Mutatox Test System

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ARTICLE NO. ES971594

38, 227–231 (1997)

Comparison of Acute Toxicity and Genotoxic Concentrations of Single Compounds and Waste Elutriates Using the Microtox/Mutatox Test System B. Hauser, G. Schrader, and M. Bahadir1 Institute of Ecological Chemistry and Waste Analysis, Hagenring 30, 38106 Braunschweig, Germany Received February 27, 1997

The Mutatox test has been developed by Microbics Corp., U.S.A., in addition to the widely used acute toxicity test Microtox. The Mutatox test indicates the presence of any material in a test sample that causes genetic damage to dark variants of the luminescent bacteria Vibrio fischeri. As the Microtox test is less timeconsuming and more cost-effective than the Mutatox test, the possibility of using the EC50 measured by Microtox for rangefinding of genotoxic concentrations for the Mutatox test was examined. Both tests were applied on single compounds and several waste elutriates. The genotoxic potential of two PAH metabolites—1-hydroxypyrene and 9-fluorenone-1-carboxylic acid—was detected. According to the present results the highest concentration of a sample in the Mutatox test should in general exceed the EC50 by about 5–10 times. Elutriates were submitted to analyses of TOC, heavy metals, phenols, and PAH; additionally GC/MS screening analyses were carried out. In most cases correlations of ecotoxicological effects with single contaminants were not possible, but it can be assumed that these effects were produced by the interaction of inorganic and organic compounds present in the elutriates. © 1997 Academic Press INTRODUCTION

The Mutatox test is a novel mutagenic bioassay which has been developed by Microbics Corporation (Carlsbad, CA) in addition to the widely known Microtox test and can be performed with the same analytical instrument. The Microtox test is an acute toxicity test measuring the ability of a test chemical to inhibit bioluminescence of the luminescent bacteria Vibrio fischeri. Several sample dilutions are incubated with luminescent bacteria for 5, 15, and/or 30 min at 15°C. The concentration of a test chemical which causes a 50% reduction of bacterial luminescence is called EC50. In contrast to the Microtox test, in the Mutatox test dark mutants of V. fischeri are used that have lost their ability to produce light. Exposure to genotoxic agents causes a reversion of dark mutants to the normal light-producing genotype. Samples are tested in two different culture media, of which one contains rat hepatic S9 enzymes 1 To whom correspondence should be addressed. E-mail: [email protected]. Internet: http://www.tu-bs.de/institute/oekochem/oekochem.html).

for metabolic activation of promutagenic compounds. Dilution series of the sample in the two media are incubated with dark mutants of V. fischeri at 27°C for 24 h. The light emission is measured after 12 h and then every 2 h. A sample is considered to be genotoxic if the dilution series contains at least two cuvettes exhibiting a light emission at least twice the average of simultaneously measured media controls. The objectives of this study were to investigate whether the EC50 measured in the Microtox test can be used for range finding of genotoxic concentrations in the Mutatox test. This would be interesting because the Microtox test is less timeconsuming and more cost-efficient than the Mutatox test. In addition to comparing genotoxic concentrations and EC50 of single compounds and waste elutriates, toxic and genotoxic effects of elutriates by analytical results are discussed as well. MATERIALS AND METHODS

The Microtox test was performed according to the manufacturer’s instructions (Microbics Microtox Manual, 1993). Samples were tested in eight dilutions; EC50 after 30 min incubation time are reported. The Mutatox test was conducted according to the Mutatox manual (Microbics Mutatox Manual, 1993); the mutagenic response of bacteria was determined after 12 h and then every 2 h until a total time of 24 h after test initiation. In order to detect growth-inhibition due to toxic effects of test chemicals or elutriates, every cuvette was diluted with 2 ml 2% NaCl after test end to reach an appropriate volume for measuring optical density at 590 nm. Optical density of samples was compared to media controls. TOC analyses in elutriates were run with a Dohrmann DC90 TOC analyzer equipped with a nondispersive infrared detector (NDIR). A 400 mg/liter potassium hydrogenphthalat standard was used for external calibration. Phenolindex was determined photometrically as blue-colored indophenolates after reaction with 2,6-dibromoquinone-4-chloroimide at pH 9.2. The metal contents of waste elutriates were investigated with ICP-OES: Jobin-Yvon 70 Plus, power 880 W, plasma gas flow 15 liters Ar/min, cross flow nebulizer, measuring by external calibration (standard solution: Alfa Products). Elutriates were

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analyzed for Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, P, Pb, S, Sn, Sr, Ti, V, and Zn. For determination of PAH and screening of organic contaminants by GC/MS 100 ml of elutriate was extracted three times with 20 ml of cyclohexane (J. T. Baker), and the pooled extracts were dried over anhydrous Na2SO4 and evaporated under reduced pressure to a final volume of 2 ml. For screening analyses the extraction was started at pH 12 of the elutriates; finally the pH of the aqueous phase was adjusted to 2. The determination of 16 EPA–PAH was conducted using a DB-5 capillary column (30 m, 0.25 mm i.d., 0.25 mm film thickness) and GC/MSD (HP 5890 II, 5970 B, Hewlett Packard) with injector KAS II (Gerstel). The analytical parameters were: injector temperature program: 80–12°C/s–300°C (2 min)– 12°C/s–350°C (2 min); oven temperature program: 60°C (4 min)–10°C/min–300°C (15 min); transfer line: 300°C; injection volume: 1 ml. Quantification was performed using a 16EPA–PAH standard (AccuStandard, New Haven) with selected ion monitoring (SIM). For screening analyses the same capillary column and GC/ MSD were used. The analytical parameters were: injector temperature: 280°C; oven temperature program: 60°C (5 min)– 10°C/min–280°C (15 min); transfer line: 280°C; injection volume: 1 ml. Screening analyses were carried out in full scan mode (60–450 amu). Samples were of different origin. Sample 153 was a filter dust from a waste incineration plant. Samples 155 and 156 both were residues of flue gas purification from a coking plant. The soil Clausthal came from a heavily PAH-contaminated ground near a tar site. From these samples, elutriates were prepared following German standard procedure DIN 38414-S4 with different elutriation media: distilled water (DEV-S4), distilled water with 2% (v/v) DMSO as carrier solvent (2% DMSO), groundwater, and two synthetically made salt solutions (Table 1). The salt solutions were adapted from naturally existing, TABLE 1 Analytical Results of Groundwater, IP9 (Q-brine), and IP21 (R-brine) Element

Groundwater (mg/liter)

IP9 (Q-brine) (mg/liter)

IP21 (R-brine) (mg/liter)

Al Ca Cu Fe K Mg Mn Na P S Zn

34.3 146 0.8 1.8 13.2 71.7 25.1 61.5 0.98 358 83.4

nd 190 nd nd 31,900 23,800 nd 90,900 nd 11,100 nd

nd 30.5 nd nd 23,000 95,700 nd 9,000 nd 7,900 nd

Note. As, B, Ba, Cd, Co, Cr, Hg, La, Mo, Ni, Pb, Rb, Sn, Sr, Ti, Tl, V, W, and Zr were not detected. nd, not detected.

TABLE 2 Analytical Results of Groundwater, IP9, and IP21 Elutriates of Sample 153

Element

Groundwater elutriate 153

IP9 elutriate 153

IP21 elutriate 153

Ba Ca Cd Cu K Mg Mo Na P Pb S Sr Zn

20.7 52,200 nd nd 5,180 nd 2.21 19,300 4.74 127 393.5 31.9 12.4

44.8 66,000 nd 3.59 55,800 2.43 2.83 71,800 11.8 2,040 99.5 58.9 57.0

16.5 31,600 19.3 2.86 27,000 76,200 3.53 17,000 8 429 19.5 13.4 73.4

Note. Al, As, B, Co, Cr, Fe, Hg, La, Mn, Ni, Rb, Sn, Ti, Tl, V, W, and Zr were not detected. nd, not detected.

geogeneous salt solutions found in salt mines (Eugster et al., 1980; Reichelt et al., 1995), representing a halit–polyhalitsaturated (invariant point IP 9: NaCl–Ca2K2Mg(SO4)4z2H2Osaturated, the so-called Q-brine) and a halit–polyhalit– carnallit–kainit–sylvin-saturated (IP21: NaCl–KCl–CaSO4– MgCl2–MgSO4-saturated, the so-called R-brine) salt solution in order to simulate elutriation media that might result from a penetration of water into a salt mine. Sample 153 was elutriated using groundwater and both salt solutions (Table 2). Samples 155 and 156 were elutriated with groundwater only (Table 3). Distilled water and distilled water containing 2% DMSO were the elutriation media for the soil Clausthal (Table 4). In addition to the waste and soil elutriates, some single compounds have been tested. Those were phenol (Merck, Darmstadt, Germany), fluoranthene (Supelco, Bellefonte, U.S.A.), and the two PAH metabolites 9-fluorenone-1-carboxylic acid (Aldrich, Steinheim, Germany) and 1-hydroxypyrene (Aldrich). RESULTS

Table 5 compares EC50 (Microtox) of samples with concentrations that gave a genotoxic response in the Mutatox test. Acute toxicity for luminescent bacteria is directly correlated with the decrease of bioluminescence and is usually expressed as EC50, whereas Mutatox results are presented as the range of concentrations which did restore bioluminescence of dark mutants. A direct correlation between these concentrations and the extent of mutagenity does not exist; in Mutatox test, results are only qualitative, not quantitative (Mutatox Manual). Authors usually refer to ‘‘yes/no’’ or ‘‘+/−’’ for mutagenity in order to indicate results of the Mutatox test. For better comparison, the concentration of single compounds or elutriates which induced

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maximum luminescence in the Mutatox test is also given (‘‘maximum luminescence at’’). This concentration does not automatically indicate the concentration of highest mutagenity. As S9 enzymes may alter ingredients of samples, only results in Mutatox medium without S9 are provided in Table 5. Further, the range of concentrations tested with Mutatox and concentrations which indicated a reduced optical density at 590 nm compared to media controls are indicated. In most cases genotoxic concentrations exceed EC50 deduced from Microtox. An exception is groundwater elutriates from samples 155 and 156 which reveal acute toxicity and genotoxicity in the same range of concentrations. Only with three of all the samples examined was detected a reduced optical density as an indication of growth-inhibition of bacteria. These samples were 1-hydroxypyrene and the IP9 and IP21 elutriates of sample 153 (Table 5). These apparently toxic concentrations of 1-hydroxypyrene and IP9 and IP21 waste elutriates largely exceed EC50, in the case of the IP9 waste elutriate by factor 190. Analytical Results Metal contents of groundwater and IP9 and IP21 elutriates of sample 153 are presented in Table 2. In these elutriates no organic contaminations have been found. Genotoxicity of elutriates is not explainable by single compound effects, as the detected heavy metals are not known to be genotoxic on their own. According to analytical results, elutriates have been remixed, demonstrating the same results as original elutriates. Obviously, genotoxic effects found in these samples are synTABLE 3 Analytical Results of Groundwater-Elutriates of Samples 155 and 156 Analytical parameter TOC (mg/liter) Element (mg/liter) Ba Ca Cd Co Cr Fe K Mg Na Ni Pb S Sn Zn Phenolindex (mg/liter) S16 EPA-PAH (mg/liter) GC/MS screening analyses

Groundwaterelutriate 155 5650

Groundwaterelutriate 156 10835

nd 42.2 nd 1.1 nd 54.1 3.1 18.6 0.25 nd nd 4987 nd nd nd

4.8 752 2.6 8.7 4.7 125 8.9 nd 22.1 4.0 4.7 5111 14.0 21.0 nd

10.5

318.9 (298.4 mg/liter naphthalene) Derivatives of naphthalenes and benzenes

Derivatives of naphthalenes and benzenes

Note. Al, As, B, Cu, Hg, La, Mn, Mo, P, Rb, Sr, Ti, Tl, V, W, and Zr were not detected. TOC, total organic carbon; nd, not detected.

TABLE 4 Analytical Results of DEV-S4-Elutriate and 2% DMSO-Elutriate of Soil Clausthal Analytical parameter TOC (mg/liter) Element (mg/liter) Al B Ba Ca Fe K Mg Mn Na S Si Sr Ti Zn Phenolindex (mg/liter) S16 EPA-PAH (mg/liter) GC/MS screening analyses

DEV-S4elutriate 224

2% DMSOelutriate 7214

3.78 nd 1.56 55.0 5.21 3.28 2.85 0.33 5.88 30.4 11.8 0.30 0.23 1.76 1.4 108.3

4.65 0.63 1.69 45.0 5.41 2.97 2.86 0.30 5.84 nd 11.61 0.33 0.25 2.02 1.9 129.4

Derivatives of phenols, naphthalenes, pyridines, and benzenes

Derivatives of phenols, naphthalenes, pyridines, and benzenes

Note. As, Cd, Cr, Cu, La, Rb, Pb, Tl, V, W, and Zr were not detected. nd, not detected.

ergistic or additive. Further results concerning sample 153 will be published elsewhere (G. Schrader et al.). Analytical results of samples 155 and 156 are presented in Table 3. Both elutriates contained high amounts of dissolved organic carbon, but low contents of toxic metals. Determination of PAH revealed low contents of low condensed compounds. In elutriate 156, 298.4 mg/l naphthalene was found, which is known to be mutagenic in the Mutatox test. Chromatograms of GC/MS screening analyses indicated about 150 different peaks; a library search indicated the presence of derivatives of naphthalenes and benzenes in both elutriates. It can be assumed that the total of organic contaminants is responsible for toxic and genotoxic effects of the elutriates, although effects of sample 155 cannot be assigned to a single compound. Nonadditive effects have not been found. The soil Clausthal came from a heavily PAH-contaminated ground near a tar site. For better transfer of PAH into the aqueous phase an additional elutriate was prepared with addition of 2% (v/v) DMSO as carrier. Both elutriates revealed very similar ecotoxicological and analytical results, except TOC (Table 4). The contents of toxic metals consisted in Al, Fe, and Zn, but concentrations were too low to explain an acute toxicity. A variety of organic contaminants was present. Phenols and PAH were quantified, and derivatives of naphthalenes, pyridines, and benzenes were detected from GC/MS screening analyses. No compound could be detected in such a high concentration that it could be the only cause of ecotoxicological effects. But as substances like phenols and PAH are present in the elutriates that have been proven to be genotoxic

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TABLE 5 Comparison of EC50 (Microtox) and Genotoxic Concentrations (Mutatox) of Single Compounds and Waste Elutriates

Compound/elutriate

EC50 (30 min) in Microtox test

Concentrations with reduced optical density in Mutatox test

Range of concentrations in Mutatox test

Phenol

29.5 mg/liter

—

0.39–200 mg/liter

Low acute toxicity

—

1.25–20 mg/liter

13.76 mg/liter

—

0.39–50 mg/liter

0.68 mg/liter

5 mg/liter

Fluoranthene 9-Fluorenone-1carboxylic acid 1-Hydroxypyrene DEV-S4-elutriate soil Clausthal 2% DMSO-elutriate soil Clausthal IP21-elutriate sample 153 IP9-elutriate sample 153 Groundwater elutriate sample 155 Groundwater elutriate sample 156

0.039–5 mg/liter

9.2 ml/liter

—

0.97–500 ml/liter

9.8 ml/liter

—

0.97–500 ml/liter

0.46 ml/liter

25–50 ml/liter

0.1–50 ml/liter

0.26 ml/liter

50 ml/liter

0.39–50 ml/liter

7.7 ml/liter

—

0.1–50 ml/liter

—

0.39–50 ml/liter

14 ml/liter

Genotoxic concentrations in Mutatox test 12.5–200 mg/liter (100 mg/liter) 1.25–10 mg/liter (5 mg/liter) 25–50 mg/liter (50 mg/liter) 0.313–2.5 mg/liter (2.5 mg/liter) 15.6–250 ml/liter (125 ml/liter) 15.6–250 ml/liter (250 ml/liter) 12.5–25 ml/liter (25 ml/liter) 25 ml/liter (25 ml/liter) 1.56–25 ml/liter (12.5 ml/liter) 3.13–25 ml/liter (12.5 ml/liter)

Note. Soil Clausthal, heavily PAH-contaminated soil from a tar site; sample 153, filter dust from a waste incineration plant; samples 155/156, residues of flue gas purification from a coking plant; OD, optical density. The concentration which induced maximum luminescence in the Mutatox test is given (maximum luminescence at). This concentration does not automatically indicate the concentration of highest mutagenity.

in the Mutatox test, the total of contaminants is likely to exert a genotoxic effect. DISCUSSION

1-Hydroxypyrene and 9-fluorenone-1-carboxylic acid have been isolated as metabolites of PAH-degrading bacteria and fungi cultures (Lange et al., 1994; Lambert et al., 1994; Kelley et al., 1991; Wunder et al., 1994; Langbehn et al., 1995). The genotoxic potential of these metabolites indicates that the disappearance of contaminants in the environment does not necessarily mean a less ecotoxicological hazard but that toxic or mutagenic properties of metabolites should be considered. Notably, 1-hydroxypyrene indicated a strong genotoxic response both with and without addition of S9 enzymes, whereas pyrene is known to be a promutagenic agent which needs metabolic activation to exert a genotoxic effect. Therefore, in this case degradation to 1-hydroxypyrene might lead to an increased risk of mutagenic effects at least on bacterial populations. The discrepancy between acute toxicity detected with Microtox and growth inhibition of bacteria in the Mutatox test may have different reasons. The reduction of luminescence in the Microtox test must not be tantamount to death of bacteria; also, nonlethal metabolic disturbances are possible. In addition both bioassays are performed under different conditions. The Microtox test is carried out at 15°C with an incubation time of 30 min. Sample dilutions are prepared with 2% NaCl solution

for osmotic protection of marine bacteria, without addition of nutrients. These conditions ensure maximum sensitivity of bacteria toward toxicants. In contrast to this the test temperature for the Mutatox test is 27°C, bacteria are incubated for 24 h, and all necessary nutrients and salts for growth are included in the culture media. The influence of temperature on sensitivity of luminescent bacteria has been reported (Krebs, 1985; Johnson, 1993). The presence of nutrients and higher salt contents (3% instead of 2% NaCl in Microtox test) also might increase the tolerance of bacteria toward toxicants. Performance of the Microtox test in Mutatox medium instead of 2% NaCl solution reduced acute toxicity of phenol by about 40– 50% (unpublished data). The luminescence reaction constitutes a branch of the electron transport chain at the level of flavin. In this reaction, reduced flavin mononucleotide and a long-chain aldehyde are oxidized by molecular oxygen to give an electronically excited flavin and the corresponding fatty acid. Measurement of bacterial luminescence assesses the flow of electrons in the respiratory chain and the metabolic state of the cell. For this reason it can be used for monitoring toxic effects (Nealson, 1992; Hastings, 1978). Quantitative structure–activity relationships of the EC50s of several organic chemicals with structural parameters have been carried out. The inhibition of bioluminescence can be correlated successfully with the partition coefficient between n-octanol and water (POW) as a model of the

ACUTE TOXICITY AND GENOTOXIC CONCENTRATIONS OF WASTE ELUTRIATES

influence of hydrophobicity of molecules on the permeation of the bacterial membrane. Introduction of molar refractivity (MR)—a function of the index of refraction, density, and molecular weight—and molar volume (MW/d) improved the quality of the relationships. The influences of MR or MW/d could be related with an interaction of the tested chemicals to the enzyme luciferase or the reduced Flavin mononucleotide (FMNH2)-donating system (Hermens, 1985). The EC50s of 55 chlorinated aromatic compounds were correlated with log POW and the hydrophilic effect parameter (VH) by Kaiser et al. (1985). The mode of action of genotoxic agents in the Mutatox test has not been fully elucidated yet. Probably, the transduction of the luminescence operon in dark mutants is under continuous repression. Restoring the luminescence of the repressed dark mutant can theoretically be achieved by three independent events: (1) blocking the formation of the repressor; altering its or the operator’s site structure; (2) inactivating the repressor of the luminescence system; and (3) changing the physical configuration of the DNA, thus allowing unrepressed transcription of the luciferase operon. Three groups of genotoxic agents have been found to be active in the Mutatox test: (1) direct mutagens being either base-substitution or frame-shift agents, (2) DNA-damaging agents and DNA synthesis inhibitors, and (3) DNA-intercalating agents (Ulitzur, 1986; Bulich, 1992). Considering the different levels at which the luminescence reaction can be influenced by toxic or genotoxic compounds in the Microtox or Mutatox test, respectively, the different concentration ranges at which these effects are exerted become understandable. According to the current investigations EC50 deduced from Microtox cannot be used for exact prediction of the concentration of a substance that will have a genotoxic effect in the Mutatox test. Nevertheless, the determination of the EC50 of a sample is useful for a rough estimation of the range of concentrations that should be tested with Mutatox. CONCLUSIONS

The determination of EC50 of samples in the Microtox test is useful for range-finding of possible genotoxic concentrations for the Mutatox test. According to current results the highest test concentration of a sample in the Mutatox test should exceed EC50 by about 5–10 times and the sample should be tested first in 8–10 dilutions of a 1:2 dilution scheme. As waste elutriates often represent complex mixtures of organic and inorganic contaminants, correlations of ecotoxicological effects with single compounds are often impossible. Further, the concentrations of single substances that exert toxic or genotoxic effects in the Microtox/Mutatox test system are in the range of mg/liter. The presence of such high concentrations of organic compounds in environmental samples is unlikely; for this reason, in most cases ecotoxicological effects will be the result of the interaction of all present contaminants.

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ACKNOWLEDGMENTS The authors thank Martina Koch and Marc Mu¨ller for analyses of metals by ICP-OES. These studies were supported by grants from the German Federal Ministry for Education and Research (BMBF), Grant 02C04159. Elutriates of samples 153, 155, and 156 were prepared by the German Society of Reactor Safety.

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