88 Fluoride Vol. 37 No. 2 88–95 2004 Research Report

FLUORIDE AND ALUMINIUM TOLERANCE IN PLANKTONIC MICROALGAE Gamila Alia Cairo, Egypt

SUMMARY: Interactive effects of fluoride (F) and aluminium (Al) toward four species of microalgae at pH 7.3, 6.0, and 4.5, along with the accumulation of these elements by the algal cells, were the focus of this investigation. The species studied were Scenedesmus obliquus, Microcystis aeruginosa, Anabaena sphaerica, and Nitzschia linearis. At a concentration of 4 mg F/L, essentially no toxic effects were observed in any of the algal species at the three different pH values. The toxicity of Al, however, increased with decreasing pH. Interestingly, the combination of F + Al significantly ameliorated the toxic effect of Al at pH 6.0 toward Scenedesmus and Microcystis. With some dependence on pH, accumulation of Al was greater in Microcystis and Nitzschia, whereas accumulation of F was greater in Scenedesmus and about the same in Anabaena. Keywords: Aluminum accumulation; Fluoride accumulation; Microalgae; Phytoplankton; Phytoplankton tolerance to Al and F. INTRODUCTION

The continuing growth in global population has greatly increased the demand for freshwater. Human activities alter water quality not only by changing hydrologic pathways but also by the addition of substances and wastes to the landscape.1 Aluminium is an abundant element in the earth’s crust, constituting about 8% of the soil minerals. Aluminium is present in many manufactured foods and medicines and is added to drinking water to remove turbidity. It has been proposed that aluminium is a contributing factor to several neurodegenerative disorders such as Alzheimer’s disease. However, this view remains controversial primarily because of the unusual properties of aluminium and a lack of information concerning its cellular sites of action.2 Fluorine (F) occurs in natural waters as the fluoride ion (F–), undissociated hydrofluoric acid (HF), and as various complexes. It is also known to inhibit a large number of biological processes including photosynthesis, respiration, protein synthesis, and enzyme activities of higher plants,3 green algae, cyanobacteria, and bacteria,4-6 at levels encountered from industrial fluoride pollution, but generally not at levels encountered in municipal fluoridation or in seawater.7 aFor

Correspondence: Dr Gamila Ali, Water Pollution Research Department, National Research Center, Dokki, Cairo, Egypt, PC 12311. E-mail: [email protected] or [email protected]

Fluoride and aluminium tolerance in microalgae 89

Since phytoplanktons are primary producers of organic compounds in aquatic systems, there is a need to determine the interactive effects of Al and F on different species of microalgae. Although some reports have indicated that fluoride can ameliorate aluminium toxicity by the formation of fluoridealuminium complexes, other studies suggest that fluoride can act synergistically to enhance aluminium toxicity.8,9 The purpose of this research was to investigate the interactive effect of Al and F toward several isolated species of microalgae at different pH values and to measure the accumulation of these elements in them. MATERIALS AND METHODS

Organisms and growth conditions: Four microalgal strains were isolated from the phytoplankton community of the River Nile: Scendesmus obliquus (green algae), Microcystis aeruginosa (colonial, blue-green algae), Anabaena sphaerica (filamentous and heterocysts blue-green algae), and Nitzschia linearis (diatoms). These organisms were grown in BG1110 containing (g/L): NaNO3 (1.5); K2HPO4 (0.04); MgSO4·7H2O (0.075); CaCl2 (0.036); citric acid (0.006); Na2CO3 (0.02); Na2EDTA (0.001); and ferric ammonium citrate (0.006); plus minor elements (µg/L) added to 1.0 L of the BG11 solution as 1 mL containing (g/L): H3BO2 (2.86); MnCl2·4H2O (1.81); ZnSO4·7H2O (0.222); Na2MoO4·2H2O (0.39); CuSO4·5H2O (0.097); and Co(NO3)2·6H2O (0.0494). Some modifications were made according to the type of species, e.g., in the case of Scenedesmus obliquus, the sodium nitrate content was reduced to 1/5 the above amount, while in the case of Anabaena sphaerica it was omitted from the media. Also, in the case of Nitzschia linearis, 0.05 mg/L of Na2SiO3·5H2O was included. All strains were grown at optimum temperature (24 ± 2 ºC) with continuous illumination by white fluorescent lamps of ~2500 Lux. The stock cultures were continuously recultivated under optimum growth conditions and introduced into the experimental systems at logarithmic phase. Initial equivalent chlorophyll “a” concentrations for all experiments ranged from 25-30 µg/ L. Bioassay flasks (conical 1-L capacity containing 500 mL of algal media) were incubated under optimum growth conditions. Flasks (in triplicate series) were shaken once each day to prevent clumping of the cells. Each experiment was run for 10 to 14 days to allow good growth without causing nutrient shortages. Growth in the cultures was determined by daily measurements of equivalent chlorophyll “a” content.11 At the end of each experiment, the algal mass was collected to determine fluoride and aluminium accumulation. The results presented are the averages of three experiments.

Stock solutions of sodium fluoride (NaF) and aluminium sulphate (Al2(SO4)3 ·16H2O) (ANALAR) were prepared before use to supplement

Fluoride 37 (2) 2004

90 Ali

the culture media. Final fluoride and aluminium dose levels of 4 mg/L, which were found to be non-inhibitory at pH 7.3 in the BG11 media, were achieved by using stock solutions containing 1 mg F or Al/mL. All experiments were performed in triplicate at pH 7.3, 6.0, and 4.5 and repeated at least twice to verify reproducibility. Fluoride and aluminium accumulation: To determine the amount of fluoride and aluminium accumulated by the cells, the algal mass at the end of each experiment was collected by centrifugation and washed three times with distilled water. The algal cells were dried at 105 ºC (5 g after dryness) and then digested for Al and F accumulation according to APHA.12 The SPADNS colorimetric method was used for F determination, and the Eriochrome Cyanine R method was used for Al determination12 RESULTS

Response of the blue-green algae species to fluoride and aluminium: Figure 1 shows that the growth of Microcystis aeruginosa was not appreciably affected by the 4 mg/L dose of F at different pHs. However, the Al toxicity to Microcystis was greatly increased with decreasing pH of the media, the percentage reduction in algal biomass being 49, 98, and 98% at pH 7.3, 6.0, and 4.5, respectively. At pH 6.0, but not at pH 4.5, F at 4 mg/L ameliorated the toxicity effect of Al to Microcystis.

Figure 1. Response of blue-green alga (Microcystis aeruginosa) to fluoride and aluminium at different pHs.

Fluoride 37 (2) 2004

Fluoride and aluminium tolerance in microalgae 91

In the case of the blue-green alga Anabaena sphaerica, the most conspicuous result was that decreasing pH has an injurious effect on the alga growth (Figure 2). The maximum algal biomass in the control culture at pH 7.3 was 820 µg/L of chlorophyll “a” equivalent, while at pH 4.5 it was only 64 µg/L Chlorophyll “a” equivalent in the control culture at pH 4.5. The ameliorating effect of F toward Al toward the algal growth was observed at pH 7.3 and 6.0, with only a slight difference between the effect of Al alone or in combinationwith F. .

Figure 2. Response of blue-green alga (Anabaena sphaerica) to fluoride and aluminium at different pHs.

Response of the green alga to fluoride and aluminium: The response of Scenedesmus obliquus towards fluoride and aluminium alone or in combination is shown in Figure 3. On treatment with 4 mg F/L, slight inhibition can be detected at decreasing pH. The toxicity of Al, however, increased greatly with decreasing pH. In addition, the ameliorating effect of F towards Al toxicity under all three pH conditions was readily apparent. Response of the diatoms alga to fluoride and aluminium: As in the Ana-

baena sphaerica culture (Figure 2), an acidity of pH 4.5 has a pronounced injurious effect on the growth of Nitzschia linearis in the control culture (Figure 4). At the same time, the presence of added fluoride has essentially no ameliorating effect on the toxicity of Al to this alga. Fluoride and aluminium accumulation: As seen in Figure 5, the algal species studied here differ greatly in their ability to take up and accumulate fluoride and aluminium. The blue-green species (Microcystis aeruginosa and Anabaena sphaerica) tended to accumulate more F in their cells than the other Fluoride 37 (2) 2004

92 Ali

two species. In the same time, Nitzschia linearis (diatoms) has the ability to accumulate Al more than the other species of algae.

Figure 3. Response of green alga (Scenedesmus obliquus) to fluoride and aluminium at different pHs.

Figure 4. Response of diatoms (Nitzschia linearis) to fluoride and aluminium at different pHs.

Fluoride 37 (2) 2004

mg / kg

mg / kg

4

0

0.5

1

1.5

2

2.5

3

3.5

0

0.5

1

1.5

2

2.5

3

3.5

F

F

pH 6.0

pH 7.3

Al

pH 6.0

Al

F

F

pH 4.5

Al

F+Al

F+Al

pH 4.5

Al F F+Al

F

F+Al

pH 7.3

Al

pH 6.0

F+Al

F+Al

pH 6.0

Al

Anabaena sphaerica F F Al

pH 7.3

Al

Nitzschia linearis

Figure 5. Fluoride and aluminium accumulation inside the algal cells

F+Al

F+Al

F+Al

Scenedesmus obliquus F F

pH 7.3

Al F+Al

Al

Microcystis aeruginosa

F

F+Al

F

pH 4.5

Al

pH 4.5

Al

F+Al

Fluoride and aluminium tolerance in microalgae 93

Fluoride 37 (2) 2004

94 Ali

DISCUSSION

The continuing rapid growth in the requirement for potable water, has increased the importance for studying and understanding the interactions between chemical substances used in the treatment of drinking water. In the present study, the most important results are that species belonging to bluegreen algae differ between each other in their tolerance to tested dose levels of F and Al at various pHs. The results also showed that the green algae can tolerate and are fairly resistant to the toxicity of F and Al at different pHs with the percentage reduction in algal biomass increasing with increasing acidity of the algal culture. This dependence of toxicity on pH may be a consequence of pH-transformed chemical forms of fluoride and aluminium in solution.13-17 In addition, it has been hypothesized that F transport through biological membranes occurs primarily through non-ionic diffusion of HF which increases at the acidic pH. Thus, it is the HF concentration and not the total quantity of fluoride that governs toxicity. The results of this study also support the hypothesis that aluminium toxicity against the green alga (Scenedesmus obliquus) is enhanced in an acidic environment.17-19 This can also be seen in the growth inhibition of Nitzschia linearis at pH 4.5, which may be attributed to the acidity rather than to the toxic effect of fluoride or aluminium. On the other hand, a change in pH plays an important role in the toxicity of Al, especially in the presence of F–, where it can form complexing ligands. The formation of AlF4–, being a PO43– analogue, might compete with PO43– for binding sites of ATPase. Inhibition of ATPase affects the uptake of vital ions including NO3– and PO43–. Therefore, a reduced nutrient uptake in the presence of test chemicals at acidic pH seems to be a major cause of inhibition of growth.8 The amounts of Al and F accumulated in the algal cells show that different species of algae differ in their ability to accumulate F and Al inside their cells. The accumulation rate depends on the type of algae,17-23 since Nichol et al23 isolated both resistant and tolerant forms of Synechococcus leopoliensis and observed that resistant cells show passive permeation of both F– and HF across the cell membrane. The sensitive cells are permeable only to HF and thus accumulate fluoride to a toxic concentration. REFERENCES 1 Peters NE, Meybeek M. Water quality degradation effects on freshwater availability: impacts of human activities. IWRA Water International 2000;25:185-93. 2 Levesque L, Mizzen CA, McLachlan DR, Fraser PE. Ligand specific effects aluminium incorporation and toxicity in neurons and astrocytes. Brain Res 2000;2:191-202.

Fluoride 37 (2) 2004

Fluoride and aluminium tolerance in microalgae 95

3 Giannini JL, Miller GW, Pushnich JC. Effects of NaF on biochemical processes of isolated soybean chloroplasts. Fluoride 1985;18:72-9. 4 Nichol BE, Budd K, Palmer GR, MacArthur JD. The mechanism of fluoride toxicity and fluoride resistance in Synechococcus leopoliensis (Cyanophyceae). J Phycol 1987;23:535-41. 5 Pettersson A, Bergman B. Effect of aluminium on ATP pools and utilization in the cyanobacterium Anabaena cylindrica: A model for the in vivo toxicity. Physiol Plant 1989;76:527-34. 6 Sturr MG, Marquis RE. Inhibition of protontranslocating ATP ases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminium. Arch Microbiol 1990;155:22-7. 7 Camargo JA. Fluoride toxicity to aquatic organisms: a review. Chemosphere 2003;50:251-64. 8 Rai LC, Husaini Y, Mallick N. Physiological and biochemical responses of Nostoc linckia to combined effects of aluminium, fluoride and acidification. Environ Exp Bot 1996;36(1):1-12. 9 Radic N, Bralic M. Aluminium fluoride complexation and its ecological importance in the aquatic environment. Sci Total Environ 1995;172:237-43. 10 Carmichael WW. Isolation, culture and toxicity testing of toxic freshwater cyanobacteria (bluegreen algae). In: Fundamental Research in Homogenous Catalysis. V Shilo, editor. New York: Gordon & Breach;1986. p. 1249-62. 11 Fitzgerald GP. A manual on methods for measuring primary production in aquatic environment. Handbook No 12. Oxford and Edniburgh: Blackwell Scientific Pub; 1971. 12 APHA. Standard methods for the examination of water and wastewater. 20th ed. Washington, DC; 1998. 13 Bhatnagar M. Fluoride tolerance in microalgae and its ecological implications (Dissertation). New Delhi: Indian Agric Res Inst;1997. 14 Chow CWK, Drikas M, House J, Burch MD, Velzeboer RMA. The impact of conventional water treatment processes on cells of the cyanobacterium Microcystis aeruginosa. Wat Res 1999;33:3253-62. 15 Lee KH, Lustigman B, Chu IY, Jou HL. Effect of aluminium and pH on the growth of Anacystis nidulans. Bull Environ Contam Toxicol 1991;46:720-6. 16 Smith AO, Woodson BR. The effects of fluoride on the growth of Chlorella pyrenoidosa. Virginia J Sci 1965;16:1-8. 17 Rai LC, Husaini Y, Mallick N. pH-altered interaction of aluminium and fluoride on nutrient uptake, photo-synthesis and other variables of Chlorella vulgaris. Aquatic toxicol 1998;42:6784. 18 Kinross JH, Read PA, Christofi N. The influence of pH and aluminium on the growth of filamentous algae in artificial streams. Arch Hydrobiol 2000;149:67-86. 19 George DB, Berk SG, Adams VD, Ting RS, Roberts RO, Parks LH, Lolt RC. Toxicity of alum sludge extracts to a freshwater algae, protozoan, fish and marine bacterium. Arch Environ Contam Toxicol 1995;29:149-58. 20 Joy CM, Balakrishnan KP. Effect of fluoride on axenic cultures of diatoms. Water Air Soil Pollut 1990;49:241-49. 21 Oliveira L, Antia NJ, Bisalputra T. Culture studies on the effects from fluoride pollution on the growth of marine phytoplanktons. J Fish Res Board Can 1978;36:1500-4. 22 Le Blanc GA. Interspecies relationships in acute toxicity of chemicals to aquatic organisms. Environ Toxicol Chem 1984;3:47-60. 23 Nichol BE, Budd K, Palmer GR, MacArthur JD. The mechanism of fluoride toxicity and fluoride resistance in Synechococcus leopliensis (Cyanophyceae). J Phycol 1987;23:535-41.

Published by the International Society for Fluoride Research http://homepages.ihug.co.nz/~spittle/fluoride-journal.htm Editorial Office: 727 Brighton Road, Ocean View, Dunedin 99051, New Zealand

Fluoride 37 (2) 2004