A ~ h i v e s

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Arch. Environ. Contam. Toxicol. 20, 505-508 (t991)

9 1991 Springer-Verlag New York Inc.

The Effects of pH on the Growth of Chlorella vulgaris and Its Interactions with Cadmium Toxicity Joseph W. Rachlin and Albania Grosso Department of Biological Sciences, Lehman College of the City University of New York, Bedford Park Boulevard West, Bronx, New York 10468-1589, USA Abstract. The effects of pH alone, and in combination with exposure to 0.89 tzM cadmium, on the growth response of the green alga Chlorella vulgaris were evaluated. Acidic (3.0-6.2) and alkaline (8.3-9.0) pH values retarded the growth of this alga. Optimal growth occurred when the pH of the medium was adjusted to values of 7.5 and 8.0. When the cells were exposed to pH adjusted medium plus the presence of 0.89 ~M Cd, a value known to reduce population growth by 50% at the control pH of 6.9, the affects were additive at the acidic (3.0-5.0) pH ranges. At alkaline pH values of 8.3-9.0 all toxicity responses could be explained by pH adjustment alone, indicating that additional cadmium toxicity was absent. At pH values of 7.5 and 8.0, cadmium toxicity was mitigated against, and resultant growth at pH 8.0 was at the same enhanced rate as this pH without cadmium.

The influence of pH on the toxicity of cations to the various components of the aquatic ecosystem is a recent concern in many ecotoxicological studies (Boudou and Ribeyre 1989a, 1989b; Rai et al. 1990). This concern developed with the recognition that man's industrial activities can cause a lowering of pH in these ecosystems, either through direct acidic discharge, or by the production of atmospheric gases which can result in acid precipitations. While it is common to think of the lowering of pH by these activities, it should be remembered that, as in the case of soda ash discharge, industrial activities can result in a shift in environmental pH values towards the alkaline side of the range. That these changes in pH can affect the speciation of, and the bioavailability of cations, is well documented (Strum and Morgan 1981). Alterations in cation speciation and availability can have profound influence on their toxicity (Astruc 1989; Campbell and Tessier 1989). Of particular concern in this regard is the influence of pH on cation toxicity to the algal base of the aquatic food web (Puiseux-Dao 1989). That pH alterations actually do influence cation toxicity in algae has been shown in a recent study of pH-altered cadmium toxicity in the cyanobacterium Anabaena flus-aquae (Rai et al. 1990). Our concern in the present study is to document the influence of

pH changes on the toxicity of cadmium to the eukaryotic green alga Chlorella vulgaris. To accomplish this, we elected to use the parameter of population growth as the measured end point (Rosko and Rachlin 1977; Rachlin et aL 1982a, 1983, 1984; Warkentine and Rachlin 1986). It therefore becomes necessary to first evaluate the effects of pH on population growth, and then examine the combined affects of altered pH and cadmium toxicity.

Materials and Methods The alga, Chiorella vuIgaris (UTEX 30) was obtained as a pure isolate from the Star Culture Collection of Algae, University of Texas at Austin, and was maintained in the laboratory in sterile modified chelator-free Bristol's medium (Bold 1949). Stock algal cultures were maintained in 50 ml of Bristol's medium in 125 ml glass Erienmeyer flasks, kept in log phase growth by removal of 20 ml of medium and cells every four days and replacing this with an equal volume of fresh sterile medium. Stock and test cultures were incubated in a Sherer-Gillett RI-24 LTP growth chamber illuminated with Sylvania cool white fluorescent lamps supplemented with a 25 watt incandescent light bulb. This provided a mean light intensity around the flasks of 5.5 Klux (500 ft. candles). Total energy at the flasks was measured, using an Eppley Precision Pyranometer Model PSP and found to be 14 Watts M -2. Photosynthetically active radiation (400-700 nm) received by the flasks was measured with a Li-Cor Quantum Sensor Model LI-190SB and gave a reading of 60 tzEM-2sec -1. The day/night program within the chamber was 16:8 h and the incubation temperature was maintained at 19 -+ I~ Test solutions consisted of the modified Bristol's medium as control (pH 6.9) or as pH adjusted solutions, either with or without cadmium. To examine the affect of acidity and alkalinity on algal growth a pH test range between 3.0 and 9.0 was selected. To achieve the desired test pH, the Bristol's medium was adjusted with appropriate amounts of either a 1 M solution of HC1 or a t M solution of KOH before autoclaving. However, for solutions of pH greater than 8.0, addition of KOH prior to autoclaving resulted in the formation of a precipitate during the autoclaving procedure. To circumvent this problem, all test solutions requiring a pH greater than 8.0 involved separate sterilization of the I M KOH and sterile addition of appropriate amounts of this solution to previously autoclaved Bristol's medium. For pH plus cadmium trials, a concentration of 0.89 p~M Cd was selected. This cadmium concentration was previously determined (Rosko and Rachlin 1977) to reduce the

506

J.W. Rachlin and A. Grosso

growth of Chlorella vulgaris cultures by 50% under the above mentioned experimental conditions. Stock solutions of CdClz at a concentration of 89.0 ~M Cd in Bristol's medium were prepared. A 1:100 dilution in appropriately pH adjusted Bristol's medium was then used to obtain the desired test concentration. Sterilization was achieved as previously described. All tests were run, using log phase algal cultures, at a cell concentration of approximately 1 x 105 cells ml- ~. Cell concentrations were determined using a brightline hemocytometer counting chamber (Rachlin and Farran 1974) which allowed us to include only viable cells in the cell count. Five ml of test medium, containing 1 x 105 cells m1-1, were inoculated into sterile Falcon 30 ml plastic tissue culture flasks and these flasks were randomly placed in the growth chamber for the 96-h exposure period. All tests were run in triplicate, and the pH of the test solutions were determined at the beginning and end of each run, using a Corning Model 10 pH meter. At the end of the 96-h exposure period the flasks were removed from the growth chamber and agitated on a vortex mixer to ensure thorough mixing for the cell counts. The ceils were then counted using the brightline hemocytometer, and since the cell counts for each flask of a triplicate run were within 10% of each other, the data for each of the triplicates was pooled for the determination of percent growth and 95% confidence intervals (Rachlin and Farran 1974).

Table 1. Percent • 95% confidence interval of control growth of Chlorella vulgaris after 96 h exposure to pH modified Bristol's Medium

Results

Initial pH

% Control Growth

Final pH

The percentage of control growth of Chlorella vulgaris incubated for 96-h under various pH conditions is shown in Table 1. It can be seen that for the acidic and neutral pH values, the pH remained relatively constant over the 96-h exposure period; but for the alkaline pH ranges (pH 7.5 and greater) cell growth seems to result in a change in pH bringing the values down to between 7.0 to 7.9, depending on the initial test pH. This was also true for the pH adjusted Bristol's medium containing 0.89 p,M cadmium, as can be seen by examination of Table 2. Alkaline pH values, in the absence of cells, remain relatively constant over the 96-h incubation period (Table 1). Based on previous studies (Rosko and Rachlin 1977), the control pH giving the reference (100%) growth is pH 6.9. Table 1 d e m o n s t r a t e s that u n d e r acidic conditions, pH 3.0-5.0, the growth of Chlorella vulgaris is less (27.3 +_ 0.16%-55.2 --- 0.18%) than the control value. At alkaline pH values of 8.3 to 9.0, growth was again reduced respectively to 46.1 -+ 0.18%-34.2 --- 0.17% of the control values. Of particular interest is that at the alkaline pH values of 7.5 and 8.0 growth exceeded control values, indicating an optimization of growth conditions within this narrow pH range. The combined effects of exposure to pH and cadmium are shown in Table 2. Under acidic conditions the growth of the cultures are reduced below control values (32.3 --- 0.16%44.1 +- 0.18%). The range of reduction of growth is similar to that for acidic pH values alone, with only slight additional reduction because of the presence of the cadmium at pH values 4.0 and 5.0 (Table 1). At pH 6.9 we expect a 50% reduction in growth in the presence of 0.89 p~M Cadmium (Rosko and Rachlin 1977). Table 2 shows that the actual growth reduction under these conditions was 44.8% (55.2 -+ 0.18% of control growth), a finding within 5.2% of our expectation. At the alkaline pH values of 8.3 to 9.0 growth in this cadmium containing medium was reduced respectively

3.0 4.0 5.0 6.9 7.5 8.0 8.3 8.5 9.0

32.3 • 0.16 27.7 +- 0.16 44.1 • 0.18 55.2 -+ 0.18 79.4 • 0.14 122.4 +- 0.19 44.2 • 0.18 40.6 • 0.18 37.1 -+ 0.17

3.0 4.2 4.6 6.9 7.1 7.0 7.4 7.5 7.6

Initial pH

% Control Growth

Final pH

3.0 4.0 5.0 6.2 6.9 7.5 8.0 8.3 8.5 9.0

27.3 • 0.16 36.3 _+ 0.17 55.6 • 0.18 91.9 • 0.10 100.0 124.9 • 0.20 120.0 _+ 0.18 46.1 ___0.18 49.7 • 0.18 34.2 _ 0.17

2.8 4.0 4.9 6.2 6.9 6.9 7.8 7.5 7.8 7.9

8.5 9.0

In The Absence of Cells In The Absence of Cells

8.1 8.5

Table 2. Percent _+95% confidence interval of control growth of Chlorella vulgaris after 96 h exposure to pH modified Bristol's Medium containing 0.89 ~M Cd

to 44.2 +- 0.18%-37.1 +- 0.17%, but again, Table 1 indicates that these reductions are consistent with pH affects alone rather than reductions resulting from cadmium. In the narrow pH range of 7.5 to 8.0, the effects of cadmium toxicity are mitigated against. The respective percents of control growth were 79.4 ___ 0.14 and 122.4 ___ 0.19 rather than the expected 50% reduction resulting from this cadmium treatment alone. A graph of the effects of pH alone and pH plus cadmium on the growth of Chlorella vulgaris is shown in Figure 1.

Discussion

In evaluating the effects of multiple toxicants in the aquatic environment it is first necessary to evaluate the action of each toxicant alone, on the target species. Following this, the toxicants should be tested in combination. These combination studies are used to determine if the toxicants act in either a synergistic, additive, or antagonistic fashion. An additional possibility is that they have no influence on each other's toxic action. In this study the toxic action of altering the pH of the medium on the growth of Chlorella vulgaris

Effects of Cadmium on Chlorella vulgaris

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Fig. 1. The effects of pH alone and pH plus 0.89 p,M cadmium on the growth of the green alga Chloreh'a vulgaris

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Adjusted pH of The Bristol's Medium

was first determined, and followed by evaluating the affects of altered pH in combination with a challenge of the heavy metal cadmium. The dosage of cadmium selected (0.89 ~M) had previously been determined (Rosko and Rachlin 1977) to reduce population growth of this alga, at a control pH of 6.9, by approximately 50%. It is clear, from the data presented in both Table 1 and Figure 1, that pH has a dramatic effect on the growth of Chlorella vulgaris; acid pH values in the range of 3.0 to 5.0 retard population growth by 72.3-44.4%. Even a pH of 6.2 results in a growth reduction of approximately 9%. Mackie (1989) demonstrated this effect of acidic pH on aquatic biota by showing that an increase in the hydrogen ion content of water had a significant effect on benthic invertebrates (Hyalella azteca, Amnicola limosa, Enallagma sp., Pisidium casertanum, and P. compressum). Further, a marked reduction in the hydrogen ion content or an increase in hydronium ion (increased alkalinity) of the water column should result in a negative affect on population growth. At pH values 8.3-9.0, population growth of Chlorella vulgaris cultures were reduced by 53.9-65.8%. However, the pH values of 7.5 and 8.0 actually stimulated population growth by approximately 25 to 20% over control values, indicating that slight increases in alkalinity might actually favor this organism. Combined effects of increased hydrogen ion content and heavy metal toxicity should provide greater toxicity than only acidic conditions. This results from increased acidity ("acid precipitation") causing mobilization of metal ions from the bound sequestered state to the active free ion state (Rai et al. 1981; Titus and Pfister 1982; Xian and Shokohifard 1989; Mosello et al. 1989). This increase in the avail-

ability of free ions should resuIt in greater toxicity to the aquatic biota. However, this does not take into account any effect of the increased hydrogen ion concentration itself, nor does :it address the question of whether this increased toxicity is manifest under conditions in which all, or most, of the metal ions are already in a "free" available state. The data presented in Table 2 and Figure I directly bears upon this latter concern. Here a concentration of the heavy metal cadmium, known to reduce the population growth of Chlorella vulgaris by 50%, was applied to these algal cultures simultaneously with adjustments in the hydrogen or hydronium ion content of the growth medium. The results confirm the earlier finding (Rosko and Rachlin ~977) that at the control pH of 6.9 an approximate 50% reduction in population growth is obtained (the actual value being 55,2% of control growth). If the effects of increased acidity is either synergistic or additive to that of metal toxicity alone, then under these combined conditions reductions in population growth greater than 50% would be anticipated. The results show that at acid pH values of 3.0, and 4.0, combined with 0.89 ~tM Cd, resulted in growth reductions of 67.7% and 72.3%. This concentration of cadmium when tested in medium whose pH was adjusted to 5,0 resulted in a growth reduction of 55.9%; at the control pH we achieve a reduction in growth of only 44.8%. The acid pH values of 3.0, 4.0, and 5.0 appear to be either synergistic or additive to the toxic actions of the cadmium alone. The anticipated joint toxicity for these acid pH values in combination with this level of cadmium would result in an average reduction in population growth of 78.1% if these toxicants were synergistic (Visviki and Rachlin 1991). Since the actual results, although greater than for cadmium alone, were less than this

508 predicted value, we should actually assume an additive rather than synergistic response. In a similar manner, if this level of cadmium were synergistic with the action of the alkaline pH values of 8.3, 8.5 and 9.0, we would calculate an average reduction in growth of 76.1%. Since the actual reductions in population growth were respectively 55.8%, 59.4%, and 62.9%, we again suggest an additive rather than a synergistic response. However, examination of Tables 1 and 2, and Figure 1 shows that the toxicity responses of the algal populations to c a d m i u m and either the acidic pH values of 3.0 and 4.0, or the alkaline pH values of 8.3, 8.5, and 9.0 can be explained by pH alone. That is the results of the combination of metal and pH mimic the results of pH alterations alone. This can be explained as the result of respective competition between H + and O H - ions and free Cd 2§ cations for critical cellular binding sites (Peterson et al. 1984). This then leaves the combination of pH 5.0 and 0.89 IxM Cd as the only remaining additive interaction. Of particular interest is the observation that within the narrow pH range of 7.5-8.0, there is mitigation against cadmium toxicity. At pH 7.5 plus the cadmium exposure population growth is actually 79.4% of control, a reduction of only 20.6% rather than the expected 50% anticipated from this level of cadmium exposure. At a pH value of 8.0, there is no longer any evident cadmium toxicity. Whether these results represent some form of ionic competition for critical cellular binding sites, or some other cellular detoxification mechanism (Rai et al. 1981, 1990; Rachlin et at. 1982b, 1984, 1985) is at present unknown. The observation that increased acidity enhanced copper toxicity to the blue-green alga Oscillatoria redekei, but retarded its effect to Aphanizomenon gracile (Luderitz and Nicklisch 1989) might even indicate that the actual effect of pH on cation toxicity is species specific.

Acknowledgments. This research was supported in part by PSCCUNY Grant #669185, and National Institutes of Health Minority Biomedical Research Support Program Grant #GRS 3SO 6 RRO 8225/05 $2/442-664 to J. W. Rachlin.

References

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J.W. Rachlin and A. Grosso Mackie GL (1989) Tolerances of five benthic invertebrates to hydrogen ions and metals (Cd, Pb, A1). Arch Environ Contam Toxicol 18:215-223 Mosello R, Calderoni A, Tartari GA (1989) pH-related variations in trace metal concentrations in Lake Orta (Italy). Science Total Environ 87/88:255-268 Peterson HG, Healey FP, Wagemann R (1984) Metal toxicity to algae: A highly pH dependent phenomenon. Can J Fish Aquat Sci 41:974-979 Puiseux-Dao S (1989) "Phytoplankton model" in ecotoxicology. In: Boudou A, Ribeyre F (eds) Aquatic ecotoxicology: Fundamental concepts and methodologies. Vol II. CRC Press Inc, Boca Raton, FL, pp 164-185 Rachlin JW, Farran M (1974) Growth response of the green algae Chlorella vulgaris to selective concentrations of zinc. Water Res 8:575-577 Rachlin JW, Warkentine B, Jensen TE (1982a) The growth responses of Chlorella saccharophila, Navicula incerta and Nitzschia elosterium to selected concentrations of cadmium. Bull Torr Bot Club 109:129-135 Rachlin JW, Jensen TE, Baxter M, Jani V (1982b) Utilization of morphometric analysis in evaluating response of Plectonema boryanum (Cyanophyceae) to exposure to eight heavy metals. Arch Environ Contam Toxicol 11:323-333 Rachlin JW, Jensen TE, Warkentine B (1983) The growth response of the diatom Navicula incerta to selected concentrations of the metals: cadmium, copper, lead and zinc. Bull Torr Bot Club 110:217-223 (1984) The toxicological responses of the alga Anabaena flos-aquae (Cyanophyceae) to cadmium. Arch Environ Contam Toxicol 13:143-151 , - - , (1985) Morphometric analysis of the response of Anabaena flos-aquae and Anabaena variabilis (Cyanophyceae) to selected concentrations of zinc. Arch Environ Contam Toxicol 14:395-402 Rai LC, Gaur JP, Kumar HD (1981) Phycology and heavy metal pollution. Biol Rev 56:99-151 Rai LC, Jensen TE, Rachlin JW (1990) A morphometric and X-ray energy dispersive approach to monitoring pH-altered cadmium toxicity in Anabaena flos-aquae. Arch Environ Contam Toxicol 19:479-487 Rosko JJ, Rachlin JW (1977) The effect of cadmium, copper, mercury, zinc and lead on cell division, growth, and chlorophyll a content of the chlorophyte Chlorella vulgaris. Bull Tort Bot Club 104:226-233 Strum W, Morgan JJ (1981) Aquatic chemistry: An introduction emphasizing chemical equilibria in natural waters. 2nd ed. John Wiley & Sons, New York Titus JA, Pfister RM (1982) Effects of pH, temperature and Eh on the uptake of cadmium by bacteria and an artificial sediment. Bull Environ Contam Toxicol 28:697-704 Visviki I, Rachlin JW (1991) The toxic action and interactions of copper and cadmium to the marine alga Dunaliella minuta, in both acute and chronic exposure. Arch Environ Contam Toxicol 20:271-275 Warkentine BE, Rachlin JW (1986) A test of a proposed organizational framework for the ordering of algal toxicity responses. Bull Tort Bot Club 113:12-15 Xian X, Shokohifard GI (1989) Effect of pH on chemical forms and plant availability of cadmium, zinc, and lead in polluted soils. Water Air Soil Pollut 45:265-273

Manuscript received October 4, 1990 and in revised form December 7, 1990.