ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

19,228-24 1 ( 1990)

Application of Bacterial Growth Kinetics to in Vitro Toxicity Assessment of Substituted Phenols and Anilines M. NENDZA,’ AND J. K. SEYDEL~ Borstel Research Institute, Med. Pharm. Chemistry, D-2061 Borstel, West Germany Received July 31,1989

Bacterialgrowth kinetics were applied to determine toxicity of substituted phenols and anilines serving as model toxicants. The effects observed on Escherichia coli can be quantified reliably. Additional information is obtained about the onset, the duration, and the time course of the toxic action. For most compounds under study a uniform mode of toxicity was observed, but multihalogen- and/or nitrosubstituted phenols differ in toxicity pattern. Possible reasons for the change in mechanism were examined. A comparison with published quantitative data for fish toxicity of halogenated phenols indicates good agreement with toxic effects observed in E. co/i cultures. This may lead to the possibility of replacing the more costly, less stable fish test system by this simple and reliable in vitro test system. 0 1990 Academic PGSS. h. INTRODUCTION

The aim of this study was to develop a simple in vitro procedure which would allow reliable testing and ranking of ecotoxic compounds. A subsequent derivation of quantitative structure-toxicity relationships should allow the estimation of the ecotoxic hazard of compounds not yet tested from the results of the “training set.” Such quantitative structure-activity relationships (QSAR) have proved to be a useful tool in estimating various types of biological activities (Seydel and Schaper, 1979). In addition exceptions to the general structure-activity pattern can be determined on the basis of QSAR analysis and more detailed studies can be undertaken. This paper deals with an adaptation of the bacterial growth kinetics approach, using Escherichia coli as a model strain. This technique has been successfully applied to study and quantify biological activities of various classes of compounds (Garrett, 1966, 197 1; Seydel, 1983). The chemicals studied were selected to serve as model toxicants occurring in the environment. Phenols and anilines are produced and applied in large scale. They are classified as “nonspecific” toxicants like more than 80% of the ecotoxicologically relevant chemicals. In order to allow the derivation of quantitative structure-toxicity relationships, derivatives have been selected from a homologous series of phenols and anilines in such a way that they differed widely in those physicochemical properties which were assumed to be related to their toxic action, e.g., lipophilicity and electronic features. The results achieved by bacterial growth kinetics can be compared with results obtained in other, mainly aquatic, biological test systems which are more complex such as fish and daphnia. These systems are more difficult to standardize, more time con’ Present address: Fraunhofer Institut fiir Umweltchemie Grafschaft, West Germany. 2 To whom all correspondence should be addressed. 0147-6513/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

228

und Gkotoxikologie,

D-5948 Schmallenberg

TOXICITY

ASSESSMENT

OF

PHENOL

AND

ANILINE

DERIVATIVES

229

suming and expensive, and less reproducible compared to the test system presented in this paper. Taking into account that the unspecific toxic action of the phenols and anilines might be due to their lipophilic and/or electronic properties, a training set of compounds was selected from homologous series, showing an optimal variation in these features from the statistical point of view. This allows the quantification of structureactivity relationships. MATERIALS

AND

METHODS

Test Organisms and Materials The bacteria used were purchased from the American Type Culture Collection (E. coli ATCC 11775). Stock cultures were maintained on agar slants at room temperature. The culture broth was dextrose-salts-casamino acids (vitamin-free), pH 6.9 (Anton, 1960). The medium was sterilized by filtration through cellulose ester membranes (0.22 u). The test chemicals were purchased from Merck or Aldrich at the highest grade of purity available. Minimal

Inhibition

Concentration

MIC values were determined according to standard procedures (Kriiger-Thiemer et al., 1965). The bacterial strains used were E. coli ATCC 11775 and Mycobacterium smegmatis M 169. Culture Systems and Conditions A broth culture was inoculated from an agar culture and the bacteria allowed to grow for 12 to 16 hr at 37°C. From this broth, cultures were prepared by dilution with medium to obtain lo4 cells/ml. Portions of 150 ml were transfered to l-liter Erlenmeyer flasks. Thirty minutes later when the bacteria were in the logarithmic growth phase the chemical was added. Samples of the incubated cultures were taken every 45 min. Particle-free saline (0.85%) and formaldehyde (2%) solution were added to stop multiplication as well as to dilute the solution to about 1,OOO- 10,000 organisms per count. Counting was achieved with a Coulter counter Model ZB equipped with a 0.030-mm orifice; counts per 0.050 ml were obtained. The instrument settings were: l/aperture current, 1; l/amplification, 4; matching switch, 40 K; gain, 10; lower threshold, 7; upper threshold, maximum. Viable Counts Along with the Coulter counter experiments samples were drawn from the cultures under toxicant treatment. Culture suspension (0.5 ml) was diluted with saline. The solution, now containing lo- 100 organisms/ml, was pipetted onto each of five petri dishes. After mixing the saline suspension with 5 ml of melted agar, the bacteria were allowed to multiply for 48 hr at 37°C. The resulting colonies were counted. Laser Microprobe

Mass Analysis (LAMMA)

The LAMMA instrument has been described in detail elsewhere (Heinen et al., 1980). Briefly the laser microprobe mass analyzer represents a combination of a laser

230

NENDZA

AND

SEYDEL

microscope with a time-of-flight (TOF) mass spectrometer. The detection limit reaches 10p2’ g for some elements (Na, K) in an analyzed sample volume of 3 X lo-l3 cm3 for organic matrix material prepared as thin sections (thickness 0. l-l pm) (Seydel and Lindner, 198 1, 1986; Seydel et al., 198 1). RESULTS The minimal inhibitory concentrations (MIC) of the training set chemicals versus E. coli ATCC 11775 and M. smegmatis M. 169 are listed in Table 1. For most chemicals parallel results were found with gram-negative bacteria and mycobacteria. Differences were only observed for compounds of extremely high or low hydrophobicity, probably due to different cell wall structures of the organisms. The toxicants were studied consecutively in the bacterial growth kinetics test in order to obtain more reliable and informative data. E. coli cultures were exposed to graded concentrations of the toxicants. Every 45 min samples were taken and the number of organisms per milliliter was counted. The generation rate curves were obtained by plotting the logarithm of the counts versus time (Fig. 1). K. is assigned to the generation rate constant of the untreated control culture and K, to the generation rate constant of the culture treated with toxic agent. The KJK, ratios for various toxicant concentrations were used to determine the 1,, value, i.e., the concentration of a chemical reducing the generation rate to 50% of the value of an untreated control. The nonlinear fitting of the dose-response curves (Fig. 2) was carried out on a Wang 2200 VP computer using standard software. For all chemicals under study, except 4-hydroxybenzoic acid, a concentration-dependent effect on the generation rate of E. coli was observed. Increasing toxicant concentrations resulted in a decrease in multiplication rate. The onset of the bacteriotoxic action occurred with a lag phase of 2-3 generations (1 hr). The effect was maintained during the entire phase of logarithmic growth of the germs up to 24 hr. The bacteriotoxic activity of the compounds is given in Table 1. Analogous inhibition experiments were conducted using test bacteria differing in their cell wall structure. The growth curves obtained from cultures treated with phenolic toxicants show the same time course as that for E. coli ATCC 11775. Minor differences were observed in the quantitative but not in the qualitative effects on E. coli DC 0, E. coli DC 2, and E. coli ATCC 11775. For some phenol derivatives the studies of kinetics were accompanied by LAMMA investigations of their effects on the intracellular Na+/K+ ratio of E. coli. The toxicants caused a change in the average Na+/K+ ratio which paralleled the degree of inhibition of the bacterial growth (i.e., a significant increase in Na+ and a decrease in K+ intensity). The MIC and ICso of combinations of phenols alone or together with anilines were studied. An equipotent mixture of 2,4,5-trichlorophenol(O.045 mmol/ liter) and 3,5-dimethoxyphenol (4 mmol/liter) was tested in the standard MIC test. The inhibitory potency of the mixtures is lower than that of the single substances. Depending on the mixture ratio some combinations show antagonism. They seem not to act in an independent, additive manner. For reasons of comparison, mixtures of phenols and anilines were also tested. Mixtures of equipotent doses of 3,5-dimethoxyphenol(2.1 mmol/liter) and 2,4-dimethylaniline ( 1.8 mmol/liter) were tested in the E. coli kinetic test. These mixtures caused additive effects on the bacterial growth; i.e., toxicity is not changed if one compound is partially replaced by an equipotent amount of a second chemical.

TOXICITY

ASSESSMENT

OF

PHENOL TABLE

BACTERIOTOXIC

ACTIVITY

OF PHENOL

AND

ANILINE

1

AND ANILINE

MIC Phenol 4-Cl-phenol 2.3-C&-phenol 2,4-C&-phenol 2,6-Cl,phenol 2,4.5-Q-phenol 2.4,6-Cl,-phenol 2,3.4,5-Q-phenol C&-phenol 2-Br-phenol 3-NHz-phenol 4-NH,-phenol 3-N(CHz)Z-phenol 2-NO,-phenol 4-NO,-phenol 2.4,6-(N02)I-phenol 4-CH,-phenol 2,4,6-(CH1)X-phenol 3,5-(OCH3)2-phenol 4-OH-phenol 4-COOH-phenol 4-CH,CONH-phenol 4-Cl-3,5-(CH,),-phenol 2-NH,-4-CH3-phenol 4-CH,-2-NO*-phenol 2-CH,-4.6-(N02)2-phenol 3-NHZCONH-phenol 3-CF3-phenol 4-CN-phenol 3,5-(tert. But)Z-phenol 2,4,6-JX-phenol 4-OH-3-CH3-phenol 3-Cl-4-OH-uhenol 4-NHZ-3-CH3-phenol 2-NHz-4-Cl-phenol Aniline 2-Cl-aniline 3-Cl-aniline 4-Cl-aniline 2,4-C&-aniline 2.6-Cl,-aniline 2-Br-aniline 2,4-(N02)Z-aniline 4-CH,-aniline 2,4-(CHj)2-aniline 2-C2H,-aniline 4-C2H,-aniline Benzidine 3-CF,-aniline 5-Cl-2,4-(OCH,)Z-aniline 4-Cl-2-NO?-aniline 3-Cl-4-CH;-aniline 3-CONHNHz-aniline 4-OCH,-aniline 4-CO&,-aniline 2-NO,-aniline 4-NO1-aniline

231

DERIVATIVES

DERIVATIVES

(mmol/liter)

MIC

IS0

E. coli

M. Ml69

E. coli

12 2.0 0.36 0.36 1.4 0.045 0.36 0.016 0.13 1.4 32 0.090

5.7 0.72 0.26 0.36 0.72 0.045 0.13

7.0 1.0 0.28 0.33 0.72 0.042 0.19 0.045 0.12 0.75 37 0.13 0.78 0.80 0.37 5.3 3.8 1.3 3.0 0.3 1

-

0.36 0.51 4.1 5.1 2.0 4.1 0.016 8.2 32 0.51 0.18 0.51 0.51 23

1.0 2.0 2.9 2.0 0.064 0.18 0.045 0.72 23 5.7 4.1 2.8 2.0b 5.7h 2.8 I.0 8.2 8.2 8.2 8.2 4.1h 5.1h 4.1 0.72 2.0

0.011 0.006 1.4 16 0.18 I.0 0.36 0.51 4.1 5.7 2.0 4.1 0.064 8.2 32 0.26

1.0 0.51 0.72 16 0.72 2.0 0.032 0.032

0.13 0.36 0.090 0.13

11 4.1

2.0 0.51 0.72 8.2h 4.1 0.51 4.1 0.72 4.1 0.72 I.0 4.lh 0.36 0.36 0.26

llh

11”

2.9 5.7 4.1 1.4

0.51 5.1h 2.0 5.7

” Value extrapolated from dose-response curves. ’ Chemical not completely in solution during experiment.

14 0.31 0.23 1.3 0.51

I1 0.35 1.9 0.5" 0.12 0.017 0.26 0.045 0.60 26 2.2 2.7 3.0 0.38 0.62

1.1 0.41 12 2.9 3.6 2.8 1.9 0.90 1.7 0.20 0.95 15

10 8.8 1.8 3.1

232

NENDZA

total

‘I

AND SEYDEL

counts/ml OH

106,

105:

addition of toxicant

lo4

0

2

I

I

I

r

4

6

1

0 t [hr]

FIG. 1. Multiplication rate of E. cofi ATCC 11775 in the presence of graded concentrations of 3,5dimethoxyphenol (concentrations in mmol/liter; (x) control, (0) 0.6, (0) 1.2, (A) 1.8, (A) 2.4, (0) 3.0, (V) 3.6, (v) 4.2).

DISCUSSION The aim of our study was to assessthe toxicity of model toxicants in a bacterial in of a test system with low complexity for ranking of hazardous compounds has been examined. The bacteria used for the study of growth kinetics were selected to meet the following criteria: vitro test system. The suitability

(a) The microorganisms must be sensitive to low doses of toxic agents. (b) Strains used should be nonpathogenic. (c) To facilitate the application of the test in any laboratory and to ensure the comparability of the results, genetically standardized cultures must be used. (d) A generation rate should not exceed 1 hr to allow rapid screening of compounds. Only rapidly multiplying bacteria can be exposed to toxicants for many generations, allowing the study of quasi-chronic effects.

TOXICITY

ASSESSMENT

OF PHENOL

AND ANILINE

DERIVATIVES

233

K/HO

0

150 = 3013.6 2 130.7pmolll 0

I 1000

0

FIG.

r 2000

I 3000 c [pmol/lJ

I 4000

I 5000

2. Dose-response curve of bacteriotoxic activity (1,: K/Ko) of 3,5-dimethoxyphenol.

(e) Aggregate-forming requires single cells.

bacteria are not suitable because Coulter counter technique

Among the available test strains E. coli ATCC 11775 showed the best agreement with the criteria stated above (Table 2). The total number of bacteria in the culture suspension was counted by Coulter counter technique. Additional monitoring of the number of viable bacteria by a plate count procedure differentiated between total and viable microorganism. For the toxicants under study both experiments resulted in identical counts. Hence substituted phenols and anilines act in a bacteriostatic but not in a bactericidal manner in the concentration range studied. Upon exposure to TABLE SELECTION

CRITERIA FOR BACTERIA IN GROWTH KINETICS

2 SUITABLE AS TEST ORGANISMS EXPERIMENTS

E. coli E. coli Lactobacterium Micrococcus Staphylococcus ATCC DC2 jlavus luteus aureus High sensitivity High generation rate Single cells in fluid

culture Genetically

standardized

0 ++

+ +

+

+ +

0 +

+

+

--

--

+

+

+

+

+

-

Note. The suitability is rated ++ (excellent), + (good), 0 (sufficient), -(low), --(insufficient).

234

NENDZA AND SEYDEL TABLE 3 PHENOLSEXPOSINGA BIPHASIC INHIBITION OF E. coli MULTIPLICATION

2,4,5Trichlorophenol 2,4,6-Trichlorophenol 2,3,4,5Tetrachlorophenol Pentachlorophenol 4-Methyl-2-nitrophenol 2,4-Dinitro-6-methylphenol 2,4,6-Trinitrophenol 2,4,6-Triiodophenol

these toxic agents the growth rate of the bacteria is reduced. Most of the chemicals examined exhibit a concentration-dependent, continuous reduction of the generation rate of E. coli. The growth curves indicate a monophasic effect of the toxicants during the logarithmic growth of the bacteria. These compounds are regarded as nonspecific toxicants. Some phenol derivatives show a different time course in the kinetic test. These compounds lead to a complete stop of bacterial multiplication for a concentrationdependent period of time. Thereafter they seem not to interfere with the bacteria which multiply at almost the same rate as the control cultures. This biphasic growth is observed for multihalogen- and/or nitrosubstituted phenols having low pK, values (Table 3). Whereas 2,6-dichlorophenol (Fig. 3A) acts in a monophasic manner, 2,4,6trichlorophenol with one additional chlorosubstituent results in biphasic activity toward E. coli (Fig. 3B). The compounds acting biphasically obviously have a different mode of toxic action. These derivatives should be studied by more detailed tests because they could be more hazardous to the environment. Possible reasons for the change in mechanism were examined. Assumptions such as (a) selection of resistant mutants of E. coli, (b) inactivation of the toxic agent, and (c) influence of the degree of ionization were tested. (a) The time course of the toxic action of the multihalogenand/or nitro-substituted phenols in the kinetic assay with a loss of activity in the second phase suggested selection of resistant bacteria. The lack of sensitivity in the second phase was however not due to selection. Recultivation experiments demonstrate that the test organisms retained their sensitivity to the chemicals. (b) Monitoring of the toxicant concentration level by HPLC excluded loss of toxic agents by metabolism or evaporation as a cause of the second phase. (c) The biphasic inhibition was observed for phenols having low pK, values. Hence the influence of the degree of ionization of the test chemicals on their bioactivity was studied. The fraction of ionized compound was varied by variation in pH of the culture medium. If the ionized portion of the toxicant was decreased, its inhibitory activity increased. The time course of the toxic action was not changed. Therefore the unionized molecule is thought to be the active principle mediating bioactivity. In addition both types of inhibition were followed by laser microprobe mass analysis (LAMMA). The intracellular Na+/K+ ratio is a very sensitive indicator of the viability of the bacteria and the extent of cell damage caused by chemicals. Untreated cells show a low Na+ and a high K+ concentration. Toxic effects on the bacterial membrane result in an increase in Na+ and a decrease in K+ concentration. Both the

TOXICITY

ASSESSMENT

OF

PHENOL

AND

ANILINE

DERIVATIVES

235

[counts/ml]

ltountslmll

0

10 t [hl

FIG. 3. Multiplication rate of E. coli ATCC 11775 in the presence of graded concentrations of (A) 2,6dichlorophenol (t) drug addition; concentrations in mmol/liter; (x) control, (0) 0.2, (0) 0.4. (A) 0.7, (A) I .O) and (B) 2,4,6-trichlorophenol ((x)control. (0) 0.06, (0)0.075, (A) 0.09, (A)O. 105, (0) 0.12, (V) 0.135).

compounds with monophasic and those with biphasic modes of action change the Na+/K+ ratio in parallel to the decrease in the generation rate of the cultures (Figs. 4 and 5). The biphasic-acting phenols cause a severe increase in the average Na+/K+ ratio only during the first, inhibited phase in the growth test. As soon as the bacteria multiply again, the membrane damage seems to be restored. It became obvious that the unchanged toxic agent was still present in the second, restored growth phase when the cultures were exposed to repeated dosage. The toxicants were applied again when the onset of the second multiplication phase in the kinetic assay was observed. A second small dose of the toxicant proved to be fatal for the test cultures (Fig. 6). Bacterial multiplication is inhibited for an equally long period either if a total dose is added at once or if the dose is fractionated and the second portion is added after the “recovery” of the cultures. The partial dosages seem to accumulate. Obviously the overcoming of the inhibitory effects in the second phase of bioactivity is only apparent. For differentiation of the two types of phenols their activity in combination was studied. If compounds having the same mode of toxicity are applied together, their combined action should be the sum of their individual responses, i.e., it should show additivity (Garrett, 197 1; Seydel, 1972). Equipotent mixtures of these compounds therefore are as toxic as the corresponding doses of the chemicals given alone (Freim, 19 13). 3,5-Dimethoxyphenol and 2,4,5-trichlorophenol, two phenols differing in their toxicity pattern in the bacterial growth kinetic test, do not act independently if administered in combination (Fig. 7). A different mode of action for the two compounds with cells can be assumed. This argument is sup-

NENDZA

236

AND SEYDEL

1 2 3

I

10

L

12

4

6

10

1.74 2.40 6.70

20

1 2 3 *

1

02

-

30

hr

30

hr

2.40 4.00 11.2

3

10

20

FIG. 4. Comparison of the impact of 2,6-dichlorophenol (concentrations: 1, control; 2.05 mmol/liter; 3, 1.Ommol/liter) on the multiplication rate of E. coli ATCC 11775 assessedby Coulter counter technique (A) and the degree of cell wall damage expressed as the distribution of the Na+/K+ ratio obtained by LAMMA for a population of 30 cells (B). Samples for LAMMA were taken at times @,a, 0.

ported by the finding that for equipotent mixtures of 3,5-dimethoxyphenol and 2,4dimethylaniline, both monophasic-acting derivatives, the equivalence of effects can indeed be demonstrated (Fig. 8). The results of these experiments imply that in general a uniform mode of toxic action can be assumed for both classes of compounds studied. Most phenols and anilines seem to interfere with the same targets in the bacterial cells.

A

10

20

1 2 3

10

-

30

hr

1.30 12,) 6.60

20

30

hr

FIG. 5. Comparison of the impact of 2,4,5-trichlorophenol (concentrations: 1, control; 2, 0.03 mmol/ liter; 3, 0.05 mmol/liter) on the multiplication rate of E. coli ATCC 11775 assessedby Coulter counter technique (A) and the degree of cell wall damage expressed as the distribution of the Na*/K+ ratio obtained by LAMMA for a population of 30 cells (B). Samples for LAMMA were taken at times @,a, 0.

TOXICITY

ASSESSMENT

OF

PHENOL

AND

ANILINE

DERIVATIVES

231

[counts/ml]

OH N

Cl 106

105

104,

0

1

2

3

4

5

t hl

FIG. 6. Effects of 2.4.6-chlorophenol on the multiplication rate of E. coli ATCC 11775 pretreated with the toxicant (2) compared to primary dosing (1,3). (Concentrations: 1, 0.025 mmol/liter at t = 0.5 hr: 2,0.025 mmol/Iiter at t = 0.5 hr and 0.025 mmol/liter at t = 2.75; 3,0.05 mmoI/Iiter at t = 0.5).

When assessing the fish toxicity of substituted phenols, Veith (1987) also observed a difference in mechanism for polyhalogen- and/or nitrosubstituted derivatives. Whereas he found “nonspecific” toxicity for most phenols, he observed decoupling of oxidative phosphorylation for these compounds. The decoupling potency over-

MIC

100 0

MIC

-%

TCP -0 % DMP -100

FIG. 7. Bacteriotoxic activity (MIC) of a series of equipotent mixtures of a monophasic (DMP: 3,5dimethoxyphenol4 mmol/liter) and a biphasic (TCP: 2,4,5-trichloropheno10.045 mmoI/liter)-acting phenol. (0) Experimentally determined toxicity; (-) toxicity calculated for independently equivalent-acting mixtures.

238

NENDZA

AND SEYDEL

--0.5

0.5.-

0

I

I

I

100 0

1

I

1

1

r

I

% DMP ‘/a DMA -100

0 0

FIG. 8. Bacteriotoxic activity (I, = K/f& kinetic test) of a series of equipotent mixtures of 3,5-dimethoxyphenol (DMP 1.4 mmol/liter) and 2,4-dimethylaniline (DMP 1.9 mmol/liter).

rules their nonspecific effects. Similar explanations might be reasonable for the observations in the bacterial test system. The apparent mutation of toxic effects in the second phase is not yet explained. The observations made about changes in mode of toxic action support the assumption that even problem toxicants can be recognized using the E. coli growth kinetic test system. Finally, the ISo values obtained by bacterial growth kinetics techniques were compared with fish toxicity data (Riibelt, 1982; Saarikoski and Viluksela, 1982; Devillers and Chambon, 1986). Regression analysis reveals a high intercorrelation between the effects of phenols on bacteria and fish (Eqs. (l)-(6), Fig. 9, and Table 4): 1%

1 /LDso

golden

orfe

=

1.008 (kO.64)log

n = 8 r = 0.84

s = 0.46

l/Z,,,

rolj + 1.49 (kO.48)

F= 14.7

(1)

log 1/=%o guppy= 0.981 (*0.41)lOg 1/150E.co/i + 1.14 (kO.34) n=9 r=0.90 s=O.347 F=31.2 1%

l/L&O

zebra fish brachydanio

n=9

=

1 a!

r=0.95

1 W.Wlog

s=O.238

l/I50

E. co/i +

F=71.7

1.10

(2)

(f0.24)

(3) In this statistically significant regression, equation compound No. 9, the pentachlorophenol, deviates more than 2 standard deviations from the calculated regression line. The reason for this is that this most lipophilic derivative does not follow the generally linear correlation between toxic effect against E. coli and lipophilic properties (log Z’) of the phenols (Nendza, 1987). These highly lipophilic phenols are less toxic than predicted from the linear relationship because of limited permeability of the hydrophilic score of E. coli membrane for highly lipophilic compounds. If the LD50 found for fish is correlated with the MIC values determined against M. smegmatis, a significant improvement in the statistics is observed (Eqs. (3)-(6); Fig. 10).

TOXICITY

log l/LD

ASSESSMENT

OF

PHENOL

AND

ANILINE

239

DERIVATIVES

50 GUPPY -9

2.6-

6,

2.2 1.8 1.4 1.0 0.6 i 0.2+ -1

I -0.2 log

I -0.6

I 0.2 1/150E.coli

I 0.6

I 1

1.4

FIG. 9. Relation between toxicity of phenols toward fish (log l/LDSOguppy)and toward E. co/i (log l/I,,, Ol,). For statistics seeEq. (4).

This can be expected because of the lipophilic nature of the mycobacterial cell wall which allows even the highly lipophilic phenols (compound No. 9) to partition into the membrane: 1’22 1 /J&o

golden orfe

= 0.876 (+O. 17)lOg l/MICM,

n=8

t-=0.98

s=O.166

TABLE TOXICITY

(LD,,)

OF HALOGENATED AND VILUKSELA,

PHENOLS

smegmatis + 1.36 (+O. 18) F= 156

(4)

4 AGAINST FISH [RUBELT,’ AND CHAMBON,~

1982; DEVILLERS

1982; SAAROKOSKI’ 19861

LD50

Zebra fish3 Phenol 4-Cl-phenol 2,3-C&-phenol 2,4-Cl*-phenol 2,6-Cl*-phenol 2,4,5-C13-phenol 2,4,6-C&-phenol 2,3,4,5-C&-phenol C&-phenol

Golden orfe’

GUPPY*

(Brachydanio rerio)

0.170 0.030 0.02 1 0.028 0.02 1 0.020 0.096

0.457 0.066 0.062 0.034 0.048 0.063 0.0115 0.0033 0.00 17

0.815 0.0675 0.0286 0.029 1 0.0446 0.067 0.0129 0.0037 0.0029

0.0004

240

NENDZA

log l/LD~

AND SEYDEL

Guppy 7

2.62.21.81.4-

0.2

I -0.5

-1

I 0

I 0.5

I 1.5

I 1

109 l/MIC

guppy = 0.78 (+O. 15)log l/MIC,

n=9 1%

~/L&O

zebra

fish brachydanio

n=9

r=0.978

2.5

and toward M. smegmatis

smegmalis+ 1.05 (&O. 17)

s=O.166

F=160

= 0.76 (*O. 18)lOg l/MIC, r = 0.965

-

thmcgmatis

FIG. 10. Relation between toxicity of phenols toward fish (log 1/LD50 .& 169 (log I/MICw sme8,,,&. For statistics see Eq. (5). log 1 /JJbo

I 2

s = 0.209

(5)

smegmatis+ 1.04 (kO.22)

F = 94.8

(6)

These test systems result in an identical ranking of toxicants. This gives evidence that the bacterial growth kinetics system, which is faster and cheaper than methods of classical toxicology, may allow reliable classification of ecologically relevant chemicals. CONCLUSION Our results indicate that it is possible to estimate an ecotoxic potential of chemicals based on the E. culi kinetic test system. The estimation of the bacterial growth rate under exposure to toxicants allows reliable quantification of toxic effects. One may speculate that this procedure yields not only acute but also chronic toxicity data, as 10 to 15 generations of the germs are exposed to the toxicants. Additional information about the mode of inhibition can be deduced from the generation rate curves which reveal the onset, the time course, and the duration of the toxic action. The phenols and anilines under study damage the bacterial cells. This is documented by the decrease in growth rate and the change in Na+/K+ ratio. The achieved classification of potentially hazardous chemicals using the E. culi growth kinetics procedure and fish tests are in good agreement. The ranking of ecotoxic potency as well as observations about the toxicity pattern conform. These results give evidence that simple and reliable in vitro tests may replace more complex

TOXICITY

ASSESSMENT

OF PHENOL

AND ANILINE

DERIVATIVES

241

systems like fish and hence can lead to the reduction in the number of laboratory animals needed for the assessment for hazardous chemicals. ACKNOWLEDGMENTS This study was supported by a grant of the Umweltbundesamt, Biophysics ofthe Borstel Research Institute for conducting LAMMA of G. Rohwer is kindly acknowledged.

Berlin. We thank the Department of experiments. The technical assistance

REFERENCES A. H. ( 1960). The relation between the binding of sulfonamides to albumin and their antibacterial efficacy. J. Pharmacol. Exp. Ther. 129,282-290. DEVILLERS, J., AND CHAMBON, P. ( 1986). Toxicite aigue des chlorophenols sur Daphnia magna et Brachydanio rerio. J. Fr. Hydrol. 17, 1 I 1-l 20. FREIM, W. ( 19 13). Versuche iiber die Kombination van Desinfektionsmitteln. 2. Hygiene Znfekz. 75,433ANTON,

496.

GARRETT, E. R. (1966). The use of microbial kinetics in the quantification of antibiotic action. Arzneim.Forsch. 16, 1364- 1369. GARRETT, E. R. (197 1). Drug action and assayby microbial kinetics. Prog. Drug Res. 15,27 l-352. HEINEN, H. J., HILLENKAMP, F., KAUFMANN, R.. SCHROEDER, W., AND WECHSUNG, R. (1980). LAMMA: A new laser microprobe mass analyser for biomedicine and material analysis. In Recent Developments in Mass Spectrometry in Biochemistry and Medicine (A. Frigerio and M. McCamish, Eds.), Vol. 6, pp. 435-45 1. Elsevier. Amsterdam. KRUGER-THIEMER, E., WEMPE, E., AND T~~PFER,M. (1965). Die antibakterielle Wirkung des nicht eiWeiBgebundenen Anteils der Sulfanilamide im menschlichen Plasmawasser. Arzneim.-Forsch. 15, 1309-1317. NENDZA, M. (1987). Toxizitatsbestimmungen von umwehrelevanten Chemikalien mit einem neuen Biotestsystem, Ermittlung physikochemischer Eigenschaften und Ableitung quantitativer Struktur-Toxizitats-Beziehungen unter Anwendung von Multiregressions- und Hauptkomponenten-Analyse. Dissertation, Math.-Nat. Fakultat, Universitat Kiel, 1987. R~~BELT,C. (Ed.) ( 1982). Schad.ytoffe im Wasser ZZ: Phenole Bold, Boppard. SAARIKOSKI, J., AND VILUKSELA, M. (1982). Relation between physicochemical properties of phenols and their toxicity and accumulation in fish. Ecotoxicol. Environ. SaJ: 6,501-5 12. SEYDEL, J. K., WEMPE, E., MILLER, G. H., AND MILLER, L. (1972). Kinetics and mechanism of action of trimethoprim and sulfonamides, alone or in combination upon E. coli. Chemotherapy 17,2 17-258. SEYDEL,J. K., AND SCHAPER,K. J. (1979). Chemische Struktur und Biologische Wirkung. Verlag-Chemie, Weinheim. SEYDEL, J. K. (1983). Kinetics of antibacterial effects. In Handbook of Experimental Pharmacology (G. H. Hitchings, Ed.), Vol. 64. Springer-Verlag, Heidelberg. SEYDEL, U.. LINDNER, B., SEYDEL, J. K., AND BRANDENBURG, K. (198 1). Detection ofexternally induced impairments in single bacterial cells by laser microprobe mass analysis. Znt. J. Lepr. 50,90-95. SEYDEL. U.. AND LINDNER, B. (198 I). Application of the laser microprobe mass analyzer (LAMMA) to qualitative and quantitative single cell analysis. Znt. J. Quant. Chem. 20,505-5 12. SEYDEL, U., AND LINDNER, B. (1986). Single bacterial cell mass analysis: A rapid test method in leprosy therapy control. Lepr. Rev. 57,(3), 163- 170. VEITH, G. (1987). Personal communication.