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A comparison of microbial bioassays for the detection of aquatic toxicants
Water Res. Vol. 17, No. 12, pp. 1757-1762, 1983 Printed in Great Britain
0043-1354/8353.00+0.00 Pergamon Press Ltd
A COMPARISON OF MICROBIAL BIOASSAYS FOR
THE DETECTION OF AQUATIC TOXICANTS GORDON A. McFETERS I*, PAMELA J. BOND I, SUSAN B. O L s o N l t
and Y. T. TCHAN2 tDepartment of Microbiology, Montana State University, Bozeman, MT 59717, U.S.A. and 2Department of Microbiology, University of Sydney, Sydney NSW, Australia 2006 (Received February 1983) A~tract--The toxicity of 35 test chemicals was analyzed using two microbial bioassay systems. The
commercially available Microtox Toxicity Analyzer SystemTM and the two-organism procedure of Tchan were used to determine the concentration of test chemicals resulting in a 50% reduction in response (ECs0). Both of the tests employed a luminescent bacterium while the procedure of Tchan also utilized an alga. Results from the two microbial tests were compared with available data obtained with fish toxicity bioassays and with each other. The MicrotoxTM procedure was somewhat more sensitive than the Tchan bioassay in detecting most of the test chemicals and fish bioassays were generally more sensitive than either of the microbial tests. As a notable exception, photosynthesis-inhibiting herbicides were detected at remarkably lower concentrations with the procedure of Tchan than any of the other bioassays. Potential applications for these tests are discussed. Key words--toxicity bioassay, algae, bacteria, luminescence
INTRODUCTION
A variety of bioassays using fish and other aquatic organisms are presently applied to detect toxic chemicals (Maciorowski et al., 1980). Microorganisms have been used to evaluate various toxicological concerns in aquatic systems. The Ames Test, using bacteria, has been applied to the detection of mutagens in wastewater, drinking water and surface water in South Africa (Grabow et al., 1980). Workers in Germany (Hertkorn-Obst and Frank, 1979; Bringman and Kuhn, 1975; Axt, 1973) have used physiological measurements of bacteria to monitor toxic materials in water. The activities of bacterial populations in chemostat cultures (Mayfield et al., 1980) and in fresh water microcosms (Ferebee and Guthrie, 1973) were studied to evaluate the effects of toxic materials, including metals and herbicides, on microorganisms. In situ microbial processes have also been investigated using Chesapeake Bay water and sediments (Mills and Colwell, 1977). Algae have also proved useful in detecting metals (Wong and Beaver, 1980), crude oil compounds (Armstrong and Calder, 1978) and a range of materials including herbicides, pesticides and metals (Turbak et al., 1979). Recently, luminous bacteria have been used in toxicity testing. For example, in 1979 the Microtox Toxicity AnalyzerT M (Beckman Instruments, Inc.) was placed on the market. This system employs
lyophilized preparations of a luminous marine bacterium as the test organism and a specially designed photometric instrument. The test developed by Tchan, which employs both a marine alga and a luminous bacterium (Tchan et al., 1975; Tchan and Chiou, 1977) has potential to rapidly screen for the presence of toxic chemicals. Because this procedure uses both a photosynthetic eukaryote and a heterotrophic prokaryote, it offers the additional advantage of biological diversity while retaining the simplicity and rapidity of microbial test procedures. The results obtained using the microbial test systems need to be correlated with those of other established bioassays employing a wide range of representative toxic substances. Some published reports of this type have evaluated the sensitivity of the Microtox T M system in determining the toxicity of pure compounds and complex effluents. It has also been reported that the sensitivity of a microbial toxicity bioassay system was generally less than fish bioassays (Chang et al., 1981). However, a systematic comparison of the Microtox T M test and the procedure of Tchan using a wide spectrum of test compounds that are potential toxicants in aquatic environments has not been done. For that reason, the purpose of the present investigation was to carry out such a correlation and relate that comparative data base to similar information, where available, from fish toxicity bioassays.
*Author to whom all correspondence should be addressed. tPresent address: Biospherics, 4928 Wyaconda, Rockville, MD 20852, U.S.A. 1757
MATERIALS AND METHODS Compounds and substances that were examined in this
1758
GORDON A. MCFETERSet al.
study were analyzed for toxicity by two bioassay methods. The Microtox Model 2055 Toxicity Analyzer SystemTM (Beckman Instruments Inc., Carlsbad, CA) system utilizing a luminous test bacterium (Bulich and Isenberg, 1980) and the two organism procedure proposed by Tchan (1977) were both used. The Microtox TM system
The Microtox TM test was performed as specified by Beckman Instruments, Inc. Dilutions of test chemicals were added to suspensions of the bacterium and incubated for 5 min at 15 + 0.1°C prior to the measurement of luminous activity in the model 2055 MicrotoxTM instrument. These values were compared with the luminous activity of identical control aliquots of the bacterial suspension not exposed to test compounds. The resulting numerical expression is referred to as the percent normalized light level. Analyses of all substances tested in this manner were repeated 3-5 times. The two-organism test system
The bacterium used in this test system (Photobacterium phosphorium, NZl 1D, obtained from K. Nealson, Scripps Institute of Oceanography, La Jolla, CA) was cultivated on sea water-yeast-peptone medium (SWYP) (Tchan and Chiou, 1977), pH 7.4. Frequent reisolation of this bacterium on SWYP agar was required to maintain a culture with a high level of luminescence since reversion to the "dark" form was a constant problem. Cultures were grown at 24°C and stored on SWYP agar medium at 5°C. Bacterial cultures (18 h) were grown in sterile SWYP broth at 24°C to an absorbancy (Ar00) of 0.30 prior to use. The alga, Dunaliella tertiolecta, provided by T. Shiroyama (EPA, Corvallis), was cultivated with continuous shaking in F/2 broth (Guillard and Ryther, 1962), pH 6.8 at 24°C under continuous illumination from four cool white fluorescent lights to an absorbancy (A660) of 0.30. In the performance of most such bioassays, five concentrations of the test chemical were prepared in sterile NaC1 (2.348~o) solutions. Equal volumes (11.25 ml) of standardized bacterial and algal suspensions were mixed and 2.5 ml of a given concentration of the test chemical added. This was repeated at the time each analysis was performed with 2.5 ml sterile saline added to the controls. These were allowed to stand for 1 or 24 h at 24°C following complete mixing. After the 1 or 24-h exposure period, both control and experimental suspensions were used to fill glass scintillation vials. The vials were sealed with tight-fitting rubber stoppers. A hypodermic needle was inserted from the top in such a way as to prevent the retention of any air bubbles within the vials. The needles were then removed. The remainder of the bioassay was performed in a specially designed instrument called the Photobioluminometer (Tchan and Chiou, 1977). This instrument in its simplest form consisted of two light-tight sample compartments with ports in each that both open into a central compartment with a double mirror that rotated, exposing the chambers alternately to either a light source or a photomultiplier (pm) tube. A 100-W microscope lamp was used as the light source and the pm-tube output was monitored after amplification on a strip chart recorder. When one of the two vials containing suspensions of the test organisms was being exposed to the light, the other sample was exposed to the pro-tube. The position of the mirror was reversed every 10 s so that the test organisms were alternately illuminated or monitored for luminescence. In this way the algae generated oxygen by photosynthesis during the illumination phase that was used to generate bacterial luminescence during the measurement phase. The absolute oxygen requirement for bacterial luminescence (Nealson and Hastings, 1979) dictates that the fully functional photosynthetic mechanism of the algae and the oxidative metabolism of the bacteria are both required for the production of luminescence. The perturbation of any aspect of the
photosynthetic metabolism of the algae or the relevant metabolism of the bacteria by an added toxicant reduced the luminescence observed. The cycling of the instrument was continued for 20 min to allow the response of both control and experimental cell suspensions to stabilize. A mean response of the last five cycles was used to measure the response of the sample exposed to a test chemical and compared to that of the control; these values are referred to as percent control response. The tests of most chemicals were repeated 3-5 times. An improved bioluminometer has been constructed at the University of Sydney to analyze 10 samples simultaneously (Y. T. Tchan, personal communication). This instrument will greatly expedite the performance of the two-organism toxicity bioassay. Data reduction
The comparative bioluminescence response measured with each concentration of the chemicals used was plotted as one point on a semilogarithmic graph with the percent normalized luminescence or percent control response as the ordinate and the concentration of the test chemical as the log-scale abscissa. The concentration of the test chemical at the 50~ point of the resultant plot is referred to as the ECs0.
RESULTS AND DISCUSSION
Toxicity bioassays applied to aquatic environments are becoming more diversified and the use of microorganisms in aquatic toxicity tests has recently received increasing prominence (Maciorowsky et al., 1980). A m o n g the advantages are the relative simplicity, rapidity and cost of such procedures (Bulich and Isenberg, 1980; Chang et al., 1981). The use of algae (Wong and Beaver, 1981; Armstrong and Caldeer, 1978; Turbak et al., 1979) has the additional advantage of providing toxicity information relative to the phototrophic segment of aquatic communities. Inclusion of algae into a test procedure with bacteria (Tchan and Chiou, 1977) therefore, presents a unique toxicological probe. This approach includes a procaryotic heterotroph and a eukaryotic phototroph in which the physiological integrity of both organisms may be simultaneously determined in the presence of test chemicals or effluents. This system might be considered a synergistic microcosm. A comprehensive evaluation of this and similar bacterial procedures has not been performed despite the potential represented by these new measurements of toxicity. The data in Table 1 provide such a comparison with a wide range of purified compounds and agrichemical formulations assessed by both the two organism system of Tchan (Tchan and Chiou, 1977) and the Microtox T M procedure (Bulich and Isenberg, 1980). The EC50 values (the concentration of test compounds causing a 50~o reduction in bacterial light production) obtained for the battery of test compounds using the procedure of Tchan are compared with that from the Microtox T M assay in Table 1 and Fig. 1. Such comparisons are inherently complicated by differences between the two test systems; Microtox T M utilizing luminescent bacteria that were reconstituted from lyophilized preparations while the
Comparison of microbial bioassays for the detection of aquatic toxicants
1759
Table 1. Comparative effects of test compounds on toxicity bioassays Tchan assay ECs0* (1 h)
Test compounds Diuron (ARS)~ Diuron (ACG)¶ Monuron (AGC) Bufencarb (ARS) Simezine (AGC) HgCl., Sodium lauryl sulfate NaN 3 lsotox (ARS) Hydroquinone ZnSO4.7H20 CuSO4 CdCI 2 Malathion (AGC) 2-4D (ARS) BeSO4. 4H20 2,4,5-T (AGC) Cytrole-amitrole (AGC) K2CrO4 Toluene Phenol Formaldehyde NiCI2' 6H;O Benzene Ethyl alcohol DMSO Aroclor 1016 (ARS) Bromacil (ARS) Dichlorobenzene-O AgNO s (ARS) Heptachlor-epoxide (ARS) Roundup (AGC) 2,4,5-T (ARS) Acetone Methyl alcohol
Tchan assay ECs0* (24 h)
Tchan 1 h CVt
0.028 0.018 0.102
0.43 1.26 0.67 -0.05 0.36 . . 0.19
0.76 1.43 6.64 19.50 23.75 135.8 173.33 246.25 159.38 160.22 2600.0 227.0 375.0 461.67 740.0 880.0 3150.0 6342.0 12,500.0 19,400.0 56,833.33 .
0.035 -0.063 -1.115 -.
1.98 30.05 29.25 76.0 492.50 -17.88 246.5 3400.0 --163.75 19,000.0 326.6 -27,000.0 <5000.0 44,666.6
0.20 0.82 0.28 0.28 1.71 0.46 0.24 0.23 0.27 0.38 .
.
.
.
476.27 24.96 416.0 59.7 61.6 252.5 78.75 181.0 2412.5 33,833.0 39.5 904.17 22,900.0 4.11 55,575.0 103,400.0 2.05 6.65 10.25 20.62 25.0 17.56 157.0 18,250.0 56,715.0
. -----380.0 --
--
>200.0 -UA[ -.
16.38 UAI[ 228.67 0.256 238.33 0.06 1.19 112.60 UArl
.
0.40 0.29 0.29 0.29 0.36
--
Microtox Assay EC5o
.
.
Microtox CV? 0.13 -0.21 0.06 0.13 0.20 0.33 0.63 -0.58 0.57 0.48 0.71 0.21 0.10 0.63 0.24 0.08 0.10 0.24 1.67 0.61 0.11 0.15 0.05 0.62 1.14 0.35 0.06 0.06 0.15 0.75 0.13 0.02
Fish assays LDs0:~ 1.0-10.0 i.(~10.0
5.0 0.014).9 5.0~6.0 0 980 < 1.0 0.24-7.20 0.1-10.7 1.0 100.0 0.07-19.5
1.0-10.0 -29-133.0 23.0 50--100.0 10.(~100.0 ~ 10.~100.0 13,500.0
0.0002 -0.56 > 1000.0 > 1.0
*Concentration of test compound (mg 1-~) causing a 50% reduction in assay response. tCoefficient of variation; standard deviation/mean. :~LDs0 (rag I-I) results obtained from the literature. §Analytical Reference Standards obtained from Quality Assurance Section, U.S.EPA, Research Triangle Park, NC 27711, U.S.A. ¶Agrichemical grade. IIUnattainable; highest concentration available produced no measurable effect.
system of Tchan employed freshly cultured luminescent bacteria and algae. Despite these differences, the design and conditions of these bioassays are similar enough to warrant a direct comparison. Most
of the data used in this comparison are clustered along the line of equality in Fig. 1, suggesting good correlation of the two tests. The compounds that lie outside this pattern of grouping are the herbicides
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1760
GORDON A. McFEavats et al.
Simazine, Monuron, and Diuron with much lower ECs0 values obtained using the procedure of Tchan. This observation is reasonable since these are herbicides which inhibit photosynthesis so would be more readily detected with Tchan's procedure. These findings agree with earlier published reports by Tchan (Tchan et al., 1975; Tchan and Chiou, 1977) that the two-organism ~oassay procedure is highly sensitive in the detection of photosynthetic inhibiting herbicides. The organisms used in the procedure of Tchan were also exposed to many of the test chemicals for an extended period (24 h) in an effort to determine if the sensitivity of the procedure could be increased. It was proposed that this approach might satisfy some of the criteria of a chronic toxicity bioassay while using microorganisms. The results (Table 1) reveal that lower ECs0 values averaging a 44~ reduction, were attained with 7 of the 17 test chemicals examined in this way while some increased. It was also of interest to compare the precision of data obtained with the 1-h Tchan assay and the Microtox TM since most of the determinations were repeated at least three times. Coefficients of variation (CV = standard deviation divided by mean) for each of the tests that were repeated are shown in Table 1. The mean CV values for the 1-h Tchan results are 0.42 while they are 0.34 for the Microtox TM data, based on 21 and 32 samples respectively. This is understandable since Microtox TM is a highly formalized procedure utilizing standardized reagents and bacteria, hence less susceptible to variation. It can also be seen that the percentage of the CV values that are greater than 0.50 are approximately equal for the two procedures ( T c h a n = 19~; MicrotoxTM= 21%). It was somewhat surprising that several of the test compounds were detected at lower concentrations with Microtox TM since both microbial test procedures used essentially the same bacterium. This observation might be explained by the instruments used since the photobioluminometer was constructed with field use in mind and the electronics are not highly sophisticated, resulting in somewhat reduced sensitivity. Also, damage caused by the lyophilization process might render the Microtox TM bacteria more susceptible to the subsequent effects of toxic test chemicals than the freshly-cultured cell suspensions used in the Tchan procedure. Sub-lethal stress-injury has been described resulting in bacteria with a greater susceptibility to commonly used selective compounds than freshly grown cells (McFeters et al., 1982). The comparison of toxicity data obtained with different systems must be approached with great caution as noted by Brown (1980). For example, extrapolations from microbial systems to fish or from fish to humans are quantum jumps where different trophic levels are represented and physiological dissimilarities are large. However, such correlations are attempted here to support the development of an expanded toxicity data base in a cost and effort
efficient manner. The ECs0 results obtained in this study using the 1 h Tchan procedure are graphically compared with LDs0 values for fish bioassays (24--48 h) reported in the literature (Beckman, 1979; NIOSH, 1980; Handbook of Toxicology, 1956; The Merck Index, 1976; U.S. EPA, 1981). This comparison is seen in Fig. 2. The fish LDs0 data are represented as bars because these results were obtained from different laboratories, and the ranges of values available are large, sometimes spanning two orders of magnitude. Most of the test compounds were detected at lower concentrations by fish bioassays, hence appear above the lines of equality in Fig. 2. The herbicide Simazene had an ECs0 value obtained with the assay of Tchan significantly lower than fish LDs0 data for the same compound. However, it should be noted that the fish LDs0 values for another herbicide (2,4,5-T) were lower than the ECs0 from the Tchan assay. A similar comparison of Microtox TM ECs0 and fish LDs0 data is shown in Fig. 3. It can be seen that the data points are generally above the line of equality indicating somewhat lower fish LDs0 values. These findings suggest that the microbial bioassays employed are less sensitive in the detection of most of the compounds examined. At the same time, it should be pointed out that the magnitude of this difference is not as large, in most cases, as the range of fish data obtained from different laboratories and with different fish species. Considering the various factors that can cause significant variation in toxicity bioassays, such as water chemistry, it is reasonable that discrepancies in bioassay data from different laboratories are seen. For example, our ECs0 for Diuron as monitored with
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Comparison of microbial bioassays for the detection of aquatic toxicants
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REFERENCES
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Acknowledgements--We express our appreciation to Dr Martin Knittel for his suggestions concerning this study and Dr Evan Smouse for statistical consultations. The clerical and technical assistance of Jerrie Beyrodt, Debbie Powell and Marie Martin is also gratefully acknowledged. This study was funded by U.S. EPA grant No. R80725601 from the Environmental Research Laboratory, Corvallis, Oregon.
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1761
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103
104
Fish LD5o Fig. 3. Comparison of bioassay toxicity data for a variety of test chemicals using the Microtox TM procedure and fish test systems. The ECs0 values are expressed as mg l-1. The fish data were obtained from the literature. The ranges of fish LDs0 data were broad for the compounds represented as bars and narrow for those shown as dots. the two organism system was approximately one order of magnitude less than that found by Tchan (Tchan et al., 1975; Tchan and Chiou, 1977). Also, published Microtox TM data (Bulich and Isenberg, 1980) were somewhat lower than those reported here while inconsistent discrepancies are seen when comparing ECs0 results from Chang et al. (1981) with the data reported here and that of Bulich and Isenberg (1980). These comparisons provide evidence that microbiological test systems, including Microtox TM, are subject to some variation from laboratory to laboratory and so are not unlike fish bioassays in that regard. The findings reported here indicate that microbial test systems represent a potentially useful approach to the bioassessment of aquatic environments with regard to toxic substances. Furthermore, the detection of the photosynthetic inhibitors is a task where the two-organism assay system of Tchan was consistently more sensitive. The role of microbial toxicity bioassays is presently unclear. The greater sensitivity o f traditional fish toxicity tests for many compounds must be weighed against the cost and time effectiveness of microbial methods. Perhaps wider acceptance of these relatively new approaches will be gained in the future since they appear well suited for applications such as comprehensive multi-level testing schemes and as rapid screening tools. Greater sensitivity with these tests might also be achieved if toxic substances in water are suitably concentrated. This is a feasable approach since the quantity of water needed for microbial bioassays is small.
Armstrong J. E. and Calder J. A. (1978) Inhibition of light-induced pH increase and 02 evolution of marine microalgae by water-soluble components of crude and refined oils. Appl. envir. Microbiol. 35, 858-862. Axt G. (1973) Continuous toxicity measurements with bacteria. Vom Wasser 41, 409-414. Beckman (1979) Bulletin 6984. Beckman Microbics, 6200 El Camino Real, Carlsbad, CA. Bringman G. and Kuhn R. (1975) Pseudomonas bacteria inhibition test in the determination of water toxicity. Vom Wasser 44, 119-129. Brown V. K. (1980) Acute Toxicity in Theory and Practice, 159 pp. Wiley, Chichester. Bulich A. A. and Isenberg D. L. (1980) Use of the luminescence bacterial system for the rapid assessment of aquatic toxicity. Adv. Instrum. 80, 35--40. Chang J. C., Taylor P. G. and Leach F. R. (1981) Use of the Microtox assay system for environmental samples. Bull. envir, contain. Toxic. 26, 150-156. Ferebee R. N. and Guthrie R. K. (1973) The effects of selected herbicides on bacterial populations in an aquatic environment. Water Res. Bull. 9, 125-1135. Grabow W. O. K., Denkhaus R. and Van Rossum P. G. (1980) Detection of mutagens in wastewater, a polluted river and drinking water by means of the Ames Salmonella/Microsome Assay, S. Afr. J. Sci. 76, 118123. Guillard R. R. L. and Ryther J. H. (1962) Studies of marine planktonic diatoms, I. Cyclotella nana Husted and Detonula contervacae (Cleve) Gran. Can. J. Microbiol. 8, 229-239. Handbook of Analytical Toxicology (1969) (Edited by Sunshine I.). The Chemical Rubber Co. Handbook of Toxicology (1956) Vol. 1, Acute toxicities (Edited by Specter R. L.). Saunders Co, New York. Hertkorn-Obst V. U. and Frank H. K. (1979) Inhibition of nitrate reduction in B. stearothermophilus in vivo and the enzyme urease in vitro as a toxicological bioassay for surface water. Arch. HydrobioL 86, 523-532. Maciorowski A. G., Sims J. L., Little L. W. and Gerrard E. D. (1980) Bioassays--procedures and results. J. Wat. Pollut. Control Fed. 53, 974-993. Mayfield C. I., Inniss W. E. and Spain P. (1980) Continuous culture of mixed sediment bacteria in the presence of mercury. Water, Air Soil Pollut. 13, 335-349. McFeters G. A., Cameron S. C. and LeChevallier M. W. (1982) Influence of diluents, media and membrane filters on detection of injured waterborne coliform bacteria. Appl. envir. Microbiol, 43, 97-103. Mills A. L. and Colwell R. R. (1977) Microbiological effects of metal ions in Chesapeake Bay water and sediments. Bull. envir, contain. Toxic. 18, 99-103. Nealson K. H. and Hastings J. W. (1979) Bacterial bioluminescence: Its control and ecological significance. Microbiol. Rev. 43, 496-518. Niosh (1980) 1979 Registry o f Toxic Effects of Chemical Substances (Edited by Lewis R. S. and Tatke R. L), Vols I and II. U.S. Department of Health and Human Services. Tchan Y. T. and Chiou C. M. (1977) Bioassay of herbicides by bioluminescence. Acta Phytopath. Acad. Sci. Hung. 12, 3-11. Tchan Y. T., Roseby J. E. and Funnell J. R. (1975) A new
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rapid specific bioassay method for photosynthesis inhibiting herbicides. Soil Biol. Biochem. 7, 39-44. The Merck Index (1976) 9th Edition. Merck & Co., Inc. Turbak S. C., Olson S. B. and McFeters G. A. (1979) Developmental evaluation of rapid microbial bioassays for herbicides, pesticides and heavy metals present in the aquatic environment. Report No. R805383, 45 pp. U.S. Environmental Protection Agency, Corvallis, OR.
U.S. Environmental Protection Agency (1981) EPA analytical reference standards and supplemental data for pesticides and other organic compounds. EPA-600/2-81011. Wong S. L. and Beaver J. L. (1980) Algal bioassays to determine toxicity of metal mixtures. Hydrobiologia 74, 199-208.
0043-1354/8353.00+0.00 Pergamon Press Ltd
A COMPARISON OF MICROBIAL BIOASSAYS FOR
THE DETECTION OF AQUATIC TOXICANTS GORDON A. McFETERS I*, PAMELA J. BOND I, SUSAN B. O L s o N l t
and Y. T. TCHAN2 tDepartment of Microbiology, Montana State University, Bozeman, MT 59717, U.S.A. and 2Department of Microbiology, University of Sydney, Sydney NSW, Australia 2006 (Received February 1983) A~tract--The toxicity of 35 test chemicals was analyzed using two microbial bioassay systems. The
commercially available Microtox Toxicity Analyzer SystemTM and the two-organism procedure of Tchan were used to determine the concentration of test chemicals resulting in a 50% reduction in response (ECs0). Both of the tests employed a luminescent bacterium while the procedure of Tchan also utilized an alga. Results from the two microbial tests were compared with available data obtained with fish toxicity bioassays and with each other. The MicrotoxTM procedure was somewhat more sensitive than the Tchan bioassay in detecting most of the test chemicals and fish bioassays were generally more sensitive than either of the microbial tests. As a notable exception, photosynthesis-inhibiting herbicides were detected at remarkably lower concentrations with the procedure of Tchan than any of the other bioassays. Potential applications for these tests are discussed. Key words--toxicity bioassay, algae, bacteria, luminescence
INTRODUCTION
A variety of bioassays using fish and other aquatic organisms are presently applied to detect toxic chemicals (Maciorowski et al., 1980). Microorganisms have been used to evaluate various toxicological concerns in aquatic systems. The Ames Test, using bacteria, has been applied to the detection of mutagens in wastewater, drinking water and surface water in South Africa (Grabow et al., 1980). Workers in Germany (Hertkorn-Obst and Frank, 1979; Bringman and Kuhn, 1975; Axt, 1973) have used physiological measurements of bacteria to monitor toxic materials in water. The activities of bacterial populations in chemostat cultures (Mayfield et al., 1980) and in fresh water microcosms (Ferebee and Guthrie, 1973) were studied to evaluate the effects of toxic materials, including metals and herbicides, on microorganisms. In situ microbial processes have also been investigated using Chesapeake Bay water and sediments (Mills and Colwell, 1977). Algae have also proved useful in detecting metals (Wong and Beaver, 1980), crude oil compounds (Armstrong and Calder, 1978) and a range of materials including herbicides, pesticides and metals (Turbak et al., 1979). Recently, luminous bacteria have been used in toxicity testing. For example, in 1979 the Microtox Toxicity AnalyzerT M (Beckman Instruments, Inc.) was placed on the market. This system employs
lyophilized preparations of a luminous marine bacterium as the test organism and a specially designed photometric instrument. The test developed by Tchan, which employs both a marine alga and a luminous bacterium (Tchan et al., 1975; Tchan and Chiou, 1977) has potential to rapidly screen for the presence of toxic chemicals. Because this procedure uses both a photosynthetic eukaryote and a heterotrophic prokaryote, it offers the additional advantage of biological diversity while retaining the simplicity and rapidity of microbial test procedures. The results obtained using the microbial test systems need to be correlated with those of other established bioassays employing a wide range of representative toxic substances. Some published reports of this type have evaluated the sensitivity of the Microtox T M system in determining the toxicity of pure compounds and complex effluents. It has also been reported that the sensitivity of a microbial toxicity bioassay system was generally less than fish bioassays (Chang et al., 1981). However, a systematic comparison of the Microtox T M test and the procedure of Tchan using a wide spectrum of test compounds that are potential toxicants in aquatic environments has not been done. For that reason, the purpose of the present investigation was to carry out such a correlation and relate that comparative data base to similar information, where available, from fish toxicity bioassays.
*Author to whom all correspondence should be addressed. tPresent address: Biospherics, 4928 Wyaconda, Rockville, MD 20852, U.S.A. 1757
MATERIALS AND METHODS Compounds and substances that were examined in this
1758
GORDON A. MCFETERSet al.
study were analyzed for toxicity by two bioassay methods. The Microtox Model 2055 Toxicity Analyzer SystemTM (Beckman Instruments Inc., Carlsbad, CA) system utilizing a luminous test bacterium (Bulich and Isenberg, 1980) and the two organism procedure proposed by Tchan (1977) were both used. The Microtox TM system
The Microtox TM test was performed as specified by Beckman Instruments, Inc. Dilutions of test chemicals were added to suspensions of the bacterium and incubated for 5 min at 15 + 0.1°C prior to the measurement of luminous activity in the model 2055 MicrotoxTM instrument. These values were compared with the luminous activity of identical control aliquots of the bacterial suspension not exposed to test compounds. The resulting numerical expression is referred to as the percent normalized light level. Analyses of all substances tested in this manner were repeated 3-5 times. The two-organism test system
The bacterium used in this test system (Photobacterium phosphorium, NZl 1D, obtained from K. Nealson, Scripps Institute of Oceanography, La Jolla, CA) was cultivated on sea water-yeast-peptone medium (SWYP) (Tchan and Chiou, 1977), pH 7.4. Frequent reisolation of this bacterium on SWYP agar was required to maintain a culture with a high level of luminescence since reversion to the "dark" form was a constant problem. Cultures were grown at 24°C and stored on SWYP agar medium at 5°C. Bacterial cultures (18 h) were grown in sterile SWYP broth at 24°C to an absorbancy (Ar00) of 0.30 prior to use. The alga, Dunaliella tertiolecta, provided by T. Shiroyama (EPA, Corvallis), was cultivated with continuous shaking in F/2 broth (Guillard and Ryther, 1962), pH 6.8 at 24°C under continuous illumination from four cool white fluorescent lights to an absorbancy (A660) of 0.30. In the performance of most such bioassays, five concentrations of the test chemical were prepared in sterile NaC1 (2.348~o) solutions. Equal volumes (11.25 ml) of standardized bacterial and algal suspensions were mixed and 2.5 ml of a given concentration of the test chemical added. This was repeated at the time each analysis was performed with 2.5 ml sterile saline added to the controls. These were allowed to stand for 1 or 24 h at 24°C following complete mixing. After the 1 or 24-h exposure period, both control and experimental suspensions were used to fill glass scintillation vials. The vials were sealed with tight-fitting rubber stoppers. A hypodermic needle was inserted from the top in such a way as to prevent the retention of any air bubbles within the vials. The needles were then removed. The remainder of the bioassay was performed in a specially designed instrument called the Photobioluminometer (Tchan and Chiou, 1977). This instrument in its simplest form consisted of two light-tight sample compartments with ports in each that both open into a central compartment with a double mirror that rotated, exposing the chambers alternately to either a light source or a photomultiplier (pm) tube. A 100-W microscope lamp was used as the light source and the pm-tube output was monitored after amplification on a strip chart recorder. When one of the two vials containing suspensions of the test organisms was being exposed to the light, the other sample was exposed to the pro-tube. The position of the mirror was reversed every 10 s so that the test organisms were alternately illuminated or monitored for luminescence. In this way the algae generated oxygen by photosynthesis during the illumination phase that was used to generate bacterial luminescence during the measurement phase. The absolute oxygen requirement for bacterial luminescence (Nealson and Hastings, 1979) dictates that the fully functional photosynthetic mechanism of the algae and the oxidative metabolism of the bacteria are both required for the production of luminescence. The perturbation of any aspect of the
photosynthetic metabolism of the algae or the relevant metabolism of the bacteria by an added toxicant reduced the luminescence observed. The cycling of the instrument was continued for 20 min to allow the response of both control and experimental cell suspensions to stabilize. A mean response of the last five cycles was used to measure the response of the sample exposed to a test chemical and compared to that of the control; these values are referred to as percent control response. The tests of most chemicals were repeated 3-5 times. An improved bioluminometer has been constructed at the University of Sydney to analyze 10 samples simultaneously (Y. T. Tchan, personal communication). This instrument will greatly expedite the performance of the two-organism toxicity bioassay. Data reduction
The comparative bioluminescence response measured with each concentration of the chemicals used was plotted as one point on a semilogarithmic graph with the percent normalized luminescence or percent control response as the ordinate and the concentration of the test chemical as the log-scale abscissa. The concentration of the test chemical at the 50~ point of the resultant plot is referred to as the ECs0.
RESULTS AND DISCUSSION
Toxicity bioassays applied to aquatic environments are becoming more diversified and the use of microorganisms in aquatic toxicity tests has recently received increasing prominence (Maciorowsky et al., 1980). A m o n g the advantages are the relative simplicity, rapidity and cost of such procedures (Bulich and Isenberg, 1980; Chang et al., 1981). The use of algae (Wong and Beaver, 1981; Armstrong and Caldeer, 1978; Turbak et al., 1979) has the additional advantage of providing toxicity information relative to the phototrophic segment of aquatic communities. Inclusion of algae into a test procedure with bacteria (Tchan and Chiou, 1977) therefore, presents a unique toxicological probe. This approach includes a procaryotic heterotroph and a eukaryotic phototroph in which the physiological integrity of both organisms may be simultaneously determined in the presence of test chemicals or effluents. This system might be considered a synergistic microcosm. A comprehensive evaluation of this and similar bacterial procedures has not been performed despite the potential represented by these new measurements of toxicity. The data in Table 1 provide such a comparison with a wide range of purified compounds and agrichemical formulations assessed by both the two organism system of Tchan (Tchan and Chiou, 1977) and the Microtox T M procedure (Bulich and Isenberg, 1980). The EC50 values (the concentration of test compounds causing a 50~o reduction in bacterial light production) obtained for the battery of test compounds using the procedure of Tchan are compared with that from the Microtox T M assay in Table 1 and Fig. 1. Such comparisons are inherently complicated by differences between the two test systems; Microtox T M utilizing luminescent bacteria that were reconstituted from lyophilized preparations while the
Comparison of microbial bioassays for the detection of aquatic toxicants
1759
Table 1. Comparative effects of test compounds on toxicity bioassays Tchan assay ECs0* (1 h)
Test compounds Diuron (ARS)~ Diuron (ACG)¶ Monuron (AGC) Bufencarb (ARS) Simezine (AGC) HgCl., Sodium lauryl sulfate NaN 3 lsotox (ARS) Hydroquinone ZnSO4.7H20 CuSO4 CdCI 2 Malathion (AGC) 2-4D (ARS) BeSO4. 4H20 2,4,5-T (AGC) Cytrole-amitrole (AGC) K2CrO4 Toluene Phenol Formaldehyde NiCI2' 6H;O Benzene Ethyl alcohol DMSO Aroclor 1016 (ARS) Bromacil (ARS) Dichlorobenzene-O AgNO s (ARS) Heptachlor-epoxide (ARS) Roundup (AGC) 2,4,5-T (ARS) Acetone Methyl alcohol
Tchan assay ECs0* (24 h)
Tchan 1 h CVt
0.028 0.018 0.102
0.43 1.26 0.67 -0.05 0.36 . . 0.19
0.76 1.43 6.64 19.50 23.75 135.8 173.33 246.25 159.38 160.22 2600.0 227.0 375.0 461.67 740.0 880.0 3150.0 6342.0 12,500.0 19,400.0 56,833.33 .
0.035 -0.063 -1.115 -.
1.98 30.05 29.25 76.0 492.50 -17.88 246.5 3400.0 --163.75 19,000.0 326.6 -27,000.0 <5000.0 44,666.6
0.20 0.82 0.28 0.28 1.71 0.46 0.24 0.23 0.27 0.38 .
.
.
.
476.27 24.96 416.0 59.7 61.6 252.5 78.75 181.0 2412.5 33,833.0 39.5 904.17 22,900.0 4.11 55,575.0 103,400.0 2.05 6.65 10.25 20.62 25.0 17.56 157.0 18,250.0 56,715.0
. -----380.0 --
--
>200.0 -UA[ -.
16.38 UAI[ 228.67 0.256 238.33 0.06 1.19 112.60 UArl
.
0.40 0.29 0.29 0.29 0.36
--
Microtox Assay EC5o
.
.
Microtox CV? 0.13 -0.21 0.06 0.13 0.20 0.33 0.63 -0.58 0.57 0.48 0.71 0.21 0.10 0.63 0.24 0.08 0.10 0.24 1.67 0.61 0.11 0.15 0.05 0.62 1.14 0.35 0.06 0.06 0.15 0.75 0.13 0.02
Fish assays LDs0:~ 1.0-10.0 i.(~10.0
5.0 0.014).9 5.0~6.0 0 980 < 1.0 0.24-7.20 0.1-10.7 1.0 100.0 0.07-19.5
1.0-10.0 -29-133.0 23.0 50--100.0 10.(~100.0 ~ 10.~100.0 13,500.0
0.0002 -0.56 > 1000.0 > 1.0
*Concentration of test compound (mg 1-~) causing a 50% reduction in assay response. tCoefficient of variation; standard deviation/mean. :~LDs0 (rag I-I) results obtained from the literature. §Analytical Reference Standards obtained from Quality Assurance Section, U.S.EPA, Research Triangle Park, NC 27711, U.S.A. ¶Agrichemical grade. IIUnattainable; highest concentration available produced no measurable effect.
system of Tchan employed freshly cultured luminescent bacteria and algae. Despite these differences, the design and conditions of these bioassays are similar enough to warrant a direct comparison. Most
of the data used in this comparison are clustered along the line of equality in Fig. 1, suggesting good correlation of the two tests. The compounds that lie outside this pattern of grouping are the herbicides
A
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1760
GORDON A. McFEavats et al.
Simazine, Monuron, and Diuron with much lower ECs0 values obtained using the procedure of Tchan. This observation is reasonable since these are herbicides which inhibit photosynthesis so would be more readily detected with Tchan's procedure. These findings agree with earlier published reports by Tchan (Tchan et al., 1975; Tchan and Chiou, 1977) that the two-organism ~oassay procedure is highly sensitive in the detection of photosynthetic inhibiting herbicides. The organisms used in the procedure of Tchan were also exposed to many of the test chemicals for an extended period (24 h) in an effort to determine if the sensitivity of the procedure could be increased. It was proposed that this approach might satisfy some of the criteria of a chronic toxicity bioassay while using microorganisms. The results (Table 1) reveal that lower ECs0 values averaging a 44~ reduction, were attained with 7 of the 17 test chemicals examined in this way while some increased. It was also of interest to compare the precision of data obtained with the 1-h Tchan assay and the Microtox TM since most of the determinations were repeated at least three times. Coefficients of variation (CV = standard deviation divided by mean) for each of the tests that were repeated are shown in Table 1. The mean CV values for the 1-h Tchan results are 0.42 while they are 0.34 for the Microtox TM data, based on 21 and 32 samples respectively. This is understandable since Microtox TM is a highly formalized procedure utilizing standardized reagents and bacteria, hence less susceptible to variation. It can also be seen that the percentage of the CV values that are greater than 0.50 are approximately equal for the two procedures ( T c h a n = 19~; MicrotoxTM= 21%). It was somewhat surprising that several of the test compounds were detected at lower concentrations with Microtox TM since both microbial test procedures used essentially the same bacterium. This observation might be explained by the instruments used since the photobioluminometer was constructed with field use in mind and the electronics are not highly sophisticated, resulting in somewhat reduced sensitivity. Also, damage caused by the lyophilization process might render the Microtox TM bacteria more susceptible to the subsequent effects of toxic test chemicals than the freshly-cultured cell suspensions used in the Tchan procedure. Sub-lethal stress-injury has been described resulting in bacteria with a greater susceptibility to commonly used selective compounds than freshly grown cells (McFeters et al., 1982). The comparison of toxicity data obtained with different systems must be approached with great caution as noted by Brown (1980). For example, extrapolations from microbial systems to fish or from fish to humans are quantum jumps where different trophic levels are represented and physiological dissimilarities are large. However, such correlations are attempted here to support the development of an expanded toxicity data base in a cost and effort
efficient manner. The ECs0 results obtained in this study using the 1 h Tchan procedure are graphically compared with LDs0 values for fish bioassays (24--48 h) reported in the literature (Beckman, 1979; NIOSH, 1980; Handbook of Toxicology, 1956; The Merck Index, 1976; U.S. EPA, 1981). This comparison is seen in Fig. 2. The fish LDs0 data are represented as bars because these results were obtained from different laboratories, and the ranges of values available are large, sometimes spanning two orders of magnitude. Most of the test compounds were detected at lower concentrations by fish bioassays, hence appear above the lines of equality in Fig. 2. The herbicide Simazene had an ECs0 value obtained with the assay of Tchan significantly lower than fish LDs0 data for the same compound. However, it should be noted that the fish LDs0 values for another herbicide (2,4,5-T) were lower than the ECs0 from the Tchan assay. A similar comparison of Microtox TM ECs0 and fish LDs0 data is shown in Fig. 3. It can be seen that the data points are generally above the line of equality indicating somewhat lower fish LDs0 values. These findings suggest that the microbial bioassays employed are less sensitive in the detection of most of the compounds examined. At the same time, it should be pointed out that the magnitude of this difference is not as large, in most cases, as the range of fish data obtained from different laboratories and with different fish species. Considering the various factors that can cause significant variation in toxicity bioassays, such as water chemistry, it is reasonable that discrepancies in bioassay data from different laboratories are seen. For example, our ECs0 for Diuron as monitored with
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Fig. 2. Comparison of bioassay toxicity data for a variety of test chemicals using the procedure of Tchan and with fish test systems. EC~ values are expressed as mg l-J. The fish data were obtained from the literature. The ranges of fish LD~ data were broad for the compounds represented as bars and narrow for those shown as dots.
Comparison of microbial bioassays for the detection of aquatic toxicants
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REFERENCES
/
DiuronARS / /
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Acknowledgements--We express our appreciation to Dr Martin Knittel for his suggestions concerning this study and Dr Evan Smouse for statistical consultations. The clerical and technical assistance of Jerrie Beyrodt, Debbie Powell and Marie Martin is also gratefully acknowledged. This study was funded by U.S. EPA grant No. R80725601 from the Environmental Research Laboratory, Corvallis, Oregon.
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1761
SOS
2 I
I
I
I
i
i
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102
103
104
Fish LD5o Fig. 3. Comparison of bioassay toxicity data for a variety of test chemicals using the Microtox TM procedure and fish test systems. The ECs0 values are expressed as mg l-1. The fish data were obtained from the literature. The ranges of fish LDs0 data were broad for the compounds represented as bars and narrow for those shown as dots. the two organism system was approximately one order of magnitude less than that found by Tchan (Tchan et al., 1975; Tchan and Chiou, 1977). Also, published Microtox TM data (Bulich and Isenberg, 1980) were somewhat lower than those reported here while inconsistent discrepancies are seen when comparing ECs0 results from Chang et al. (1981) with the data reported here and that of Bulich and Isenberg (1980). These comparisons provide evidence that microbiological test systems, including Microtox TM, are subject to some variation from laboratory to laboratory and so are not unlike fish bioassays in that regard. The findings reported here indicate that microbial test systems represent a potentially useful approach to the bioassessment of aquatic environments with regard to toxic substances. Furthermore, the detection of the photosynthetic inhibitors is a task where the two-organism assay system of Tchan was consistently more sensitive. The role of microbial toxicity bioassays is presently unclear. The greater sensitivity o f traditional fish toxicity tests for many compounds must be weighed against the cost and time effectiveness of microbial methods. Perhaps wider acceptance of these relatively new approaches will be gained in the future since they appear well suited for applications such as comprehensive multi-level testing schemes and as rapid screening tools. Greater sensitivity with these tests might also be achieved if toxic substances in water are suitably concentrated. This is a feasable approach since the quantity of water needed for microbial bioassays is small.
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rapid specific bioassay method for photosynthesis inhibiting herbicides. Soil Biol. Biochem. 7, 39-44. The Merck Index (1976) 9th Edition. Merck & Co., Inc. Turbak S. C., Olson S. B. and McFeters G. A. (1979) Developmental evaluation of rapid microbial bioassays for herbicides, pesticides and heavy metals present in the aquatic environment. Report No. R805383, 45 pp. U.S. Environmental Protection Agency, Corvallis, OR.
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