Toxicity testing using immobilized algae

Aquatic Toxicology, 14 (1989) 345-352 Elsevier

345

AQT 00360

Toxicity testing using immobilized algae J o h n Bozeman, Ben K o o p m a n and Gabriel Bitton Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL, U.S.A. (Received 19 September 1988; revision received 27 January 1989; accepted 27 January 1989)

Alginate-immobilized and free Selenastrum capricornutum Printz cells were compared for use in algal toxicity assays. Chemicals investigated were cadmium, copper, Glyphosate, Hydrothol, Paraquat, pentachlorophenol, and sodium dodecyl sulfate. Toxicity was characterized in terms of growth inhibition, with growth measured by in vitro chlorophyll fluorescence. Sensitivity of the immobilized and free algal assays to copper, cadmium and pentachlorophenol was similar. However, immobilization substantially reduced the toxicity of Hydrothol, Paraquat, and Glyphosate to the algae. Sodium dodecyl sulfate disrupted the alginate matrix and therefore its toxicity could not be determined successfully with the immobilized algal assay. Key words: Toxicity; Bioassay; Algae; Selenastrum; Herbicide; Heavy metal; Immobilization; Surfactant

INTRODUCTION

Interest in immobilized cell technology has grown rapidly in the past fifteen years. While most efforts have been directed toward bacteria, considerable attention has also been given to eukaryotic animal and plant cells, including algae (Mattiason, 1983). Applications proposed for immobilized algae include hydrocarbon production for fuels (Bailliez et al., 1985), removal of nitrogen and phosphorus from wastewater (Chevalier and de la Noiie, 1985), and photoproduction of ammonia and hydrogen (Brouers and Hall, 1986; Jeanfils and Loudeche, 1986). A previously unexplored application for immobilized algae is in ecotoxicity testing. Protocols employing free algal ceils have been extensively used for toxicity assessment, particularly for heavy metals and herbicides (Turbak et al., 1986). Immobilized systems could expand the utility of algal toxicity assays. For example, a test alga could be encapsulated and exposed to toxic effluent under field conditions, then retrieved for laboratory analysis of growth or other physiological

Correspondence to." B. Koopman, Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL 32611, U.S.A. 0166-445X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

346 characteristics. Endpoints of immobilized algae assays may be determined directly (without dissolving the solid matrix) by chlorophyll extraction or physiological measurements such as oxygen evolution or 14C uptake. Matrices of calcium alginate can be readily dissolved by exposure to hexametaphosphate, which has a high affinity for calcium. In this manner, the entrapped cells are freed, allowing cell counts to be carried out. Another feature of immobilized cells is the ability to overcome problems with colored or turbid effluents. Cells could be contacted with a toxic sample in the dark, then transferred to standard media under illumination for a period of growth. The favorable storage characteristics of immobilized algae (Tamponnet et al., 1985) could allow inocula to be distributed from central culturing facilities, relieving smaller laboratories of the burden of culture maintenance and also promoting interlaboratory standardization. In the present study, we report the use of alginate-immobilized algae for testing the toxicity of cadmium, copper, pentachlorophenol, sodium dodecyl sulfate, and three herbicides (Glyphosate, Hydrothol, and Paraquat). Toxicity of the chemicals to freely suspended algal cells in the same media was also determined for comparison. MATERIALS AND METHODS The culture of Selenastrum capricornutum Printz was obtained from the USEPA, Corvallis, OR. Glyphosate (99.9°/0) and Paraquat (99o70) were obtained from the Pesticides and Industrial Chemicals Repository of the U S E P A (Research Triangle Park, NC); SDS (95o70), CuSO4 5H20 and P C P from Sigma (St. Louis, MO); CdCI2 2 . 5 H 2 0 from Fisher (Fairlawn, N J); and Hydrothol 191 (commercial grade, 53%) from Pennwalt (Philadelphia, PA). Glassware was acid washed in 0.2 N HC1. Glassware used with hydrophobic compounds was also rinsed in acetone. Algal cultures were grown to an absorbance of 0.01 at 750 nm in Bold's Basal Medium (Bold and Wynne, 1978) using 125 ml Erlenmeyer flasks stoppered with cotton plugs. This medium was chosen because it provided satisfactory growth rates and did not react with the alginate matrix. E P A Algal Assay Bottle Test medium (Miller et al., 1978), T A P medium (Gorman and Levine, 1965), and Sun Nitrate medium (Israel et al., 1978) caused precipitates to form or dissolved the alginate beads. All other growth conditions were the same as in the toxicity tests (see below). The volume of algal culture harvested was determined according to the following equation: Vc -

0.05 Va, D750

(1)

where Vc = volume of algal culture harvested by centrifugation, D 7 5 o = absorbance of cell culture at 750 nm through a 1-cm path length, and Va -- volume of alginate

347 (usually 35 ml). This ensured that a constant initial concentration of algae was present in all preparations. The coefficient 0.05 was chosen empirically. Sodium alginate (Type VI, Sigma) was dissolved in warm distilled water to form a 4°7o w / v solution. The solution was autoclaved for 13 min at 121°C and 1520 kPa. Standardization of this procedure is needed as different alginate sources have different gel porosities (Tanaka et al., 1985) and ion affinities (Morris, 1986). The selected volume of algal culture was centrifuged for 15 min at 1400 x g, the supernatant discarded, and the algal pellet transferred into 35 ml of alginate solution. Aliquots of this mixture, after thorough stirring, were loaded into a 5-ml syringe fitted with a # 25-G needle, and extruded dropwise into an autoclaved solution of 2°/0 (w/v) CaCl2 2H20. Approximately 25 beads were made from each 1.0 ml of the algae-alginate mixture. The newly formed beads were held at 4°C to allow gel hardening to take place. Beads were used within 18 to 36 h of their preparation. Individual beads made in this fashion were almost colorless, but within several days under favorable growth conditions the beads became noticeably green in color. The suspension of free algal cells for toxicity testing was prepared by harvesting a volume of algal culture equal to that harvested for bead preparation. The algal pellet was transferred into 35 ml of distilled water. Thus, the same initial concentration of algae was present in both the immobilized and free cell preparations. The toxicity tests were conducted in 50 ml Erlenmeyer flasks. To each flask, 20 ml of Bold's Basal Medium was added, followed by the addition of toxicant. At this time either five beads or 200/zl of algal suspension were added. The flasks were incubated at 26°C in shaking water baths for 3.5-4.0 days. A bank of six fluorescent tubes (40 W, cool white) spaced 12 cm apart, 70 cm above the flask bottoms was used as a constant light source. The intensity and uniformity of illumination were checked with an integrating r a d i o m e t e r / p h o t o m e t e r equipped a quantum sensor (LI-188, Licor, Lincoln, NE). Light flux was 45.6 _+ 3.0/zE m - 2s - 2 over the water bath area. Four replicates were used for each toxicant concentration, as well as four controls with no toxicant. Experimental pairs (an immobilized set and a free set) were randomized on a pair of shaking water baths. At the end of the incubation period, the beads from each flask were retrieved and placed in 25 ml of methanol at r o o m temperature in the dark. Extraction of chlorophyll was continued for 24-48 h. T h o u g h freely permeable to methanol, the beads did not dissolve in this solvent, as indicated by the fact that they remained firm during the extraction period and did not add turbidity to the medium. Free algae were recovered from the appropriate flasks by filtration through W h a t m a n G F / C glass fiber filters (1.2 #m pore size). Each filter was then placed in 25 ml of methanol for chlorophyll extraction in the same manner as the beads. Chlorophyll was determined using a Sequoia-Turner model 111 fluorometer (Palo Alto, CA) equipped with a 365 nm primary filter (no. 7-60) and a 595 nm secondary filter ( # 110-820 (25)). Cuvette temperature was maintained at 20°C using a constant temperature flow-through cell. This technique (Schmidt, 1986; Benecke et al., 1982)

348 worked well for the extracts from both free and immobilized ceils. Extracted chlorophyll could also have been measured spectroscopically using absorbance (Tailing and Driver, 1963); however, the chlorophyll levels observed were at the lower limit of spectroscopic detection. Sample replicates were divided randomly into four series. The percent inhibition o f chorophyll production at each toxicant concentration was calculated according to: I, °70 -

z • t F-- c~ e AFc

× 100,

(2)

where AFc and AFe are the changes in relative fluorescence of the control and experimental flasks, respectively, during the incubation period. Inhibitions > 95°7o or <5°7o were discarded prior to further analysis of the data. Percent inhibition values were normalized using the gamma function (Johnson et al., 1974), where: % inhibition F = 1 0 0 - % inhibition

(3)

Relationships between log I' and concentration were found by least squares linear regression and used to estimate the ICso values. RESULTS AND DISCUSSION A typical set of results, showing in this case the toxicity of Glyphosate to immobilized algae, is displayed in Fig. 1. The change in relative fluorescence, AFe, is plotted along the left axis at the top of the figure. Corresponding values of percent inhibition are shown along the right axis. As indicated, the percent inhibition increased in curvilinear fashion with increasing Glyphosate concentration. The transformed data (log I') were linearly related to Glyphosate concentration (bottom Fig. 1). The median inhibitory concentration (26.3 mg/l) was found at a log I~ of zero from the line of best fit. This analysis was repeated for the other chemicals under investigation, giving the IC50 values listed in Table I. The toxicity of SDS could not be measured successfully because it disrupted the alginate matrix of the beads. There was no significant difference in copper toxicity between free and immobilized cells. This was somewhat surprising as it was expected that the copper ions would complex with the negatively charged portions of the calcium alginate polysaccharide matrix. The expected complexation may have been prevented because the ionized sites in the alginate matrix were occupied by calcium ions. Calcium binds more strongly to the alginate than does copper. This is indicated by studies of bead dissolution rates under the influence of chelating agents (Dainty et al., 1986) and by magnesium (0.072 and 0.066 nm, respectively) bind less strongly.

349

T

70

30 .-

<3 50

......... 50"~ __=

LI_

30

~

70

0.4 0.2 L o~-0.2 -0.4 -0.6 -0.8 0

o . . . . . . . . . . . . . . . . . . . . . . . . .

o

a,

1'5 ' 2St,coo Glyphosote,mg/L I

i

35 I

Fig. 1. Toxicity of Glyphosate to immobilized algae. Top: effect of Glyphosate on chlorophyll production, as measured by relative fluorescence; bottom: transformation of percent inhibition data to give a linear relationship with concentration. Error bars are _+ 1.0 S.D.

models illustrating the interaction of alginate and calcium (Rees et al., 1982). The most stable gels are formed from strontium, calcium and barium, which have ionic radii of 0.097, 0.099 and 0.134 nm, respectively. Smaller ions, such as copper and magnesium (0.072 and 0.066 nm, respectively) bind less strongly. The results for cadmium would seem to agree with this hypothesis, as the ICs0 for immobilized cells was one-fourth greater than the ICso for free cells. Like copper,

TABLE I Toxicity of several chemicals to free and immobilized algae." Toxicant

Cadmium Copper Glyphosate Hydrothol Paraquat Pentachlorophenol

IC5o, mg/1 a Immobilized cells

Free cells

12.9 6.0 26.3 0.39 7.8 0.19

10.5 6.7 7.8 0.055 1.8 0.17

_+ 1.3 ___ 0.60 b _+ 1.5 _+ 0.10 _+ 2.4 _ 0.008 b

aMean + S.D. bMeans not significantly different at P < 0 . 0 2 5 .

_+ 0.87 _+ 0.15 _+ 1.5 _+ 0.036 + 0.29 _+ 0.041

350

the cadmium ion has a 2 + charge. At 0.097 nm, its ionic radius is similar to that of calcium, possibly giving it an affinity for the alginate matrix similar to that of calcium. However, the greater concentration of calcium in the medium may limit cadmium binding to the alginate. The organic toxicants Glyphosate, Hydrothol and Paraquat were substantially less toxic to the immobilized cells than to the free cells. These compounds are relatively hydrophilic. Paraquat carries a 2 + charge whereas the other two compounds exist mostly as 2- anions. In contrast, pentachlorophenol showed only a slight decrease in toxicity toward the immobilized cells. Pentachlorophenol has the highest molecular weight of the compounds tested and is relatively hydrophobic. It has a pKa of 4.74 (Westall et al., 1985) and thus carries a negative charge (1-) at pH 7, the initial pH of the assays. The sensitivity of the immobilized algal assay is compared to that of alternative toxicity assays in Table II. Immobilized S. capricornutum, while less sensitive to T A B L E 11 Sensitivity of immobilized algae assay in comparison to other toxicity assays. Toxicant

Method

1C50, m g / l

Reference

Cadmium

Immobil. S. capricornutum S. capricornutum S. capricornutum

10.5 0.04 0.04 12-106 6.6 0.048-0.058 0.04-0.06 0.03-0.04 0.032-2 21.8 0.004 127 0.37 1.5 2.4 300- > 1000 4 5 - > t000 6.8 > 5000 50- > 5000 20-2500 0.19 0.001 1-1.5 1.2

This study Walker, 1987 Blaise et al., 1986 Walker, 1987 This study Turbak et al., 1986 Walker, 1987 Blaise et al., 1986 Walker, 1987 This study Turbak et al., 1986 Walker, 1987 This study Mudge et al., 1986 Unpublished data b Verschueren, 1983 Verschueren, 1983 This study Unpublished data b Verschueren, 1983 Verschueren, 1983 This study Verschueren, 1983 Walker, 1987 Unpubl. data b

Copper

Microtox Immobil. S. capricornutum

S. capricornutum S. capricornutum S. capricornutum Microtox Glyphosate Glyphosate a Hydrothol

Paraquat

S. capricornutum S. capricornutum Microtox Immobil. S. capricornutum Mixed algal culture Microtox Chlorococcum sp.

Dunaliella tertiolecta Immobil. S. capricornutum Microtox

PCP

Chlorococcum sp. Dunaliella tertiolecta Immobil. S. caprieornutum C. pyrenoidosa Microtox Microtox

aAs R o u n d u p . bVoiland, G., G. Bitton and B. K o o p m a n , Univ. of Florida.

351 H y d r o t h o l t h a n free cells, a r e still m o r e sensitive t h a n o t h e r a s s a y t e c h n i q u e s such as M i c r o t o x ( V o i l a n d , B i t t o n a n d K o o p m a n , u n p u b l i s h e d d a t a , U n i v e r s i t y o f F l o r i d a ) a n d 0 2 p r o d u c t i o n o r g r o w t h i n h i b i t i o n in algae o r o t h e r species ( V e r s c h u e r e n , 1983). This is also t h e case for P a r a q u a t . In the case o f c a d m i u m , P C P , a n d G l y p h o s a t e , the i m m o b i l i z e d algal test was less sensitive t h a n o t h e r algal assays, h o w e v e r , this a s s a y was still m o r e sensitive t h a n M i c r o t o x . IC5o values f o r c o p p e r are h i g h e r t h a n for m o s t o t h e r tests, algal o r b a c t e r i a l . It has b e e n s h o w n in the p r e s e n t s t u d y t h a t i m m o b i l i z e d algae can be used successfully for a s s a y o f a q u a t i c t o x i c i t y d u e to h e a v y metals a n d o r g a n i c s . E n c a p s u l a t i o n o f the algal cells in a l g i n a t e reduces their sensitivity, b u t this d r a w b a c k m a y be o f f s e t b y the a d v a n t a g e s o f i m m o b i l i z e d systems in m a n y cases. F u r t h e r m o r e , the i m m o b i l i z e d a l g a e c o m p a r e d f a v o r a b l y to a l t e r n a t i v e m i c r o b i a l toxicity tests. W h i l e this test c o u l d easily be m o d i f i e d for interspecies c o m p a r i s o n o f the effects o f a q u a t i c t o x i c a n t s , algal species p r o d u c i n g large a m o u n t o f e x t r a c e l l u l a r p r o d u c t s (as f o u n d in s o m e c y a n o p h y t e s such as Nostoc) m i g h t require m o d i f i c a t i o n o f the test p r o c e d u r e . Survival o f m i c r o b e s in toxic e n v i r o n m e n t s (e.g., c o n t a m i n a t e d soils) m a y be e n h a n c e d b y i m m o b i l i z a t i o n , e n h a n c i n g their p o t e n t i a l f o r b i o d e g r a d a t i o n of chemical pollution. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y the N a t i o n a l Science F o u n d a t i o n t h r o u g h G r a n t no. CES-8619073. T h e assistance o f G e o r g e V o i l a n d a n d W i l l i a m Davis is g r a t e f u l l y acknowledged.

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