95% of the sediment was silt-sized (o63 mm). 2.4. Influence of particle size distribution on growth of the employed microalgae In order to check the influence of particle-size distribution on growth of C. closterium, different percentages of sand (463 mm) were mixed with ground sand (silt-sized, o63 mm) on a final weight of 5 g, which was disposed in sterile borosilicate Erlenmeyer flasks of 125 mL capacity, and topped with artificial cotton (Perlon). The weight of 5 g was chosen because it was the minimum amount of material able to cover the bottom of the flasks. An amount of 50 mL of C. closterium culture (104 cells mL 1), enriched with the nutrients described above, was added to each flask. Experiments were carried out by triplicate. A first wide range (100% sand; 75% sand and 25% sand-sized silt; 50% sand and 50% sand-sized silt; 25% sand and 75% sand-sized silt; and 0% sand and 100% sand-sized silt) and then a fine range (100% sand; 95% sand and 5% sand-sized silt; 90% sand and 10% sand-sized silt; 85% sand and 15% sand-sized silt; 80% sand and 20% sandsized silt; and 75% sand and 25% sand-sized silt) were assayed. Cellular density was measured after 72 h by counting in a Neubauer chamber.
2.2. Media
2.5. Experimental contamination of sediments and toxicity assays
Artificial substitute ocean water (ASTM, 1975) was used in all the experiments. For routine cultures, Guillard’s f =2 medium (Guillard and Ryther, 1962) enriched with 500 mg L 1 SiO2 was used. For toxicity experiments, only SiO2 (50 mg L 1), NO3 (6 mg L 1), and PO34 (6 mg L 1) were added as nutrients. This procedure was developed in order to avoid the effect of the chelator EDTA, included in the Guillard’s f =2 formulation, on the toxic effect of heavy metals. The nutrient concentrations in this light medium represent average natural concentrations of the same substances in actual locations in the Ca´diz Bay (Establier et al., 1990). Satisfactory growth of control cultures of C. closterium during 72 h in this simplified medium was checked before experiments.
Aliquots of 10 g of ground (silt-sized) sand were mixed for each metal with 1 L of ASTM-SOW containing 10 mg L 1 of Cu, Cd, or Pb for 7 days in 2 L spherical borosilicate glass flasks, providing orbital agitation (120 cpm) at 201C711C. It is assumed that after this time adsorption equilibrium is reached between sediment and water (Sa´enz, 1998). After this time, water was eliminated by decantation and the sediment was dried at 601C in an oven and washed with 100 mL of distilled water in order to remove the remaining salt. This process was repeated. This spiked sediment was used in toxicity experiments, and it was performed in triplicate: Assuming an experimental particle distribution of 90% clean sand and 10% ground sand, the latter (0.5 g in each flask) was composed in
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each case with clean and contaminated ground sand, resulting in increasing percentages of contaminated ground sand (for the final 5 g of sediment per flask) of 0, 2%, 4%, 6%, 8%, and 10% (w/w). Afterward 50 mL of C. closterium culture, enriched as described above, was added to each flask. Initial cellular density was 104 cells mL 1 and cellular density was measured every 24 h, for 72 h, by counting in Neubauer chamber. Growth curves were plotted, and the area under them was calculated as described in OECD (1998) protocols, and the percentage of growth inhibition as well as EC50%72 h was calculated following Hampel et al. (2001). Flasks were shaken only before counting (one time each day). 2.6. Measurement of metals For the analysis of heavy metals in the sediment, the total decomposition method proposed by Loring and Rantala (1992) has been followed. In the case of Cd and Pb, the heavy metal concentration in sediment was measured by GFAAS with a Zeeman background correction (Perkin–Elmer 4100 ZL). In the case of Cu, the heavy metal concentration in sediment was measured by flame spectrophotometry (Perkin–Elmer 2100). Summarized operating conditions for the analysis of the heavy metals in digested sediment extracts are shown in Table 1. 2.7. Reagents and glassware Metal concentration in the seawater for sediment spiking processes was obtained by addition of adequate volumes of standard solutions (Merck). All glassware (borosilicate) was cleaned with nitric acid (60%, Panreac) and rinsed with ultrapure water (Milli-Q) several times before use. All salts employed in substitute ocean water as well as silicate, nitrate, and phosphate were of analytical grade.
case, initial cellular concentration (104 cells mL 1) increases more than 40 times in 72 h considered as 100% growth for comparison with growth in assays. When increasing the percentage of silt-sized material in the sediment, growth of populations of C. closterium decreases if compared with optimal growth. Fisher’s LSD procedure was used in order to determinate significant differences between treatments. Population growth of C. closterium cultured in flasks with experimental clean (unpolluted) sediment constituted by 90% sand-sized sediment and 10% silt-sized sediment did not differ from growth of the same species cultured in flasks with experimental clean sediment constituted by 100% sand-sized particles. The results of experiment performed in order to establish the influence of the distribution of sediment size on the growth of C. closterium are shown in Fig. 1. 3.2. Toxicity assays Toxicity assays were designed taking into account the results obtained from the experiment described above. For this reason 90% sand-sized sediment and 10% siltsized sediment were added to all flasks. From this latter 10%, different percentages of silt-sized polluted sediment were mixed with silt-sized nonpolluted sediment. In this case, complete growth curves were taken into account in order to compare growth of populations exposed to sediments with increasing concentrations of Cd, Cu, or Pb. Resulting growth curves are shown in Fig. 2. Comparing obtained growth of microalgal populations, expressed as the area under the growth curve following OECD (1998), exposed to increasing levels of these heavy metals with growth of control populations, curves of the percentage of growth inhibition versus dose of each heavy metal were obtained (Fig. 3). In the case of sediment spiked with Cd or Pb, 100% of
2.8. Statistical analysis of results Analysis of variance (ANOVA) was applied on the obtained results in order to determine significant differences between treatments. The method used was the 95% Fisher’s least significant difference (LSD). All experiments were carried out in triplicate. 3. Results 3.1. Influence of particle size distribution on growth of the cultured microalgae Optimal growth of C. closterium populations occurs at about 100% sand content in the sediment. In this
Fig. 1. Growth percentage of C. closterium populations on sediment with different granulometric composition. Silt-sized percentage of particles in experimental sediment is equal to 100% sand-sized percentage of particles. Crossed circles indicate samples statistically different from 100% sand.
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Table 1 Summarized operating conditions for the analysis of the heavy metals in digested sediment extracts Metal
l (nm)
S (nm)
Method
Flame composition
Tp (1C)
Ta (1C)
Cd Cu Pb
324.8 228.8 213.6
0.7 0.7 0.7
Graphite furnace Flame Graphite furnace
— Air-acetylene —
700 — 1300
1400 — 2500
The flame atomic absorption spectrophotometer was a Perkin–Elmer 2100 model. Graphite furnace atomic absorption spectrophotometer was a Perkin–Elmer 4100 ZL model; l is the incident radiation wavelength, S is the slit setting; and Tp and Ta are the pyrolysis and atomization process temperatures, respectively.
Fig. 2. Growth curves of C. closterium populations exposed to increasing concentrations (expressed as mg kg 1) of metal in sediment. For Cd, (white circles) 0.05; (gray squares) 19.75; (black triangles) 39.45; (white inverted triangles) 59.15; (gray rhombus) 78.85; (dark gray hexagons) 98.50. For Cu, (white circles) 10.00; (gray squares) 25.94; (black triangles) 41.88; (white inverted triangles) 57.82; (gray rhombus) 73.76; (dark gray hexagons) 89.70. For Pb, (white circles) 7.00; (gray squares) 20.54; (black triangles) 34.08; (white inverted triangles) 47.62; (gray rhombus) 61.16; (dark gray hexagons) 74.70.
inhibition is not reached, but near 70% inhibition and around 80% inhibition were obtained for the highest concentration, which made possible the calculation of EC50 values. The Arc tangent sigmoid model, as described in Hampel et al. (2001), was used in order to fit triplicate data of growth inhibition versus heavy metal concentration in experimental sediment. Deviation of EC50 is expressed as the standard error and is supplied by fitting graphic software (Sigma Plot 5.0).
Fig. 3. Growth inhibition percentages of C. closterium populations exposed to increasing concentrations of the selected heavy metals in sediment.
4. Discussion The influence of initial cellular density on sensitivity of toxicity tests with microalgae has been demonstrated (Moreno-Garrido et al., 2000). As consequence, environmentally realistic concentrations should be employed in toxicity testing, which is possible using electronic particle counters (Coulter type) and, overall, flow cytometry, which permits detection and study of microalgal populations at very low cellular concentrations
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(Collier, 2000; Franklin et al., 2001). However, if the water contains suspended matter, as in the case of toxicity tests involving microalgae directly exposed to sediment, a Neubauer chamber is the most suitable procedure in order to study the evolution of an experimental microalgal population by using brightfield microscopy. Due to the characteristics of the Neubauer counting chamber, cellular densities lower than 104 cells mL 1 cannot be accurately detected; initial cellular densities were employed in the performed assays. Data from natural locations were reported showing concentrations of epipelic diatoms ranging from 3 103 to 397 103cells cm 2 in the Ebro river delta (northeast of Spain) (Delgado, 1989). In resuspension laboratory experiments of phytoplankton populations, higher cellular densities (up to 1 106 cells cm 2) have been used (Delgado et al., 1991). Assuming a 3-cm radius in the bottom of the employed conical flasks, volumes of 50 mL with initial cellular density of 104 cells mL 1, and that all cells disposed in the bottom of the flask (on the experimental sediment), an initial surface cellular concentration of 1.8 103 cells cm 2 is achieved, being that this concentration is within the range of the cellular concentrations found in natural locations. The effect of particle-size distribution on the growth of experimental populations of C. closterium could be explained by a slower settling of the fine (silt-sized, o63 mm) sediment than the cells (and thus on the cells). An initial shadowing effect occurs, inhibiting photosynthesis. Additionally, algal cells will spent more energy in digging up, increasing this effort if the layer of silt-sized sediment over the cells is larger. It is remarkable that even in the case of 100% silt-sized sediment cellular population increases 4-fold in 72 h (data not shown), which implies, as shown in Fig. 1, that less than 20% of the optimal growth (20-fold in 72 h) occurs around 100% sand-sized sediment. The effect of the particle-size distribution could be an important handicap to sediment toxicity test with microalgae. The problem should be avoided by performing one single cell count after 72 h of incubation. This will eliminate the daily covering of the cells by the smaller, slow-settling particles of sediment. Other alternative is the use of smaller species (i.e., Navicula spp.) inhabiting the same biotope, but with a minor settling rate than C. closterium. The performed experiments revealed EC50 values for the employed species of 79 mg kg 1 for Cd; 26 mg kg 1 for Cu, and 29 mg kg 1 for Pb. Blasco et al. (2000) found values from 10 to 20 mg kg 1 of Cu in Barbate River salt marshes (a low polluted area), values from 20 to 40 mg kg 1 of Cu in different depths at the bay of Ca´diz, and values from and more than 3 g kg 1 of Cu in the Odiel River salt marshes (a very highly heavy-metalpolluted area). Other data from sediments collected in
different locations at the gulf of Ca´diz (SW of Spain) indicate Cd concentrations ranging from 0.39 to 1.24 mg kg 1, Cu concentrations between 13 and 92 mg kg 1, as well as Pb concentrations ranging from 7 to 65 mg kg 1 in the same areas (DelValls et al., 1998). In experiments assessing toxicity of sediments on larvae of Sparus aurata (Teleostea), no effects were found at values below 0.94 mg kg 1Cd, 49.4 mg kg 1 Cu, and 51.2 mg kg 1 Pb. In general, unicellular organisms are not very suitable organisms to be used in determination of general toxicity of Cd, as the doses, which have shown to be sublethal to these organisms, can be deleterious for others (Moreno-Garrido et al., 1999). Cladocerans are particularly sensitive to this element, showing 48-h LC50 at values as low as 34–60 mg L 1 (Wren et al., 1995). Perhaps bioaccumulation and biomagnification processes are important items to be taken into account in relation to Cd and microalgae. These microorganisms demonstrated to accumulate Cd and other heavy metals (Moreno-Garrido, 1997). As the excretion rate of biologically incorporated Cd is slow, mammals and birds will accumulate high concentrations of this metal in livers and kidneys (target organs). In relation to Cu and Pb, C. closterium was found to be an adequate species for monitorizing contamination effects, as well as other pelagic microalgal species (Maeda and Sakaguchi, 1990; Moreno-Garrido, 1997), but more work should be done in benthic microalgal species. Further efforts must be also done in order to adapt the toxicity test procedure described above to natural samples. Different microalgal species should be assayed to check sensitivity or adaptation capacity to toxicants. 5. Conclusion Cylindrotheca closterium is a suitable organism to be used in toxicity tests in surface sediments, presenting a good growth rate and sensitivity to heavy metals. The present toxicity test is the first experimental design to expose microalgae to contaminated sediment (not to elutriates). The lack of information about this type of bioassay prevents comparisons with other results. References Absil, M.C.P., Berntssen, M., Gerringa, L.J.A., 1996. The influence of sediment, food and organic ligands on the uptake of copper by sediment-dwelling bivalves. Aquat. Toxicol. 34, 13–29. Absil, M.C.P., Van Scheppingen, Y., 1996. Concentrations of selected heavy metals in benthic diatoms and sediment in the Westerchelde estuary. B. Environ. Contam. Tox. 56, 1008–1015. ASTM (American Standard for Testing and Materials). 1975. Standard Specification for Substitute Ocean Water. Designation D 1141-75.
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Maeda, S., Sakaguchi, T., 1990. Accumulation and detoxification of toxic elements by algae. In: Akatsuka, I. (Ed.), Introduction to Applied Phycology. SPB Academic, The Hage, The Netherlands. Matthiesen, P., Bifield, S., Jarrett, F., Kirby, M.F., Law, R.J., McMinn, W.R., Sheahan, D.A., Thain, J.E., Whale, G.F., 1998. An assessment of sediment toxicity in the River Tyne estuary, UK by means of bioassays. Mar. Environ. Res. 45 (1), 1–15. Moreno-Garrido, I., 1997. Toxicidad y Acumulacio´n de Metales Pesados en Microalgas Marinas Usadas en Acuicultura, Ph. D. thesis. University of Ca´diz, Spain. Moreno-Garrido, I., Lubia´n, L.M., Soares, A.M.V.M., 1999b. Growth differences in cultured populations of Brachionus plicatilis Mu¨ller caused by heavy metal stress as function of microalgal diet. Environ. Contam. Toxicol. B 63, 392–398. Moreno-Garrido, I., Lubia´n, L.M., Soares, A.M.V.M., 2000. Influence of cellular density on determination of EC50 in microalgal growth inhibition tests. Ecotox. Environ. Safe. 47, 112–116. Munawar, M., Munawar, I.F., 1987. Phytoplankton bioassays for evaluating toxicity of in situ sediment contaminants. Hydrobiologia 149, 87–105. OECD (Organization for the Economic Cooperation and Development), 1998. OECD Guideline for Testing of Chemicals: Alga, Growth Inhibition Test, OECD Publications, Paris. Radix, P., Le´onard, M., Papantoniou, C., Roman, G., Saouter, E., Gallotti-Schmitt, S., Thie¨baud, H., Vasseur, P., 2000. Comparison of four chronic toxicity tests using algae, bacteria and invertebrates with sixteen chemicals. Ecotox. Environ. Safe. 47, 186–194. Riethmu¨ller, R., Heineke, M., Ku¨hl, H., Keuker-Ru¨diger, R., 2000. Chlorophyll a concentration as an index of sediment surface stabilisation by microphytobentos? Cont. Shelf Res. 20, 1351–1372. Ross, I.S., 1987. The use of microorganisms for the removal and recovery of heavy metals from aqueous effluents. Int. Ind. Biotechnol. 99 (7), 6–7. Sa´enz, V., 1998. Distribucio´n Espacial y Especiacio´n de Metales Pesados en Sedimentos de Tres sistemas Litorales del Golfo de Ca´diz, Ph. D. thesis, University of Ca´diz, Spain. Servos, M.R., 1999. Review of the aquatic toxicology, estrogenic responses and bioaccumulation of alkylphenols and alkylphenol polyethoxylates. Water Qual. Res. J. Can. 34 (1), 123–177. SETAC (Society of Environmental Toxicology and Chemistry— Europe). 1993. Guidance document on sediment toxicity tests and bioassays for freshwater and marine environments. Presented at the Workshop on Sediment Toxicity Assessment, November 1993, Renesse, The Netherlands. Stauber, J.L., Florence, T.M., 1990. Mechanism of toxicity of zinc to the marine diatom Nitzschia closterium. Mar. Biol. 105, 519–524. Wong, P.T.S., Couture, P., 1986. Toxicity screening using phytoplancton. In: Toxicity Testing Using Microorganisms. CRC Press, Boca Rato´n, FL. Wren, C.D., Harris, S., Harttrup, N., 1995. Ecotoxicology of mercury and cadmium. In: Hoffman, D.J., Rattner, B.A., Burton, G.A. Jr., Cairns, J. Jr. (Eds.), Handbook of Ecotoxicology. Lewis, Boca Rato´n, FL (Chapter 17).
Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae) I. Moreno-Garrido, M. Hampel, L.M. Lubia´n, and J. Blasco Campus Rı´o San Pedro, Instituto de Ciencias Marinas de Andalucı´a (CSIC), s/n. Apdo. Oficial, 11510, Puerto Real, Ca´diz, Spain Received 31 December 2001; accepted 8 July 2002
Abstract A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC50 values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC50 values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg 1 for Cd, 26 mg kg 1 for Cu, and 29 mg kg 1 for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Sediment toxicity tests; Cylindrotheca closterium; cadmium; copper; lead, microphytobenthos
1. Introduction Benthic microalgae (microphytobenthos) are quantitatively very important components of aquatic environments, inhabiting diverse environments from intertidal mudflats to continental shelf systems (Light and Beardall, 2001; Blanchard et al., 2000). In certain situations, biomass of microphytobentos matches or even exceeds bacterial biomass in intertidal sediments (La Rosa et al., 2001). Epipelic species dominate in sheltered habitats, where they are not easily suspended (Absil and Van Scheppingen, 1996). The importance of microphytobenthos for sediment stability has been investigated recently (de Brower et al., 2000), finding twofold sediment stability in spring due to the increase of epipelic microalgae populations; which implies a larger amount of exopolymers released by these cells into the medium. Possibility of predicting critical erosion by measuring chlorophyll a levels (due to microphytoben
Corresponding author. Fax: 34-956-834-701. E-mail address: [email protected] (I. Moreno-Garrido).
tos) in the first millimeters of sediment has also been reported (Riethmu¨ller et al., 2000). Primary productivity and oxygen production at the upper level of intertidal sediments are, in great part, due to microphytobentos. Characterization of sediments by routine bulk chemical analysis provides no useful information about toxicity of sediment-bound contaminants (Munawar and Munawar, 1987). Sediment toxicity tests and bioassays are tools of increasing importance for regulators, scientists, and technologists involved in testing the toxicity and bioavailability of chemical compounds in the sediment on benthic organisms, but little or no effort in standardization has been made on microalgae (SETAC, 1993; Lamberson et al., 1992). In fact, sediment toxicity tests involving microalgae directly exposed to sediments have not been found in the literature: The small number of experiments involving sediment toxicity tests with microalgae were carried out on elutriates (Munawar and Munawar, 1987; Matthiesen et al., 1998; Wong and Couture, 1986). In planktonic environments, microalgae demonstrate higher sensitivity to different toxicants than other organisms
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I. Moreno-Garrido et al. / Ecotoxicology and Environmental Safety 54 (2003) 290–295
for which they should be considered in environmental safety assessment (Radix et al., 2000; Stauber and Florence, 1990; Servos, 1999). Microphytobentos (constituted mainly by diatoms) (Delgado, 1989) is the major food supply for numerous intertidal species. Thus, it is obvious that microflora might play an important role in accumulation of contaminants through the coastal food chains (Absil et al., 1996). Many studies revealed the high accumulation capacity of microalgae for heavy metals (MorenoGarrido, 1997; Ferna´ndez-Leborans and Novillo, 1996; Absil and Van Scheppingen, 1996; Fisher, 1985; Maeda and Sakaguchi, 1990; Ross, 1897). In this work, a novel approach to sediment toxicity tests is developed, taking growth inhibition as the endpoint. Populations of cultured benthic microalgae were directly exposed to laboratory-spiked sediment with three separate heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)]. 2. Materials and methods 2.1. Organisms A strain of Cylindrotheca closterium (Ehremberg) Lewin & Reimann [formerly Nitzschia closterium (Ehremberg) Wm. Smith] was isolated in May 2000 from a salt pond near the Instituto Ciencias Marinas Andalucı´ a (ICMAN) in Puerto Real (SW of Spain). Since its isolation, the strain was included in the Culture Collection of Marine Microalgae of the ICMAN (CCMM-ICMAN, BIOCISE). The strain was cultured in artificial substitute ocean water (ASTM, 1975) medium during months before its employment in the experiments in order to avoid possible adaptation problems that would increase the lag phase in experimental cultures. Organisms used for toxicity tests were always in exponential growth phase (3-day-old cultures).
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2.3. Sediment Natural marine sand obtained from a nonpolluted area (Sancti Petri, SW of Spain) was washed in laboratory with 0.1 N HNO3 and rinsed with ultrapure water (Milli Q) several times. Aliquots of this material were moulted ground in a mill (Fritsch, model Pulverisette 6) for 12 min at 380 cpm. The fraction o63 mm was not significant in the initial sediment. Initial content of silt-sized particles (o63 mm) was not significant in sand sediment. After grinding, more than 95% of the sediment was silt-sized (o63 mm). 2.4. Influence of particle size distribution on growth of the employed microalgae In order to check the influence of particle-size distribution on growth of C. closterium, different percentages of sand (463 mm) were mixed with ground sand (silt-sized, o63 mm) on a final weight of 5 g, which was disposed in sterile borosilicate Erlenmeyer flasks of 125 mL capacity, and topped with artificial cotton (Perlon). The weight of 5 g was chosen because it was the minimum amount of material able to cover the bottom of the flasks. An amount of 50 mL of C. closterium culture (104 cells mL 1), enriched with the nutrients described above, was added to each flask. Experiments were carried out by triplicate. A first wide range (100% sand; 75% sand and 25% sand-sized silt; 50% sand and 50% sand-sized silt; 25% sand and 75% sand-sized silt; and 0% sand and 100% sand-sized silt) and then a fine range (100% sand; 95% sand and 5% sand-sized silt; 90% sand and 10% sand-sized silt; 85% sand and 15% sand-sized silt; 80% sand and 20% sandsized silt; and 75% sand and 25% sand-sized silt) were assayed. Cellular density was measured after 72 h by counting in a Neubauer chamber.
2.2. Media
2.5. Experimental contamination of sediments and toxicity assays
Artificial substitute ocean water (ASTM, 1975) was used in all the experiments. For routine cultures, Guillard’s f =2 medium (Guillard and Ryther, 1962) enriched with 500 mg L 1 SiO2 was used. For toxicity experiments, only SiO2 (50 mg L 1), NO3 (6 mg L 1), and PO34 (6 mg L 1) were added as nutrients. This procedure was developed in order to avoid the effect of the chelator EDTA, included in the Guillard’s f =2 formulation, on the toxic effect of heavy metals. The nutrient concentrations in this light medium represent average natural concentrations of the same substances in actual locations in the Ca´diz Bay (Establier et al., 1990). Satisfactory growth of control cultures of C. closterium during 72 h in this simplified medium was checked before experiments.
Aliquots of 10 g of ground (silt-sized) sand were mixed for each metal with 1 L of ASTM-SOW containing 10 mg L 1 of Cu, Cd, or Pb for 7 days in 2 L spherical borosilicate glass flasks, providing orbital agitation (120 cpm) at 201C711C. It is assumed that after this time adsorption equilibrium is reached between sediment and water (Sa´enz, 1998). After this time, water was eliminated by decantation and the sediment was dried at 601C in an oven and washed with 100 mL of distilled water in order to remove the remaining salt. This process was repeated. This spiked sediment was used in toxicity experiments, and it was performed in triplicate: Assuming an experimental particle distribution of 90% clean sand and 10% ground sand, the latter (0.5 g in each flask) was composed in
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each case with clean and contaminated ground sand, resulting in increasing percentages of contaminated ground sand (for the final 5 g of sediment per flask) of 0, 2%, 4%, 6%, 8%, and 10% (w/w). Afterward 50 mL of C. closterium culture, enriched as described above, was added to each flask. Initial cellular density was 104 cells mL 1 and cellular density was measured every 24 h, for 72 h, by counting in Neubauer chamber. Growth curves were plotted, and the area under them was calculated as described in OECD (1998) protocols, and the percentage of growth inhibition as well as EC50%72 h was calculated following Hampel et al. (2001). Flasks were shaken only before counting (one time each day). 2.6. Measurement of metals For the analysis of heavy metals in the sediment, the total decomposition method proposed by Loring and Rantala (1992) has been followed. In the case of Cd and Pb, the heavy metal concentration in sediment was measured by GFAAS with a Zeeman background correction (Perkin–Elmer 4100 ZL). In the case of Cu, the heavy metal concentration in sediment was measured by flame spectrophotometry (Perkin–Elmer 2100). Summarized operating conditions for the analysis of the heavy metals in digested sediment extracts are shown in Table 1. 2.7. Reagents and glassware Metal concentration in the seawater for sediment spiking processes was obtained by addition of adequate volumes of standard solutions (Merck). All glassware (borosilicate) was cleaned with nitric acid (60%, Panreac) and rinsed with ultrapure water (Milli-Q) several times before use. All salts employed in substitute ocean water as well as silicate, nitrate, and phosphate were of analytical grade.
case, initial cellular concentration (104 cells mL 1) increases more than 40 times in 72 h considered as 100% growth for comparison with growth in assays. When increasing the percentage of silt-sized material in the sediment, growth of populations of C. closterium decreases if compared with optimal growth. Fisher’s LSD procedure was used in order to determinate significant differences between treatments. Population growth of C. closterium cultured in flasks with experimental clean (unpolluted) sediment constituted by 90% sand-sized sediment and 10% silt-sized sediment did not differ from growth of the same species cultured in flasks with experimental clean sediment constituted by 100% sand-sized particles. The results of experiment performed in order to establish the influence of the distribution of sediment size on the growth of C. closterium are shown in Fig. 1. 3.2. Toxicity assays Toxicity assays were designed taking into account the results obtained from the experiment described above. For this reason 90% sand-sized sediment and 10% siltsized sediment were added to all flasks. From this latter 10%, different percentages of silt-sized polluted sediment were mixed with silt-sized nonpolluted sediment. In this case, complete growth curves were taken into account in order to compare growth of populations exposed to sediments with increasing concentrations of Cd, Cu, or Pb. Resulting growth curves are shown in Fig. 2. Comparing obtained growth of microalgal populations, expressed as the area under the growth curve following OECD (1998), exposed to increasing levels of these heavy metals with growth of control populations, curves of the percentage of growth inhibition versus dose of each heavy metal were obtained (Fig. 3). In the case of sediment spiked with Cd or Pb, 100% of
2.8. Statistical analysis of results Analysis of variance (ANOVA) was applied on the obtained results in order to determine significant differences between treatments. The method used was the 95% Fisher’s least significant difference (LSD). All experiments were carried out in triplicate. 3. Results 3.1. Influence of particle size distribution on growth of the cultured microalgae Optimal growth of C. closterium populations occurs at about 100% sand content in the sediment. In this
Fig. 1. Growth percentage of C. closterium populations on sediment with different granulometric composition. Silt-sized percentage of particles in experimental sediment is equal to 100% sand-sized percentage of particles. Crossed circles indicate samples statistically different from 100% sand.
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Table 1 Summarized operating conditions for the analysis of the heavy metals in digested sediment extracts Metal
l (nm)
S (nm)
Method
Flame composition
Tp (1C)
Ta (1C)
Cd Cu Pb
324.8 228.8 213.6
0.7 0.7 0.7
Graphite furnace Flame Graphite furnace
— Air-acetylene —
700 — 1300
1400 — 2500
The flame atomic absorption spectrophotometer was a Perkin–Elmer 2100 model. Graphite furnace atomic absorption spectrophotometer was a Perkin–Elmer 4100 ZL model; l is the incident radiation wavelength, S is the slit setting; and Tp and Ta are the pyrolysis and atomization process temperatures, respectively.
Fig. 2. Growth curves of C. closterium populations exposed to increasing concentrations (expressed as mg kg 1) of metal in sediment. For Cd, (white circles) 0.05; (gray squares) 19.75; (black triangles) 39.45; (white inverted triangles) 59.15; (gray rhombus) 78.85; (dark gray hexagons) 98.50. For Cu, (white circles) 10.00; (gray squares) 25.94; (black triangles) 41.88; (white inverted triangles) 57.82; (gray rhombus) 73.76; (dark gray hexagons) 89.70. For Pb, (white circles) 7.00; (gray squares) 20.54; (black triangles) 34.08; (white inverted triangles) 47.62; (gray rhombus) 61.16; (dark gray hexagons) 74.70.
inhibition is not reached, but near 70% inhibition and around 80% inhibition were obtained for the highest concentration, which made possible the calculation of EC50 values. The Arc tangent sigmoid model, as described in Hampel et al. (2001), was used in order to fit triplicate data of growth inhibition versus heavy metal concentration in experimental sediment. Deviation of EC50 is expressed as the standard error and is supplied by fitting graphic software (Sigma Plot 5.0).
Fig. 3. Growth inhibition percentages of C. closterium populations exposed to increasing concentrations of the selected heavy metals in sediment.
4. Discussion The influence of initial cellular density on sensitivity of toxicity tests with microalgae has been demonstrated (Moreno-Garrido et al., 2000). As consequence, environmentally realistic concentrations should be employed in toxicity testing, which is possible using electronic particle counters (Coulter type) and, overall, flow cytometry, which permits detection and study of microalgal populations at very low cellular concentrations
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(Collier, 2000; Franklin et al., 2001). However, if the water contains suspended matter, as in the case of toxicity tests involving microalgae directly exposed to sediment, a Neubauer chamber is the most suitable procedure in order to study the evolution of an experimental microalgal population by using brightfield microscopy. Due to the characteristics of the Neubauer counting chamber, cellular densities lower than 104 cells mL 1 cannot be accurately detected; initial cellular densities were employed in the performed assays. Data from natural locations were reported showing concentrations of epipelic diatoms ranging from 3 103 to 397 103cells cm 2 in the Ebro river delta (northeast of Spain) (Delgado, 1989). In resuspension laboratory experiments of phytoplankton populations, higher cellular densities (up to 1 106 cells cm 2) have been used (Delgado et al., 1991). Assuming a 3-cm radius in the bottom of the employed conical flasks, volumes of 50 mL with initial cellular density of 104 cells mL 1, and that all cells disposed in the bottom of the flask (on the experimental sediment), an initial surface cellular concentration of 1.8 103 cells cm 2 is achieved, being that this concentration is within the range of the cellular concentrations found in natural locations. The effect of particle-size distribution on the growth of experimental populations of C. closterium could be explained by a slower settling of the fine (silt-sized, o63 mm) sediment than the cells (and thus on the cells). An initial shadowing effect occurs, inhibiting photosynthesis. Additionally, algal cells will spent more energy in digging up, increasing this effort if the layer of silt-sized sediment over the cells is larger. It is remarkable that even in the case of 100% silt-sized sediment cellular population increases 4-fold in 72 h (data not shown), which implies, as shown in Fig. 1, that less than 20% of the optimal growth (20-fold in 72 h) occurs around 100% sand-sized sediment. The effect of the particle-size distribution could be an important handicap to sediment toxicity test with microalgae. The problem should be avoided by performing one single cell count after 72 h of incubation. This will eliminate the daily covering of the cells by the smaller, slow-settling particles of sediment. Other alternative is the use of smaller species (i.e., Navicula spp.) inhabiting the same biotope, but with a minor settling rate than C. closterium. The performed experiments revealed EC50 values for the employed species of 79 mg kg 1 for Cd; 26 mg kg 1 for Cu, and 29 mg kg 1 for Pb. Blasco et al. (2000) found values from 10 to 20 mg kg 1 of Cu in Barbate River salt marshes (a low polluted area), values from 20 to 40 mg kg 1 of Cu in different depths at the bay of Ca´diz, and values from and more than 3 g kg 1 of Cu in the Odiel River salt marshes (a very highly heavy-metalpolluted area). Other data from sediments collected in
different locations at the gulf of Ca´diz (SW of Spain) indicate Cd concentrations ranging from 0.39 to 1.24 mg kg 1, Cu concentrations between 13 and 92 mg kg 1, as well as Pb concentrations ranging from 7 to 65 mg kg 1 in the same areas (DelValls et al., 1998). In experiments assessing toxicity of sediments on larvae of Sparus aurata (Teleostea), no effects were found at values below 0.94 mg kg 1Cd, 49.4 mg kg 1 Cu, and 51.2 mg kg 1 Pb. In general, unicellular organisms are not very suitable organisms to be used in determination of general toxicity of Cd, as the doses, which have shown to be sublethal to these organisms, can be deleterious for others (Moreno-Garrido et al., 1999). Cladocerans are particularly sensitive to this element, showing 48-h LC50 at values as low as 34–60 mg L 1 (Wren et al., 1995). Perhaps bioaccumulation and biomagnification processes are important items to be taken into account in relation to Cd and microalgae. These microorganisms demonstrated to accumulate Cd and other heavy metals (Moreno-Garrido, 1997). As the excretion rate of biologically incorporated Cd is slow, mammals and birds will accumulate high concentrations of this metal in livers and kidneys (target organs). In relation to Cu and Pb, C. closterium was found to be an adequate species for monitorizing contamination effects, as well as other pelagic microalgal species (Maeda and Sakaguchi, 1990; Moreno-Garrido, 1997), but more work should be done in benthic microalgal species. Further efforts must be also done in order to adapt the toxicity test procedure described above to natural samples. Different microalgal species should be assayed to check sensitivity or adaptation capacity to toxicants. 5. Conclusion Cylindrotheca closterium is a suitable organism to be used in toxicity tests in surface sediments, presenting a good growth rate and sensitivity to heavy metals. The present toxicity test is the first experimental design to expose microalgae to contaminated sediment (not to elutriates). The lack of information about this type of bioassay prevents comparisons with other results. References Absil, M.C.P., Berntssen, M., Gerringa, L.J.A., 1996. The influence of sediment, food and organic ligands on the uptake of copper by sediment-dwelling bivalves. Aquat. Toxicol. 34, 13–29. Absil, M.C.P., Van Scheppingen, Y., 1996. Concentrations of selected heavy metals in benthic diatoms and sediment in the Westerchelde estuary. B. Environ. Contam. Tox. 56, 1008–1015. ASTM (American Standard for Testing and Materials). 1975. Standard Specification for Substitute Ocean Water. Designation D 1141-75.
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