Factors Affecting the Magnitude of Toxicant Interactions in Microbial Bioassays

GLENN W. STRATTON, Environmental Microbiology Laboratory, Department of Biology, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada B2N 5E3

Abstract

A toxicant interaction method was used to study the effects of various bioassay parameters on interaction responses obtained in microbial bioassays. The fungus Pythium ultirnun was employed as the test organism, and was exposed to various combinations of the fungicide captan and several organic solvents, using a poisoned agar technique. In all cases the fungicide and solvents interacted synergistically toward culture growth. For most experiments acetone was used as the test solvent. Where pH and temperature were altered, the magnitude of the interaction response between captan and acetone increased dramatically as the pH or temperature was raised from 4.5 to 7.5, or 15 to 3WC, respectively. This corresponded to similar increases in the culture growth rate and decreases in the toxicity of captan. When the medium composition was changed, interaction magnitudes were again greatest in media eliciting the fastest growth rate. These media also yielded the lowest captan toxicity. The largest interaction magnitudes occurred with V8 juice agar, followed by corn meal agar, potato dextrose agar, and malt extract agar. When the solvent used in the interaction experiments was changed, a similar response was obtained, in that the greatest interaction magnitudes occurred in systems eliciting the lowest captan toxicity. The largest magnitudes were measured with acetone, followed by hexane, ethanol, dimethylformamide, methanol, and dimethyl sulfoxide. The significance of these data in toxicant interaction bioassays is discussed.

INTRODUCTION

Microbial bioassays are an integral part of any program designed to evaluate the environmental impact of potential toxicants. However, most microbial bioassays share a weakness with bioassays involving other organisms in that they usually test only the effects of individual toxicants rather than toxicant combinations. Any given toxicant is rarely, if ever, found in the natural environment in total isolation from other toxicants, or from other chemicals capable of interacting with it and altering its toxicity. Therefore, in order to obtain a more accurate picture of any compound’s biological impact, it must also be tested in combination with other xenobiotics likely to be found with it in the

Toxicity Assessment: An International Journal Vol. 4, 425-435 (1989) 0 1989 John Wiley & Sons, Inc. CCC 0884-8181/89/040425-011$04.00

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environment. Unfortunately, toxicity interaction studies put pressures on laboratory resources and require unique modifications in methodol- ogy and data interpretation.

Although the importance of interaction research is generally recognized (National Research Council, 19821, few attempts have been made at standardizing these bioassay procedures. There are various ways of interpreting interaction data from microbial bioassays in order to determine synergistic or antagonistic responses (Aoyama et al., 19871, but most have been adapted from other disciplines (Stratton et al., 1982). Recently, a general method has been proposed for identi- fying and classifying toxicant interactions in microbial bioassays (Stratton, 1988). This method is simple to use and can be employed to study the combination effects of both water-soluble and water-insolu- ble chemicals in most test systems. It was developed during research into the effects of solvent-pesticide interactions in fungal bioassays (Stratton et al., 1982; Stratton 1985, 1986a, 1986b, 19871, interactions that have also been well documented in other test systems (Dalela et al., 1979; Bowman et al., 1981; Mac and Seelye, 1981). One important potential use of any toxicant interaction method is determining the effects that various bioassay parameters have on the basic interaction response. This information is critical in standardizing methods and procedures associated with interaction bioassays.

To date, some preliminary information is available on the effects of various cultural and incubation parameters, such as pH, tempera- ture, and medium composition, and solvent-pesticide interaction responses in fungal bioassays (Stratton, 1986a, 1986b, 1987). Changes in these parameters have no significant effect on the basic type of interaction noted, whether synergism, antagonism, or an additive response. However, some effect on the magnitude or size of the interaction response is suggested. No other studies are available on factors affecting interaction data. The purpose of the present study was to provide more detailed information on factors affecting the magni- tude of toxicant interactions, since such data are important in stan- dardizing bioassays where it is desirable to have interaction re- sponses that are clearly defined and easily identified. This was accomplished by evaluating the effects of pH, temperature, medium composition, and solvent type on solvent-fungicide interactions in the fungus Pythium ultimum.

MATERIALS AND METHODS

Test Organism

The fungus Pythium ultimum (Mastigomycotina) was used as the test organism. Cultures were obtained from the Department of Environ-

FACTORS AFFECTING TOXICANT INTERACTIONS/427

mental Biology, University of Guelph, Guelph, Ontario, Canada, and maintained on Difco potato dextrose agar (pH 5.5 2 0.2; Difco Laboratories, Detroit, MI USA) a t 30 f 05°C.

Test Chemicals The pesticide used in all interaction experiments was the fungicide captan (3a,4,7,7a-tetrahydro-2[(trichloromethylthio~l-lH-isoindole-l,- 3(2H)-dione, technical grade 96.8% purity, Chipman Chemical Co., Stoney Creek, Ontario, Canada). Acetone was the solvent employed in pH, temperature, and medium composition studies (glass-distilled pesticide research grade, Caledon Laboratories Ltd., Georgetown, Ontario, Canada). In the solvent type study, acetone was replaced with methanol, hexane (glass-distilled pesticide research grade, Caledon Laboratories Ltd.), ethanol (absolute, Commercial Alcohols Ltd., Gat- ineau, Quebec, Canada), dimethyl sulfoxide (DMSO), and N,N-di- methylformamide (DMF; reagent grade, Fisher Scientific, Fair Lawn, NJ, USA). Solvent concentrations are given as percent (v/v) and captan concentrations as parts per million (mg L-') of active ingre- dient.

Fungal Bioassay

The fungitoxicity of solvent and solvent-captan mixtures was deter- mined using a poisoned agar technique (Stratton et al., 1982). Captan solution and solvent were added to molten agar growth medium (50"C), and then mixed on a rotary shaker for 2 min in sealed glass containers to prevent solvent loss by volatilization. The medium was dispensed as 10-mL aliquots into petri dishes and later inoculated with mycelial disks (8-mm diameter) taken from the outer margin of fresh stock culture plates. Plates were incubated in Precision Model 815 refriger- ated incubators (Precision Scientific, Chicago, IL, USA) until control growth reached a diameter of 50-70 mm, at which time all plates were examined and growth recorded as colony diameter. Growth data were analyzed as outlined below. All treatments were replicated five times and each experiment repeated at least three times.

Captan was treated at concentrations of 2.5, 5.0, 7.5, and 10.0 ppm. For pH, temperature, and medium composition studies, each concentration of captan was interacted with 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% acetone. For the solvent type study, acetone was replaced with either ethanol (0.1, 0.25, 0.5, 1.0, and 2.5%), methanol (0.1, 0.5, 1.0, 1.5, and 2.0%), hexane (1.0, 5.0, 7.5, 10.0, and 25.0%), DMSO (1.0, 1.5, 2.0, 2.5, and 3.0%), or DMF (0.5, 0.75, 1.0, 1.25, and 1.5%). In all cases, captan was dissolved in the appropriate test solvent and an

428lSTRATTON

amount of this stock solution was added to the test medium in order to reach both the captan concentration and the lowest solvent level desired for that particular solvent. Solvent concentrations greater than that were obtained by adding the appropriate volume of solvent directly to the test medium. Interaction data were analyzed as outlined below.

For the pH study, each individual captan-acetone combination was interacted with P. ultimum at four separate pH values: 4.5, 5.5, 6.5, and 7.5 (all 20.2). The medium pH was adjusted prior to the addition of test chemicals by aseptically adding filter sterilized 0.1N NaOH or 0.1N HC1 to a known volume (either 50 or 100 mL) of sterile medium. An incubation temperature of 30 2 05°C was used, and the medium employed was potato dextrose agar. Pythium ultimum was also grown at the four pH values in the absence of toxicants in order to obtain growth curve data.

For the temperature study, each individual captan-acetone com- bination was interacted toward P . ultimum using four separate incu- bation temperatures: 15,20,25, and 30°C (all 20.5"C). Potato dextrose agar, pH 5.5, was used as the test medium. Pythium ultimum was also grown at the various temperatures in the absence of toxicants in order to obtain growth curve data.

For the medium composition study, each captan-acetone mixture was tested with Pythium ultimum using four agar media: potato dextrose agar (PDA), corn meal agar (CMA), malt extract agar (MEA) (Difco Laboratories, Detroit, MI, USA), and V8 juice agar (V8A) (200 mL of V8 vegetable juice, 3.0 g CaC03, 15 g agar, and 800 mL of water). The pH of all test media was adjusted to 5.5, as outlined above. All plates were incubated a t 30°C. Pythium ultimum was also grown on each medium in the absence of toxicants in order to obtain growth curve data.

For the solvent type study, PDA (pH 5.5) was used as the test medium. All plates were incubated at 30°C.

Data Analysis

Growth curve data were obtained by measuring the mean colony diameter at various time intervals. These curves were analyzed using the linear regression analysis program of the Stat-Pac microcomputer statistics analysis package (Walonick Associates, Minneapolis, MN, USA). The growth rate for each bioassay system was then calculated as mm of growth (increase in colony diameter) per hour, and plotted against the test parameter being studied.

FACTORS AFFECTING TOXICANT INTERACTIONS/&?g

Captan-solvent interactions were analyzed using a method out- lined in detail elsewhere (Stratton et al., 1982; Stratton, 1988). In this method, growth in each individual captan-solvent mixture was com- pared to that in the appropriate solvent control (same concentration of solvent minus the captan). A net corrected percent inhibition was calculated according to the equation

Corrected inhibition = 100

x 100) Growth in captan-solvent mixture

Growth in solvent control

These net corrected inhibitions were then compared to a reference value ( t test at p = 0.05) and used to determine interaction response (Stratton et al., 1982; Stratton, 1988).

For each separate bioassay system, captan toxicity was deter- mined by taking the mean of the corrected inhibition values falling within the additive captan-solvent interaction range. Values result- ing from synergistic or antagonistic responses are unreliable and cannot be used to determine captan toxicity (Stratton et al., 1982).

The magnitude of captan-solvent interactions was determined by calculating the differences between the mean additive inhibitions (captan toxicity) and the highest net corrected inhibition recorded for that particular series, if synergism was observed, or the lowest corrected inhibition if antagonism occurred. This gives a measure of the absolute difference between the additive and nonadditive re- sponses.

RESULTS AND DISCUSSION

Captan and each of the solvents tested interacted synergistically toward growth of P. ultimum regardless of the bioassay system studied. With acetone, synergism occurred a t levels above 1.0-1.5%. There was no effect of pH, temperature, or medium composition on the basic captan-acetone interaction. With ethanol, methanol, DMF, hexane, and DMSO, synergism occurred at solvent concentrations above 0.1, 0.1, 0.75, 1.0, and 2.0%, respectively. These data are consistent with results from previous solvent-fungicide interaction studies using this organism (Stratton et al., 1982; Stratton 1985, 1986a, 1986b, 1987). Although all captan concentrations yielded the same interaction responses, results for 10.0 ppm captan were the most consistent. This is because 10.0 ppm captan causes levels of inhibition that fall within the range yielding the most reliable interaction data

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Fig. 1. Effect of pH on selected test parameters involving the interaction between acetone and 10.0 ppm captan. Growth rate (*), captan toxicity (O), and interaction magnitude (0).

(Stratton, 1988). Consequently, these data are used to illustrate the patterns discussed below.

Although pH, temperature, medium composition, and solvent type had no effect on the class of captan-solvent interaction obtained, i.e., synergism, there was a pronounced effect of these parameters on the toxicity of captan itself, and on the size or magnitude of the interaction response (both calculated as outlined above ).

The effect of pH on some aspects of the captan-acetone interaction are outlined in Fig. 1. The culture growth rate was lowest a t pH 4.5 and increased as the pH was raised to 7.5. Such a growth pattern is common in fungi (Moore-Landecker, 1982), and is probably due to pH effects on either the biochemistry of cell surfaces (Griffin, 1981), the availability of metallic ions (Moore-Landecker, 1982), and/or cellular permeability (Moore-Landecker, 1982). The increase in growth of P . ultimum was associated with a pronounced increase in the magnitude of the captan-acetone synergistic effect from a value of approximately 10% at pH 4.5 to >95% at pH 7.5 (Fig. 1). At the same time, the toxicity of 10.0 ppm captan decreased from around 90% at pH 4.5 to '<5% at pH 7.5 (Fig. 1). Therefore, captan is more toxic toward P . ultimum at lower pH values where the culture is growing poorly. This increased

FACTORS AFFECTING TOXICANT INTERACTIONS/&? 1

susceptibility is probably associated with some pH-mediated growth stress. Under these conditions, the presence of a solvent capable of enhancing captan toxicity (synergism) has a weak effect on the interaction magnitude, possibly because the culture is already se- verely stressed and is growing so poorly that further inhibition goes unnoticed.

In contrast, when the culture is growing rapidly under more ideal conditions, it is less susceptible to captan (Fig. 1). Under these condi- tions the addition of a solvent capable of enhancing captan toxicity has a far more pronounced effect on the interaction magnitude, possibly by causing membrane leakage, which could result in an increased uptake of captan into the cells. Since the culture is growing so well, any increase in inhibitory effects becomes quite noticeable and is easily measured. This is only one possible explanation for the data outlined in Fig. 1. Alternately, the pH response may be due to changes in chemical speciation of the toxicants used and/or macromolecules on the surface of fungal cells. However, as discussed below, the same response phenomenon occurs when other parameters capable of al- tering the culture growth rate are varied, which suggests that a more generalized theory, such as that outlined, is necessary to explain these data. Further research is required to adequately address this problem.

The effects of temperature on the growth rate of P. ultimum, the toxicity of 10.0 ppm captan, and the magnitude of interaction between captan and acetone are outlined in Fig. 2. The growth rate was lowest at 1 5 ° C and increased as the temperature was raised to 30°C. This is a common temperature growth pattern for fungi, and is probably related to alterations in transport phenomena and other biochemical reactions (Moore-Landecker, 1982). Again, an increase in growth rate was accompanied by an increase in the magnitude of the synergistic interaction and a decrease in the toxicity of 10.0 ppm captan (Fig. 2). The interaction magnitude increased from a value of approximately 10% at 15°C to 50% at 30"C, while the toxicity of captan decreased from a value of around 85 to 40% over the same temperature range. In this series of experiments the medium composition and pH were standard- ized (PDA, pH 5.5). When growing under optimal temperature condi- tions, P. ultimurn is again more susceptible to the addition of compounds capable of synergizing captan toxicity. This too may be due to the generalized theory presented above for pH effects, although other factors may be involved.

When the nutritional status of the culture was altered by chang- ing the medium composition, a trend in results similar to that noted for pH and temperature was obtained (Fig. 3). Media that elicited the best growth of P. ultirnum (in the other VSA, CMA, PDA, and MEA) also

*

0 1 I I I

15 20 25 30

Temperature ("C)

Fig. 2. Effect of temperature on selected parameters involving the interaction between acetone and 10.0 ppm captan. Growth rate (*), captan toxicity (O), and interaction magnitude (0).

elicited the highest interaction magnitudes but the lowest captan toxicity. In these experiments the incubation temperature and pH were held constant at 30°C and pH 5.5. Although the inhibitory effects due to 10.0 ppm captan varied from approximately 85% with MEA down to 30% with V8A, the range in interaction magnitudes was lower (Fig. 3).

It appears that anything capable of affecting culture growth, such as pH, temperature, and medium composition, has a similar effect on a xlture's toxicity response to a given toxicant and its combination with a synergizing chemical. The general pattern that emerges is that interactions are more pronounced when cultures are growing at faster rates. This has a significant implication in bioassays, since it allows a researcher to make interaction responses easier to identify by ensuring optimal growth conditions for the microorganism tested. However, it also means that toxicant interactions will be harder to identify during in situ bioassays, when microbes are seldom growing under optimal conditions. Further research is required on the mode of action of toxicant interactions before any definitive explanation can be made for these data.

The chemical used to synergize a given test toxicant seems to be unimportant. In the present study, when acetone was replaced in the

FACTORS AFFECTING TOXICANT INTERACTIONS433

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Fig. 3. Effect of medium composition on selected parameters involving the interac- tion between acetone and 10.0 ppm captan.

interaction mixture by other solvents, the same general trend in captan-solvent interaction response was evident (Fig. 4). That is, systems eliciting the lowest captan toxicity always elicited the great- est magnitude of interaction (Fig. 4). In this case, the greatest

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Fig. 4. Effect of solvent type on selected parameters involving the interaction of solvents with 10.0 ppm captan.

magnitude occurred with acetone, followed by hexane, ethanol, DMF, methanol, and DMSO. For these experiments all growth parameters were standardized using PDA, pH 5.5, and an incubation temperature of 30°C. The relationship between the toxicity of .captan and the interaction magnitude is independent of the actual toxicity of the solvents themselves. When tested alone, the solvents do not yield a toxicity sequence the same as the sequence given above for the interaction magnitude. Ethanol is the most toxic solvent to growth of P . ultimum, followed by DMF, methanol, acetone, DMSO, and hexane (Stratton, 1985). The significance of this pattern of response is hard to evaluate, due to the overall lack of information on solvent-pesticide and other toxicant interactions.

CONCLUSIONS

A bioassay system employing the fungus P . ultimum and mixtures of the fungicide captan and various organic solvents has been used to demonstrate a relationship between culture growth and the size or magnitude of toxicant interaction responses. Changes in bioassay parameters, such as pH, temperature, and medium composition, that result in increases in culture growth rate also elicit larger interaction magnitudes (Figs. 1-3) . This makes the identification and classi- fication of interaction responses easier, especially when using micro- bial bioassay procedures designed specifically for toxicant interaction studies (e.g., Stratton et al., 1982; Stratton, 1988). In the present study the size of the interaction magnitude was also affected by the solvent used in captan-solvent mixtures (Fig. 4 ) . Although these data may be partially explained by relating culture susceptibility to growth, any definitive explanations require further research into the basic mecha- nisms responsible for toxicant interactions. Such information is pres- ently unavailable.

This research was supported by the Natural Sciences and Engineering Research Council of Canada.

References

Aoyama, I., H. Okamura, and M. Yagi. 1987. The interaction effects of toxic chemical combinations on Chlorella ellipsoidea. Toxic. Assess. 2:341-355.

Bowman, M.C., W.L. Oller, and T. Cairns. 1981. Stressed bioassay systems for rapid screening of pesticide residues. Part I: Evaluation of bioassay systems. Arch. Environ. Contam. Toxicol. 10:9-24.

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Dalela, R.C., S.K. Bansal, A.K. Gupta, and S.R. Verma. 1979. Effect of solvents on in vitro pesticides inhibition of ATPase in certain tissues of Labeo rohitn. Water Air Soil Pollut. 11:201-205.

Griffin, D.H. 1981. Fungal Physiology. John Wiley & Sons, New York, 131 p. Mac, M.J., and J.G. Seelye. 1981. Potential influence of acetone in aquatic bioassays

testing the dynamics and effects of PCBs. Bull. Environ. Contam. Toxicol. 27:359-367. Moore-Landecker, E. 1982. Fundamentals of the Fungi (2nd ed.) Prentice-Hall Inc.,

Eaglewood Cliffs, NJ 275 p. National Research Council. 1982. The need for new directions in toxicological funding.

National Resource Council of Canada, Ottawa. Pub. no. 18983. Stratton, G.W. 1985. The influence of solvent type on solvent-pesticide interactions in

bioassays. Arch. Environ. Contam. Toxicol. 14:651-658. Stratton, G.W. 1986a. The effect of pH on fungitoxic interactions between a solvent and

pesticide. Water Air Soil Pollut. 28:393-405. Stratton, G. W. 1986b. Medium composition and its influence on solvent-pesticide

interactions in laboratory bioassays. Bull. Environ. Contam. Toxicol. 36807-814. Stratton, G.W. 1987. Influence of temperature on solvent-pesticide interaction effects

towards fungi. Water Air Soil Pollut. 35195-206. Stratton, G.W. 1988. Method for determining toxicant interaction effects towards

microorganisms. Toxic. Assess. 3:345-353. Stratton, G.W., R.E. Burrell, and C.T. Corke. 1982. Technique for identifying and

minimizing solvent-pesticide interactions in bioassays. Arch. Environ. Contam. Toxi- col. ll:437-445.