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Calcium chloride and calcium bromide aqueous solutions of technical and analytical grade in Lemna bioassay
Chemosphere 41 (2000) 1535±1542
Calcium chloride and calcium bromide aqueous solutions of technical and analytical grade in Lemna bioassay Marija Vujevic a, Zeljka Vidakovic-Cifrek a,*, Mirta Tkalec a, Mihovil Tomic b, Ivan Regula a a
Department of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia b CROSCO Co. (Integrated Drilling and Well Services, INA-Group), Ulica grada Vukovara 18, HR-10000 Zagreb, Croatia Received 1 October 1999; accepted 25 February 2000
Abstract Saturated water solutions of calcium chloride, calcium bromide and their 1:1 mixture are commonly used as ``high density brines'' for pressure control in oil wells. To compare the eect of these chemicals of technical grade with the eect of the chemicals of analytical grade the Lemna test was used. The multiplication rate, fresh weight, dry to fresh weight ratio, area covered by plants and chlorophyll content were measured as toxicity parameters. The concentrations of tested chemicals were 0.025, 0.05, 0.075 and 0.1 mol dmÿ3 . Generally, the chemicals of both technical and analytical grade in concentrations of 0.025 mol dmÿ3 stimulated the Lemna minor growth, while tested chemicals in concentrations of 0.05 mol dmÿ3 did not aect the growth signi®cantly. The exceptions were results obtained by measuring fresh weight. Most of tested chemicals in concentrations of 0.075 mol dmÿ3 and all chemicals in concentrations of 0.1 mol dmÿ3 reduced the growth. No major dierences between eects of tested chemicals of technical and analytical grade on plant growth were observed, except that tested chemicals of analytical grade in concentrations of 0.1 mol dmÿ3 increased dry to fresh weight ratio much stronger than chemicals of technical grade. All tested chemicals in all concentrations increased chlorophyll content. After treatment with chemicals of analytical grade much higher increase of chlorophyll a concentration in comparison to increase of chlorophyll b was noticed, while chemicals of technical grade caused more prominent increase of chlorophyll b. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Lemna minor L.; Lemna test; High density brines; Calcium chloride; Calcium bromide
1. Introduction Duckweed Lemna minor L. is a small ¯oating freshwater monocotyledon belonging to family Lemnaceae. It has been used as convenient bioassay organism in phytotoxicity evaluation due to its small size, rapid growth, vegetative reproduction, ease of culture and sensitivity to numerous pollutants (Wang, 1986; Lewis, 1995). Till now Lemna bioassay has been used in detection of
*
Corresponding author. Tel.: +385-1-48-26-262; fax: +3851-48-26-262. VidakovicÂ-Cifrek). E-mail address: [email protected] (Z.
phytotoxicity of heavy metals (Smith and Kwan, 1989; Huebert and Shay, 1991; Huebert et al., 1993; Sajwan and Ornes, 1994), phenols (Cowgill et al., 1991), herbicides (Krsnik-Rasol and Rendic, 1977; Lockhart et al., 1989; Peterson et al., 1994; Fairchild et al., 1997), surfactants (Dirilgen and Ince, 1995) and some chemical mixtures (Clement and Bouvet, 1993; Kanekar et al., 1993). Saturated calcium chloride and calcium bromide aqueous solutions of technical grade and their 1:1 mixture are commercially known as oil industry ``high density brines'' and have regularly been used in exploration and production of crude oil and natural gas (Schmidt et al., 1983). During the special operations,
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certain amounts of these solutions could be released into the environment, so animal and plant organisms living close to oil wells could become exposed to increased concentrations of salts. Tkalec et al. (1998) have already investigated the in¯uence of CaCl2 , CaBr2 and their 1:1 mixture aqueous solutions of technical grade (used as high density brines) on Lemna minor growth. They investigated these solutions in volume concentrations of 0.5, 1.0, 1.5 and 2.0% (v/v) in order to ®nd out which solution had the most prominent eect. They have noticed that all three tested solutions in 0.5, 1.0 and 1.5% (v/v) concentrations stimulated the Lemna minor growth. The highest concentration tested, 2.0% (v/v), caused the inhibition of Lemna minor growth. The in¯uence of high density brines was also investigated in other test systems. Vidakovic-Cifrek et al. (1999) noticed that lower concentrations of all three tested solutions had similar stimulative eect on the growth of green alga Chlorella kessleri. On the other hand, among solutions of the highest concentration (2.0% v/v), CaBr2 showed the most prominent inhibitory eect. Mazuran et al. (1999) investigated the eect of these chemicals in several sublethal concentrations on the fecundity of the freshwater snail Planorbarius corneus L. The tested solutions containing bromide caused more prominent reduction of fecundity in comparison to CaCl2 solutions. The objective of this work was to evaluate the in¯uence of CaCl2 , CaBr2 and their 1:1 mixture in concentrations of 0.025, 0.05, 0.075 and 0.1 mol dmÿ3 on Lemna minor growth. The chemicals of technical grade as well as chemicals of analytical grade were tested in order to compare the results and to distinguish the eect of tested salts from the eect of impurities commonly present in chemicals of technical grade.
pH value of nutrient solution was adjusted to 4.55 before autoclaving (120°C, 0.15 MPa, 20 min). Experimental cultures were started by inoculating a healthy colony with 2±3 fronds from stock cultures into the 100 ml Erlenmeyer ¯asks containing 60 ml of modi®ed HoaglandÕs nutrient solution (Krajncic and Devide, 1980) supplemented with CaCl2 , CaBr2 and their 1:1 mixture. Plants grown on modi®ed HoaglandÕs nutrient solution without tested chemicals were used as control. The pH value of nutrient solution was adjusted to 5.0. Both, the stock and experimental cultures were grown in chamber conditions under 16 h photoperiod (¯uorescent light, 80 lE sÿ1 mÿ2 ) at 24 2°C. 2.2. Tested chemicals To investigate the in¯uence of high density brines saturated solutions of CaCl2 (q 1300 g dmÿ3 ) and CaBr2 (q 1610 g dmÿ3 ), as well as their 1:1 mixture, were added into the modi®ed HoaglandÕs nutrient solution in volumes appropriate to achieve the following concentrations: 0.025, 0.05, 0.075 and 0.1 mol dmÿ3 . Atomic absorption spectrophotometry (ASTM D 51193, 1995) and volumetric method (ASTM D 512-89, 1995) were used to determine an accurate amount of calcium chloride, calcium bromide and some inorganic substances in these solutions (Table 1). Amounts of heavy metals (Cd, Cr, Ni, V, Fe and Co) were under detectable levels. Detection limits for those metals were (mg dmÿ3 ): Cd 0:0005; Cr 0:07; Ni 0:008; V 0:1; Fe 0:005 and Co 0:006. Afterwards, we repeated the experiment by addition of CaCl2 2H2 O and CaBr2 of analytical grade (Sigma) into the modi®ed HoaglandÕs nutrient solution in amounts appropriate to achieve the same concentrations of tested chemicals as before. 2.3. Lemna bioassay
2. Materials and methods
Duckweed Lemna minor was exposed to tested solutions for two weeks. The in¯uence of tested solutions on Lemna minor growth was evaluated due to the following end points (Arnon, 1949; Ensley et al., 1994): (1) relative growth of frond number, (2) relative growth of fresh weight, (3) dry to fresh weight ratio, (4) relative area
2.1. Cultures Axenic stock cultures of Lemna minor L. were maintained on the Pirson±SeidelÕs nutrient solution (Pirson and Seidel, 1950) and subcultured biweekly. The
Table 1 Chemical composition of CaCl2 and CaBr2 solutions of technical grade (commonly used as ``high density brines'') determined by atomic absorption spectrophotometry and volumetric method and expressed as mg dmÿ3 and mol dmÿ3 Solutions of technical grade
Concentrations
Ca2
Mg2
Zn2
Clÿ
Brÿ
CaCl2
mg dmÿ3 mol dmÿ3
170 000 4.24
6.0 2:47 10ÿ4
1.8 2:75 10ÿ5
301 350 8.5
± ±
CaBr2
mg dmÿ3 mol dmÿ3
218 000 5.44
195.0 8:02 10ÿ3
6.1 9:33 10ÿ5
± ±
867 500 10.85
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covered by plants and (5) chlorophyll a and chlorophyll b content and their ratio. All procedures were described by Tkalec et al. (1998). Results obtained by evaluation of growth parameters were represented as mean values of eight replicates. The control was represented as 100% and the results obtained with treated plants were represented as percentage of control. Chemicals that aected Lemna minor growth signi®cantly dierent (P < 0:05) from each other and control were marked with dierent letters according to DuncanÕs New Multiple Range Test (Duncan, 1955). Experiment for determination of chlorophyll a and chlorophyll b contents was repeated three times. Results were calculated as mean values and represented as percentage of control. 3. Results 3.1. The in¯uence of tested chemicals on frond number Tested chemicals (CaCl2 , CaBr2 and their 1:1 mixture) of both technical and analytical grade, added into the modi®ed HoaglandÕs nutrient solution in concentrations of 0.025 mol dmÿ3 signi®cantly stimulated (P < 0:05) the multiplication of plants (Fig. 1). On the
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contrary, all tested chemicals present in concentrations of 0.05 mol dmÿ3 and CaBr2 and 1:1 mixture in concentrations of 0.075 mol dmÿ3 did not signi®cantly change the frond number in comparison with the control. All tested solutions in concentrations of 0.1 mol dmÿ3 and CaCl2 in 0.075 mol dmÿ3 caused a signi®cant decrease (P < 0:05) of frond number. 3.2. The in¯uence of tested chemicals on fresh weight CaCl2 , CaBr2 and their 1:1 mixture of both technical and analytical grade in concentrations of 0.025 mol dmÿ3 did not change the relative growth of fresh weight in comparison to control plants, except CaBr2 of analytical grade that stimulated the growth (P < 0:05). CaCl2 and CaBr2 of analytical grade in concentrations of 0.05 mol dmÿ3 and CaBr2 of technical grade in concentration 0.075 mol dmÿ3 also had no signi®cant eect on fresh weight (Fig. 2). Other solutions in 0.05 and 0.075 mol dmÿ3 and all solutions in 0.1 mol dmÿ3 inhibited the growth of fresh weight (P < 0:05). 3.3. The in¯uence of tested chemicals on dry to fresh weight ratio Tested chemicals of both technical and analytical grade present in concentrations of 0.025 and 0.05 mol
Fig. 1. Relative growth of Lemna minor frond number after two week exposure to tested chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
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Fig. 2. Relative growth of Lemna minor fresh weight after two week exposure to tested chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
dmÿ3 did not in¯uence dry to fresh weight ratio signi®cantly (P < 0:05). Tested chemicals in higher concentrations (0.075 and 0.1 mol dmÿ3 ) increased observed ratio (Fig. 3). The exceptions were CaCl2 of analytical grade and CaBr2 of technical grade in concentrations of 0.075 mol dmÿ3 and CaCl2 and CaBr2 of technical grade in concentrations of 0.1 mol dmÿ3 . The most signi®cant increase was obtained by 0.1 mol dmÿ3 tested solutions of analytical grade (P < 0:05). 3.4. The in¯uence of tested chemicals on the relative area covered by plants Tested chemicals of both technical and analytical grade in concentrations of 0.025 mol dmÿ3 did not signi®cantly increase the relative area covered by plants in comparison to control plants except 1:1 mixture (P < 0:05). The relative area covered by duckweed treated with tested solutions in 0.05 and 0.075 mol dmÿ3 was similar to the relative area covered by control plants (Fig. 4). CaCl2 , CaBr2 and their 1:1 mixture of both technical and analytical grade added into the modi®ed HoaglandÕs nutrient solution in concentrations of 0.1 mol dmÿ3 decreased the relative area covered by plants (P < 0:05).
3.5. The in¯uence of tested chemicals on photosynthetic pigments content The chlorophyll a and chlorophyll b content increased correspondingly with the increase of tested chemicalsÕ concentration of both technical and analytical grade (Figs. 5 and 6). In duckweed treated with tested chemicals of analytical grade the increase of chlorophyll a content was more prominent than the increase of chlorophyll b content. The consequence was increased chlorophyll a to chlorophyll b ratio (Fig. 5). On the contrary, in duckweed grown on nutrient solution supplemented with tested chemicals of technical grade, the chlorophyll b content increased to a greater extent than chlorophyll a content, so the ratio was slightly decreased in comparison to control (Fig. 6).
4. Discussion Tested chemicals in concentrations of 0.025 mol dmÿ3 stimulated the Lemna minor growth similarly to results of Tkalec et al. (1998) obtained with 0.5% (v/v) dilution of high density brines (0.5% v/v CaCl2 solution contains 0.0218 mol dmÿ3 CaCl2 , while 0.5% v/v CaBr2
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Fig. 3. Dry to fresh weight ratio in Lemna minor plants grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
solution contains 0.0266 mol dmÿ3 CaBr2 ). This stimulative eect of tested salts in lower concentrations could be explained either by establishing better growth conditions by additional amounts of nutrients brought in with the sample (Wundram et al., 1997) or by positive eect of increased turgor (Oertli, 1975, cited by von Sury and Fl uckiger, 1983). We also noticed that the inhibition caused by chemicals in concentrations of 0.1 mol dmÿ3 corresponded to results of Tkalec et al. (1998) obtained with high density brines in 2.0% (v/v) dilution (2.0% v/v CaCl2 solution contains 0.0867 mol dmÿ3 CaCl2 , while 2.0% v/v CaBr2 solution contains 0.1066 mol dmÿ3 CaBr2 ). Since the present results as well as results of Tkalec et al. (1998) showed no signi®cant dierences between eects of CaCl2 , CaBr2 and their 1:1 mixture in concentrations of 0.1 mol dmÿ3 , the inhibition could be due to the osmotic eect. Chemical analysis (Table 1) showed that there were not signi®cant amounts of heavy metals in high density brines solutions. But, it could be possible that other impurities (of inorganic or organic nature) were present in the samples. The eect of high density brines solutions on duckweed growth showed no major dierences in comparison to the eect of chemicals of analytical grade. There were few exceptions according to the DuncanÕs
New Multiple Range Test. Some solutions of analytical grade caused stronger stimulation of growth than solutions of technical grade (P < 0:05): for example, in¯uence of mixture in concentrations of 0.025 and 0.075 mol dmÿ3 on frond number (Fig. 1) and in¯uence of 0.025 mol dmÿ3 CaBr2 on fresh weight (Fig. 2). CaBr2 and 1:1 mixture of analytical grade in concentrations of 0.1 mol dmÿ3 inhibited the increase of frond number stronger than the same substances of technical grade (Fig. 1). The most obvious dierence between chemicals of technical and analytical grade was noticed in dry to fresh weight ratio, which was signi®cantly higher (P < 0:05) after treatment with all three samples of analytical grade in concentrations of 0.1 mol dmÿ3 . The reason for such a result could be the starch accumulation in chloroplasts of treated plants due to disturbed sugar metabolism (Hillman, 1961; Severi, 1991). In our recent investigations (data not shown) we con®rmed the starch accumulation in chloroplasts of treated plants by microscopy and centrifugation of isolated chloroplasts in sucrose gradient. A few signi®cantly dierent results obtained after treatment with chemicals of analytical and technical grade could be explained by possible interactions of impurities with constituents of nutrient solution or with
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Fig. 4. Relative area covered by Lemna minor plants grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of analytical grade (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
Fig. 5. Chlorophyll a and chlorophyll b content and their ratio in Lemna minor grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of analytical grade. Values are the average of three dierent experiments represented as percentage of control.
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Fig. 6. Chlorophyll a and chlorophyll b content and their ratio in Lemna minor grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of technical grade. Values are the average of three dierent experiments represented as percentage of control.
ions of tested solutions. Interactions between the constituents of the nutrient solution are well known (Wang, 1992). As a consequence of such interactions, certain substances could be more available to plants than others and vice versa. Chemicals of both analytical and technical grade increased chlorophyll a and chlorophyll b content, but some dierences between chemicals of analytical and technical grade were noticed. Chemicals of analytical grade caused much greater increase of chlorophyll a content in comparison to increase of chlorophyll b content, so the chlorophyll a to chlorophyll b ratio was also increased (Fig. 5). On the other hand, chemicals of technical grade caused greater increase of chlorophyll b content in comparison to increase of chlorophyll a content, so the ratio was slightly decreased (Fig. 6). It is possible that the presence of impurities in chemicals of technical grade promoted the formation of chlorophyll b from the chlorophyll a, possibly by oxidising methyl group on pyrol ring no. 2 into aldehyde group (Gil et al., 1995). Only minor growth dierences caused by chemicals of analytical and technical grade support the hypothesis that eect noticed after treatment with chemicals of technical grade used as high density brines was due to the major constituents of these solutions (Ca2 , Clÿ and Brÿ ions) and was not the consequence of impurities commonly present in chemicals of technical grade.
However, chemicals of analytical and technical grade had dierent eect on chlorophyll pigments content in duckweed plants. References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1±15. ASTM D 511-93, 1995. Standard test methods for calcium and magnesium in water, Test method B, Atomic Absorption spectrophotometric. Annal Book of ASTM standards, Section 11, Water and Environmental Technology, Vol 11.01 Water (1), American Society for Testing and Materials, Philadelphia, PA. ASTM D 512-89, 1995. Standard test methods for chloride ion in water, Test method B, Silver nitrate titration. Annal Book of ASTM standards, Section 11, Water and Environmental Technology, Vol 11.01 Water (1), American Society for Testing and Materials, Philadelphia, PA. Clement, B., Bouvet, Y., 1993. Assessment of land®ll leachate toxicity using the duckweed Lemna minor. In: Proceedings of the Second European Conference of Ecotoxicology. Amsterdam. Sci. Total Environ. (Suppl.), 1179±1190. Cowgill, U.M., Milazzo, D.P., Landenberger, B.D., 1991. The sensitivity of Lemna gibba G-3 and four clones of Lemna minor to eight common chemicals using a 7-day test. Research J. WPCF 63 (7), 991±998. Dirilgen, N., Ince, N., 1995. Inhibition eect of the anionic surfactant SDS on duckweed, Lemna minor with consider-
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Schmidt, D.D., Hudson, T.E., Harris, T.M., 1983. Introduction on brine completion and workover ¯uids. Part 1 ± Chemical and physical properties of clear completion brines. Petrol. Eng. Int. August, 80±96. Severi, A., 1991. Eects of aluminium on some morphophysiological aspects on Lemna minor L. Atti. Soc. Nat. e Mat. di Modena 122, 95±108. Smith, S., Kwan, M.K.H., 1989. Use of aquatic macrophytes as bioassay method to assess relative toxicity, uptake kinetics and accumulated forms of trace metals. Hydrobiologia 188/ 189, 345±351. Regula, I., 1998. The eect of Tkalec, M., Vidakovic-Cifrek, Z, oil industry ``high density brines'' on duckweed Lemna minor. Chemosphere 37 (13), 2703±2715. Tkalec, M., Horvatic, J., Regula, I., 1999. Vidakovic-Cifrek, Z., Eect of oil industry high density brines in miniaturized algal growth bioassay and Lemna test. Phyton cAnn. Rei Bot. 39 (3), 193±197. (Special issue ± Second Slovenian Symposium on Plant Physiology. Gozd Martuljek, Slovenia). von Sury, R., Fl uckiger, W., 1983. The eect of dierent mixtures of NaCl and CaCl2 on the silver ®r (Abies alba Miller). Eur. J. For. Path. 13, 24±30. Wang, W., 1986. Toxicity tests of aquatic pollutants by using common duckweed. Environ. Pollut. B 11, 1±14. Wang, W., 1992. Use of plants for the assessment of environmental contaminants. Rev. Environ. Contam. Toxicol. 126, 87±127. Wundram, M., Selmar, D., Bahadir, M., 1997. Representative evaluation of phytotoxicity ± reliability and peculiarities. Angew. Bot. 71, 139±143. Marija Vujevic received her B.Sc. degree in 1998 from the University of Zagreb, Croatia. She is M.Sc. student at the Faculty of Science, University of Zagreb. The subject of her current research is in vitro propagation of rare and endangered Croatian plant species. Zeljka Vidakovic-Cifrek received her B.Sc. (1990), M.Sc. (1993) and Ph.D. (1999) degree from the University of Zagreb, Croatia. She is research assistant at the Department of Botany, Faculty of Science, University of Zagreb. Her research interests focus on in¯uence of various environmental factors on physiological processes in plants. Mirta Tkalec received her B.Sc. degree in 1996 from the University of Zagreb, Croatia. She is currently M.Sc. student at the Faculty of Science, University of Zagreb. The main topic of her research is evaluation of stress factors by Lemna test. Mihovil Tomic received his B.Sc. degree from the University of Sarajevo, Bosnia and Herzegovina and M.Sc. degree from the University of Zagreb, Croatia. He works in INA-Oil industry on analytical aspects in exploration and production of gas and oil as well as on pollution problems in oil industry. Ivan Regula is full professor of Plant Physiology at the Department of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Croatia. His scienti®c interests include structure and function of indolic compounds in plants as well as in¯uence of xenobiotics on plant metabolism.
Calcium chloride and calcium bromide aqueous solutions of technical and analytical grade in Lemna bioassay Marija Vujevic a, Zeljka Vidakovic-Cifrek a,*, Mirta Tkalec a, Mihovil Tomic b, Ivan Regula a a
Department of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia b CROSCO Co. (Integrated Drilling and Well Services, INA-Group), Ulica grada Vukovara 18, HR-10000 Zagreb, Croatia Received 1 October 1999; accepted 25 February 2000
Abstract Saturated water solutions of calcium chloride, calcium bromide and their 1:1 mixture are commonly used as ``high density brines'' for pressure control in oil wells. To compare the eect of these chemicals of technical grade with the eect of the chemicals of analytical grade the Lemna test was used. The multiplication rate, fresh weight, dry to fresh weight ratio, area covered by plants and chlorophyll content were measured as toxicity parameters. The concentrations of tested chemicals were 0.025, 0.05, 0.075 and 0.1 mol dmÿ3 . Generally, the chemicals of both technical and analytical grade in concentrations of 0.025 mol dmÿ3 stimulated the Lemna minor growth, while tested chemicals in concentrations of 0.05 mol dmÿ3 did not aect the growth signi®cantly. The exceptions were results obtained by measuring fresh weight. Most of tested chemicals in concentrations of 0.075 mol dmÿ3 and all chemicals in concentrations of 0.1 mol dmÿ3 reduced the growth. No major dierences between eects of tested chemicals of technical and analytical grade on plant growth were observed, except that tested chemicals of analytical grade in concentrations of 0.1 mol dmÿ3 increased dry to fresh weight ratio much stronger than chemicals of technical grade. All tested chemicals in all concentrations increased chlorophyll content. After treatment with chemicals of analytical grade much higher increase of chlorophyll a concentration in comparison to increase of chlorophyll b was noticed, while chemicals of technical grade caused more prominent increase of chlorophyll b. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Lemna minor L.; Lemna test; High density brines; Calcium chloride; Calcium bromide
1. Introduction Duckweed Lemna minor L. is a small ¯oating freshwater monocotyledon belonging to family Lemnaceae. It has been used as convenient bioassay organism in phytotoxicity evaluation due to its small size, rapid growth, vegetative reproduction, ease of culture and sensitivity to numerous pollutants (Wang, 1986; Lewis, 1995). Till now Lemna bioassay has been used in detection of
*
Corresponding author. Tel.: +385-1-48-26-262; fax: +3851-48-26-262. VidakovicÂ-Cifrek). E-mail address: [email protected] (Z.
phytotoxicity of heavy metals (Smith and Kwan, 1989; Huebert and Shay, 1991; Huebert et al., 1993; Sajwan and Ornes, 1994), phenols (Cowgill et al., 1991), herbicides (Krsnik-Rasol and Rendic, 1977; Lockhart et al., 1989; Peterson et al., 1994; Fairchild et al., 1997), surfactants (Dirilgen and Ince, 1995) and some chemical mixtures (Clement and Bouvet, 1993; Kanekar et al., 1993). Saturated calcium chloride and calcium bromide aqueous solutions of technical grade and their 1:1 mixture are commercially known as oil industry ``high density brines'' and have regularly been used in exploration and production of crude oil and natural gas (Schmidt et al., 1983). During the special operations,
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certain amounts of these solutions could be released into the environment, so animal and plant organisms living close to oil wells could become exposed to increased concentrations of salts. Tkalec et al. (1998) have already investigated the in¯uence of CaCl2 , CaBr2 and their 1:1 mixture aqueous solutions of technical grade (used as high density brines) on Lemna minor growth. They investigated these solutions in volume concentrations of 0.5, 1.0, 1.5 and 2.0% (v/v) in order to ®nd out which solution had the most prominent eect. They have noticed that all three tested solutions in 0.5, 1.0 and 1.5% (v/v) concentrations stimulated the Lemna minor growth. The highest concentration tested, 2.0% (v/v), caused the inhibition of Lemna minor growth. The in¯uence of high density brines was also investigated in other test systems. Vidakovic-Cifrek et al. (1999) noticed that lower concentrations of all three tested solutions had similar stimulative eect on the growth of green alga Chlorella kessleri. On the other hand, among solutions of the highest concentration (2.0% v/v), CaBr2 showed the most prominent inhibitory eect. Mazuran et al. (1999) investigated the eect of these chemicals in several sublethal concentrations on the fecundity of the freshwater snail Planorbarius corneus L. The tested solutions containing bromide caused more prominent reduction of fecundity in comparison to CaCl2 solutions. The objective of this work was to evaluate the in¯uence of CaCl2 , CaBr2 and their 1:1 mixture in concentrations of 0.025, 0.05, 0.075 and 0.1 mol dmÿ3 on Lemna minor growth. The chemicals of technical grade as well as chemicals of analytical grade were tested in order to compare the results and to distinguish the eect of tested salts from the eect of impurities commonly present in chemicals of technical grade.
pH value of nutrient solution was adjusted to 4.55 before autoclaving (120°C, 0.15 MPa, 20 min). Experimental cultures were started by inoculating a healthy colony with 2±3 fronds from stock cultures into the 100 ml Erlenmeyer ¯asks containing 60 ml of modi®ed HoaglandÕs nutrient solution (Krajncic and Devide, 1980) supplemented with CaCl2 , CaBr2 and their 1:1 mixture. Plants grown on modi®ed HoaglandÕs nutrient solution without tested chemicals were used as control. The pH value of nutrient solution was adjusted to 5.0. Both, the stock and experimental cultures were grown in chamber conditions under 16 h photoperiod (¯uorescent light, 80 lE sÿ1 mÿ2 ) at 24 2°C. 2.2. Tested chemicals To investigate the in¯uence of high density brines saturated solutions of CaCl2 (q 1300 g dmÿ3 ) and CaBr2 (q 1610 g dmÿ3 ), as well as their 1:1 mixture, were added into the modi®ed HoaglandÕs nutrient solution in volumes appropriate to achieve the following concentrations: 0.025, 0.05, 0.075 and 0.1 mol dmÿ3 . Atomic absorption spectrophotometry (ASTM D 51193, 1995) and volumetric method (ASTM D 512-89, 1995) were used to determine an accurate amount of calcium chloride, calcium bromide and some inorganic substances in these solutions (Table 1). Amounts of heavy metals (Cd, Cr, Ni, V, Fe and Co) were under detectable levels. Detection limits for those metals were (mg dmÿ3 ): Cd 0:0005; Cr 0:07; Ni 0:008; V 0:1; Fe 0:005 and Co 0:006. Afterwards, we repeated the experiment by addition of CaCl2 2H2 O and CaBr2 of analytical grade (Sigma) into the modi®ed HoaglandÕs nutrient solution in amounts appropriate to achieve the same concentrations of tested chemicals as before. 2.3. Lemna bioassay
2. Materials and methods
Duckweed Lemna minor was exposed to tested solutions for two weeks. The in¯uence of tested solutions on Lemna minor growth was evaluated due to the following end points (Arnon, 1949; Ensley et al., 1994): (1) relative growth of frond number, (2) relative growth of fresh weight, (3) dry to fresh weight ratio, (4) relative area
2.1. Cultures Axenic stock cultures of Lemna minor L. were maintained on the Pirson±SeidelÕs nutrient solution (Pirson and Seidel, 1950) and subcultured biweekly. The
Table 1 Chemical composition of CaCl2 and CaBr2 solutions of technical grade (commonly used as ``high density brines'') determined by atomic absorption spectrophotometry and volumetric method and expressed as mg dmÿ3 and mol dmÿ3 Solutions of technical grade
Concentrations
Ca2
Mg2
Zn2
Clÿ
Brÿ
CaCl2
mg dmÿ3 mol dmÿ3
170 000 4.24
6.0 2:47 10ÿ4
1.8 2:75 10ÿ5
301 350 8.5
± ±
CaBr2
mg dmÿ3 mol dmÿ3
218 000 5.44
195.0 8:02 10ÿ3
6.1 9:33 10ÿ5
± ±
867 500 10.85
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covered by plants and (5) chlorophyll a and chlorophyll b content and their ratio. All procedures were described by Tkalec et al. (1998). Results obtained by evaluation of growth parameters were represented as mean values of eight replicates. The control was represented as 100% and the results obtained with treated plants were represented as percentage of control. Chemicals that aected Lemna minor growth signi®cantly dierent (P < 0:05) from each other and control were marked with dierent letters according to DuncanÕs New Multiple Range Test (Duncan, 1955). Experiment for determination of chlorophyll a and chlorophyll b contents was repeated three times. Results were calculated as mean values and represented as percentage of control. 3. Results 3.1. The in¯uence of tested chemicals on frond number Tested chemicals (CaCl2 , CaBr2 and their 1:1 mixture) of both technical and analytical grade, added into the modi®ed HoaglandÕs nutrient solution in concentrations of 0.025 mol dmÿ3 signi®cantly stimulated (P < 0:05) the multiplication of plants (Fig. 1). On the
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contrary, all tested chemicals present in concentrations of 0.05 mol dmÿ3 and CaBr2 and 1:1 mixture in concentrations of 0.075 mol dmÿ3 did not signi®cantly change the frond number in comparison with the control. All tested solutions in concentrations of 0.1 mol dmÿ3 and CaCl2 in 0.075 mol dmÿ3 caused a signi®cant decrease (P < 0:05) of frond number. 3.2. The in¯uence of tested chemicals on fresh weight CaCl2 , CaBr2 and their 1:1 mixture of both technical and analytical grade in concentrations of 0.025 mol dmÿ3 did not change the relative growth of fresh weight in comparison to control plants, except CaBr2 of analytical grade that stimulated the growth (P < 0:05). CaCl2 and CaBr2 of analytical grade in concentrations of 0.05 mol dmÿ3 and CaBr2 of technical grade in concentration 0.075 mol dmÿ3 also had no signi®cant eect on fresh weight (Fig. 2). Other solutions in 0.05 and 0.075 mol dmÿ3 and all solutions in 0.1 mol dmÿ3 inhibited the growth of fresh weight (P < 0:05). 3.3. The in¯uence of tested chemicals on dry to fresh weight ratio Tested chemicals of both technical and analytical grade present in concentrations of 0.025 and 0.05 mol
Fig. 1. Relative growth of Lemna minor frond number after two week exposure to tested chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
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Fig. 2. Relative growth of Lemna minor fresh weight after two week exposure to tested chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
dmÿ3 did not in¯uence dry to fresh weight ratio signi®cantly (P < 0:05). Tested chemicals in higher concentrations (0.075 and 0.1 mol dmÿ3 ) increased observed ratio (Fig. 3). The exceptions were CaCl2 of analytical grade and CaBr2 of technical grade in concentrations of 0.075 mol dmÿ3 and CaCl2 and CaBr2 of technical grade in concentrations of 0.1 mol dmÿ3 . The most signi®cant increase was obtained by 0.1 mol dmÿ3 tested solutions of analytical grade (P < 0:05). 3.4. The in¯uence of tested chemicals on the relative area covered by plants Tested chemicals of both technical and analytical grade in concentrations of 0.025 mol dmÿ3 did not signi®cantly increase the relative area covered by plants in comparison to control plants except 1:1 mixture (P < 0:05). The relative area covered by duckweed treated with tested solutions in 0.05 and 0.075 mol dmÿ3 was similar to the relative area covered by control plants (Fig. 4). CaCl2 , CaBr2 and their 1:1 mixture of both technical and analytical grade added into the modi®ed HoaglandÕs nutrient solution in concentrations of 0.1 mol dmÿ3 decreased the relative area covered by plants (P < 0:05).
3.5. The in¯uence of tested chemicals on photosynthetic pigments content The chlorophyll a and chlorophyll b content increased correspondingly with the increase of tested chemicalsÕ concentration of both technical and analytical grade (Figs. 5 and 6). In duckweed treated with tested chemicals of analytical grade the increase of chlorophyll a content was more prominent than the increase of chlorophyll b content. The consequence was increased chlorophyll a to chlorophyll b ratio (Fig. 5). On the contrary, in duckweed grown on nutrient solution supplemented with tested chemicals of technical grade, the chlorophyll b content increased to a greater extent than chlorophyll a content, so the ratio was slightly decreased in comparison to control (Fig. 6).
4. Discussion Tested chemicals in concentrations of 0.025 mol dmÿ3 stimulated the Lemna minor growth similarly to results of Tkalec et al. (1998) obtained with 0.5% (v/v) dilution of high density brines (0.5% v/v CaCl2 solution contains 0.0218 mol dmÿ3 CaCl2 , while 0.5% v/v CaBr2
M. Vujevic et al. / Chemosphere 41 (2000) 1535±1542
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Fig. 3. Dry to fresh weight ratio in Lemna minor plants grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
solution contains 0.0266 mol dmÿ3 CaBr2 ). This stimulative eect of tested salts in lower concentrations could be explained either by establishing better growth conditions by additional amounts of nutrients brought in with the sample (Wundram et al., 1997) or by positive eect of increased turgor (Oertli, 1975, cited by von Sury and Fl uckiger, 1983). We also noticed that the inhibition caused by chemicals in concentrations of 0.1 mol dmÿ3 corresponded to results of Tkalec et al. (1998) obtained with high density brines in 2.0% (v/v) dilution (2.0% v/v CaCl2 solution contains 0.0867 mol dmÿ3 CaCl2 , while 2.0% v/v CaBr2 solution contains 0.1066 mol dmÿ3 CaBr2 ). Since the present results as well as results of Tkalec et al. (1998) showed no signi®cant dierences between eects of CaCl2 , CaBr2 and their 1:1 mixture in concentrations of 0.1 mol dmÿ3 , the inhibition could be due to the osmotic eect. Chemical analysis (Table 1) showed that there were not signi®cant amounts of heavy metals in high density brines solutions. But, it could be possible that other impurities (of inorganic or organic nature) were present in the samples. The eect of high density brines solutions on duckweed growth showed no major dierences in comparison to the eect of chemicals of analytical grade. There were few exceptions according to the DuncanÕs
New Multiple Range Test. Some solutions of analytical grade caused stronger stimulation of growth than solutions of technical grade (P < 0:05): for example, in¯uence of mixture in concentrations of 0.025 and 0.075 mol dmÿ3 on frond number (Fig. 1) and in¯uence of 0.025 mol dmÿ3 CaBr2 on fresh weight (Fig. 2). CaBr2 and 1:1 mixture of analytical grade in concentrations of 0.1 mol dmÿ3 inhibited the increase of frond number stronger than the same substances of technical grade (Fig. 1). The most obvious dierence between chemicals of technical and analytical grade was noticed in dry to fresh weight ratio, which was signi®cantly higher (P < 0:05) after treatment with all three samples of analytical grade in concentrations of 0.1 mol dmÿ3 . The reason for such a result could be the starch accumulation in chloroplasts of treated plants due to disturbed sugar metabolism (Hillman, 1961; Severi, 1991). In our recent investigations (data not shown) we con®rmed the starch accumulation in chloroplasts of treated plants by microscopy and centrifugation of isolated chloroplasts in sucrose gradient. A few signi®cantly dierent results obtained after treatment with chemicals of analytical and technical grade could be explained by possible interactions of impurities with constituents of nutrient solution or with
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Fig. 4. Relative area covered by Lemna minor plants grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of analytical grade (left) and technical grade (right). Dierent letters on the top of the column indicate signi®cant dierences between treatments at P < 0:05 by DuncanÕs New Multiple Range Test.
Fig. 5. Chlorophyll a and chlorophyll b content and their ratio in Lemna minor grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of analytical grade. Values are the average of three dierent experiments represented as percentage of control.
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Fig. 6. Chlorophyll a and chlorophyll b content and their ratio in Lemna minor grown on modi®ed HoaglandÕs nutrient solution supplemented with tested chemicals of technical grade. Values are the average of three dierent experiments represented as percentage of control.
ions of tested solutions. Interactions between the constituents of the nutrient solution are well known (Wang, 1992). As a consequence of such interactions, certain substances could be more available to plants than others and vice versa. Chemicals of both analytical and technical grade increased chlorophyll a and chlorophyll b content, but some dierences between chemicals of analytical and technical grade were noticed. Chemicals of analytical grade caused much greater increase of chlorophyll a content in comparison to increase of chlorophyll b content, so the chlorophyll a to chlorophyll b ratio was also increased (Fig. 5). On the other hand, chemicals of technical grade caused greater increase of chlorophyll b content in comparison to increase of chlorophyll a content, so the ratio was slightly decreased (Fig. 6). It is possible that the presence of impurities in chemicals of technical grade promoted the formation of chlorophyll b from the chlorophyll a, possibly by oxidising methyl group on pyrol ring no. 2 into aldehyde group (Gil et al., 1995). Only minor growth dierences caused by chemicals of analytical and technical grade support the hypothesis that eect noticed after treatment with chemicals of technical grade used as high density brines was due to the major constituents of these solutions (Ca2 , Clÿ and Brÿ ions) and was not the consequence of impurities commonly present in chemicals of technical grade.
However, chemicals of analytical and technical grade had dierent eect on chlorophyll pigments content in duckweed plants. References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1±15. ASTM D 511-93, 1995. Standard test methods for calcium and magnesium in water, Test method B, Atomic Absorption spectrophotometric. Annal Book of ASTM standards, Section 11, Water and Environmental Technology, Vol 11.01 Water (1), American Society for Testing and Materials, Philadelphia, PA. ASTM D 512-89, 1995. Standard test methods for chloride ion in water, Test method B, Silver nitrate titration. Annal Book of ASTM standards, Section 11, Water and Environmental Technology, Vol 11.01 Water (1), American Society for Testing and Materials, Philadelphia, PA. Clement, B., Bouvet, Y., 1993. Assessment of land®ll leachate toxicity using the duckweed Lemna minor. In: Proceedings of the Second European Conference of Ecotoxicology. Amsterdam. Sci. Total Environ. (Suppl.), 1179±1190. Cowgill, U.M., Milazzo, D.P., Landenberger, B.D., 1991. The sensitivity of Lemna gibba G-3 and four clones of Lemna minor to eight common chemicals using a 7-day test. Research J. WPCF 63 (7), 991±998. Dirilgen, N., Ince, N., 1995. Inhibition eect of the anionic surfactant SDS on duckweed, Lemna minor with consider-
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Schmidt, D.D., Hudson, T.E., Harris, T.M., 1983. Introduction on brine completion and workover ¯uids. Part 1 ± Chemical and physical properties of clear completion brines. Petrol. Eng. Int. August, 80±96. Severi, A., 1991. Eects of aluminium on some morphophysiological aspects on Lemna minor L. Atti. Soc. Nat. e Mat. di Modena 122, 95±108. Smith, S., Kwan, M.K.H., 1989. Use of aquatic macrophytes as bioassay method to assess relative toxicity, uptake kinetics and accumulated forms of trace metals. Hydrobiologia 188/ 189, 345±351. Regula, I., 1998. The eect of Tkalec, M., Vidakovic-Cifrek, Z, oil industry ``high density brines'' on duckweed Lemna minor. Chemosphere 37 (13), 2703±2715. Tkalec, M., Horvatic, J., Regula, I., 1999. Vidakovic-Cifrek, Z., Eect of oil industry high density brines in miniaturized algal growth bioassay and Lemna test. Phyton cAnn. Rei Bot. 39 (3), 193±197. (Special issue ± Second Slovenian Symposium on Plant Physiology. Gozd Martuljek, Slovenia). von Sury, R., Fl uckiger, W., 1983. The eect of dierent mixtures of NaCl and CaCl2 on the silver ®r (Abies alba Miller). Eur. J. For. Path. 13, 24±30. Wang, W., 1986. Toxicity tests of aquatic pollutants by using common duckweed. Environ. Pollut. B 11, 1±14. Wang, W., 1992. Use of plants for the assessment of environmental contaminants. Rev. Environ. Contam. Toxicol. 126, 87±127. Wundram, M., Selmar, D., Bahadir, M., 1997. Representative evaluation of phytotoxicity ± reliability and peculiarities. Angew. Bot. 71, 139±143. Marija Vujevic received her B.Sc. degree in 1998 from the University of Zagreb, Croatia. She is M.Sc. student at the Faculty of Science, University of Zagreb. The subject of her current research is in vitro propagation of rare and endangered Croatian plant species. Zeljka Vidakovic-Cifrek received her B.Sc. (1990), M.Sc. (1993) and Ph.D. (1999) degree from the University of Zagreb, Croatia. She is research assistant at the Department of Botany, Faculty of Science, University of Zagreb. Her research interests focus on in¯uence of various environmental factors on physiological processes in plants. Mirta Tkalec received her B.Sc. degree in 1996 from the University of Zagreb, Croatia. She is currently M.Sc. student at the Faculty of Science, University of Zagreb. The main topic of her research is evaluation of stress factors by Lemna test. Mihovil Tomic received his B.Sc. degree from the University of Sarajevo, Bosnia and Herzegovina and M.Sc. degree from the University of Zagreb, Croatia. He works in INA-Oil industry on analytical aspects in exploration and production of gas and oil as well as on pollution problems in oil industry. Ivan Regula is full professor of Plant Physiology at the Department of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Croatia. His scienti®c interests include structure and function of indolic compounds in plants as well as in¯uence of xenobiotics on plant metabolism.