APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1990, p. 3006-3016

Vol. 56, No. 10

0099-2240/90/103006-11$02.00/0 Copyright © 1990, American Society for Microbiology

Biotoxicity of Mercury

as

Influenced by Mercury(II) Speciationt

RICHARD E. FARRELL, JAMES J. GERMIDA,* AND P. MING HUANG

Department of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada Received 14 March 1990/Accepted 25 July 1990

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolate to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50 s; mercuric chloride, on the other hand, always produced lower IC5Os. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1 % glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)22+1, chloride (HgCl+, HgCl20, HgCIOH°, and HgCI3-), phosphate (HgHPO40), ammonia (HgNH32 ), glycine [Hg(GLY)+l, alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl20, and HgCIOH° were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)22- was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.

Microbial interactions with mercury play a key role in the biogeochemical cycle of mercury and, hence, are of great practical interest. From an ecotoxicological standpoint, biotransformations of inorganic mercury into the highly toxic organometallic methyl mercury (MeHg+) species are of major concern. For example, although inorganic forms of mercury [primarily mercuric hydroxide, Hg(OH)2, and mercuric chloride, HgCl2] predominate in aquatic ecosystems (16), the mercury found in contaminated fish is almost exclusively in the form of methyl mercury (35). The occurrence of methyl mercury in aquatic environments is primarily a result of the microbial methylation of Hg(II) in sediments (10, 17). Microbial methylation, however, depends on the bioavailability and toxicity of mercury which, in turn, depend on the aqueous speciation of the mercuric ion (Hg2+). Because of the complex coordination chemistry of mercury in aqueous systems (the speciation of Hg2+ is a function of the concentrations of all potential coordinating ligands in solution), the nature of the Hg(II) species present in aquatic environments is influenced greatly by water chemistry (e.g., pH, inorganic ion composition, and dissolved organics). Consequently, the influence of environmental factors on the aqueous speciation of mercury has been the focus of much attention (5, 6, 12, 25). Nevertheless, there is very little information available regarding the influence of speciation on the bioavailability or toxicity of Hg(II) to Hg-methylating bacteria. Likewise, although

much is known about the biogeochemistry of mercury in general (11), the ecological importance of individual Hg species remains largely unclear. It is generally accepted that the free, uncomplexed metal ion is the most toxic form of a metal. Thus, the effects of environmental variables on the bioavailability and toxicity of mercury to aquatic microorganisms are usually attributed to their impact on the availability of free Hg2+ (4, 7, 8, 12, 28). Studies with copper, however, have demonstrated that in addition to the free Cu2+ ion, complexes of Cu(II) with inorganic (ammonia and hydroxyl) and low-molecularweight organic (alanine, glycine, glutamic acid, and citrate) ligands can also be toxic (9, 14, 15, 19, 31). It is likely, therefore, that the same holds true for complexes of Hg(II). For example, it has been suggested that the 3- and 4-coordinate complexes of Hg(II) with chloride and hydroxyl ions are less toxic than the 1- and 2-coordinate complexes (4, 28). The influence of competitive complexation on the bioavailability and biotoxicity of mercury in mixed ligand systems, however, has received little attention. Computer programs for calculating chemical equilibria in aqueous systems have been available for a number of years (32-34). As these programs have increased in sophistication, it has become possible to calculate the speciation of metals and ligands in complex systems and the partitioning of individual metal-ligand species among their aqueous, solid, and adsorbed forms. To date, these computer programs have been used in toxicity assessment studies only infrequently, yet the utility of this approach has been ably demonstrated (9, 15, 30, 31). The present investigation was initiated with the intent of

* Corresponding author. t Contribution no. R647, Saskatchewan Institute of Pedology.

3006

BIOTOXICITY OF Hg(II) SPECIES

VOL. 56, 1990

integrating physicochemical procedures for studying mercury speciation with microbiological procedures for evaluating the toxicity of mercury to Hg(II)-methylating bacteria. The specific objectives of the study were (i) to evaluate the effects of pH and selected ligands (chloride, citrate, and cysteine) on the acute toxicity of Hg(II) to a Pseudomonas fluorescens isolate in a chemically well-defined synthetic growth medium, (ii) to compute the effects of pH, chloride, citrate, and cysteine on the aqueous speciation of Hg(II) in this medium, and (iii) to ascertain the dependence of biotoxicity on the chemical species of Hg(II). MATERIALS AND METHODS Bacterial isolates. Surface sediments were collected from Buffalo Pound Lake in the upper Qu'Appelle River basin, Saskatchewan, Canada. Samples were collected with an Eckman dredge, transferred into sterile polyethylene bottles, and packed on ice until processed in the laboratory. Total viable counts were enumerated from a composite sample of the sediment by diluting the sample serially in sterile deionized water and spread plating (in duplicate) on tryptic soy agar (TSA) at 1/10th normal strength (i.e., 0.3% tryptic soy broth plus 1.5% Bacto-Agar; Difco Laboratories, Detroit, Mich.). TSA containing 100 ,ug of HgC2 ml-l was used to enumerate the Hg-resistant (Hg) population. Plates were incubated aerobically for 48 h at 25°C before enumeration of the CFU. Individual organisms were isolated by streaking selected CFU from the Hg-amended serial dilution plates onto fresh TSA plates which were then reincubated for 48 h at 25°C. This procedure was repeated (at least three times) until a pure culture was obtained. The bacterial isolates were tentatively identified by using the API 20E system (2). Stock cultures were prepared by streaking the isolates onto TSA slants, which were incubated for 48 h at 25°C and then stored at 4°C. Working cultures of the Hgr isolates were prepared by streaking the stock cultures onto fresh TSA-Hg (100 ,ug of HgCl2 ml-) plates which were incubated for 48 h at 25°C and then stored at 4°C. Fresh working cultures of the Hgr isolates were prepared every 6 to 8 weeks. Toxicity assessment. Bioassays based on the procedure of Alsop et al. (1) were used to evaluate the acute toxicity of mercury. The bioassays were conducted in a synthetic growth medium with a representative P. fluorescens isolate, i.e., sediment isolate BPL85-47. The synthetic growth medium (M-IIY) was the minimal salts medium of Chan (13) prepared at 1/10th normal strength and amended to contain 0.1% (wt/vol) yeast extract and 0.1% (vol/vol) glycerol. The medium was prepared without the use of Cl salts by substituting appropriate NO3 salts. The chemical composition of the medium is given in Table 1. The procedure used to evaluate the effects of pH on the toxicity of different Hg salts (i.e., mercuric nitrate, mercuric acetate, and mercuric chloride) was as follows. (i) Seed bacteria were prepared by inoculating a loopful of the

working culture of P. fluorescens into 50 ml of M-IIY

supplemented with additional yeast extract (total concentration = 0.5% [wt/vol]) and incubating for 24 h at 25°C with shaking (115 + 5 rpm); (ii) 0.1 ml of an appropriate Hg(II) standard was added aseptically (filtered through a Nalgene syringe filter; 0.45-p.m pore size; Nalge/Sybron Corp., Rochester, N.Y.) to 10 ml of M-IIY medium in test tubes (15 by 150 mm) (the pH of the medium was adjusted to the appropriate value by the addition of 0. 10 M HNO3 or 0.10 M KOH after autoclaving and adding the Hg salt); (iii) 0.1 ml of

3007

TABLE 1. Chemical composition of M-IIY medium Substance

Metals Calcium

(Ca2") .................. Potassium (K+) .................. Sodium (Na+) .................. Iron (Fe2+) .................. Copper (Cu2+) .................. Zinc (Zn2+) .................. Manganese (Mn2+) .................. Mercury (Hg2+) ..................

Magnesium (Mg2+) ..................

Total molar concn'

2.94 x 2.19 x 1.93 x 1.33x 3.60 x 1.10 x 1.99 x 5.93 x

10-4 10-4 10-2

10-3 10-5 10-7

10-6

10-5 Variableb

Ligands

Hydroxide (OH-) ..................

pH dependent 8.17 x 10-3 5.36 x 10-5 4.82 x 1O-4 6.55 x 10-3 Phosphate (PO43-) .................. Nitrate (NO3-) ...................9. 91 x 10-3 Carbonate (CO32-) .................. 7.84 x 10-4 1.09 x 10-2 Glycerol .................. 1.13 x 10-4 Glycine (GLY-) .................. 2.75 x 10-4 Glutamic acid (GLU2-) .................. 5.28 x 10-5 Arginine (ARG-) .................. 2.77 x 10-5 Histidine (HIS-) .................. 1.19 x 10-4 Aspartic acid (ASP2-) .................. 1.51 x 10-4 Serine (SER-) .................. 3.48 x 10-4 Alanine (ALA-) .................. 4.91 x 10-5 Tyrosine (TYR-) .................. 3.95 x 10-5 Methionine (MET-) .................. Valine (VAL-) .................. 1.62 x 10-4 9.17 x 10-5 Lysine (LYS-) .................. Threonine (THR-) .................. 1.05 x 10-4 1.08 x 10-4 Phenylalanine (PHE-) .................. 1.13 x 10-4 Isoleucine (ILE-) .................. 2.06 x 10-4 Leucine (LEU-) .................. 5.99 x 10-5 Proline (PRO-) ..................

Sulfate (SO42-) .................. Chloride (Cl-) .................. Ammonia (NH3) ..................

a Metal and ligand concentrations were calculated to reflect the composition of Chan's medium (13) as well as that of a 0.1% yeast extract solution

(21).

b Total Hg(II) concentrations varied from 5 x 10-6 to 1 x 1O-3 M.

the inoculum was transferred to the Hg-amended medium; (iv) the tubes were sealed with foam plugs and incubated for 24 h at 25°C with shaking (115 + 5 rpm); (v) optical density (i.e., absorbance measured at 530 nm) measurements were then made against a water blank. Inoculated controls were used to measure growth without mercury present. Absorbance values of the Hg-amended cultures were calculated as a fraction of the control and plotted against the logarithm of the total Hg(II) concentration. IC50s (i.e., the concentration of total Hg that inhibits bacterial growth by 50%) were calculated from equations obtained by regression analysis of the absorbance versus Hg(II) concentration data. The effects of various ligands (i.e., chloride, citrate, and cysteine) on the toxicity of mercuric nitrate at pH 8.0 were evaluated in a similar fashion, except that 0.5 ml of the appropriate Hg(II) and ligand standards and 0.5 ml of the inoculum were added to 50 ml of M-IIY medium in 300-ml Erlenmeyer flasks which were then incubated for 16 h at 25°C with shaking (115 + 5 rpm). Tukey's honest significant difference was used to analyze the data from the toxicity assessment studies. The initial study to evaluate the effect of pH on the toxicity of the different Hg salts had two replicates of each treatment and was repeated twice. The study to evaluate the effects of chloride, citrate, and cysteine on the toxicity of mercuric

APPL. ENVIRON. MICROBIOL.

FARRELL ET AL.

3008

TABLE 2. Formation constants used to calculate the aqueous speciation of Hg(II) in M-IIY medium Log pa

Ligand

(L) OHAc-b

S042-

Cl-

NH3

p043NO3C032-

GLY-

GLU2ARGHISASP2SERALATYRMETVALTHRPHE-

HgL

HgL2

HgL3

-3.40 4.20 2.50 7.20 8.70 7.90 0.30 5.10 10.80 6.30 5.80 7.70 6.90

-6.20 8.40 3.50 14.00 17.50

-21.10

10.20 7.00 10.40

ILELEU-

PRO-

CYS2CIT3-

14.40 10.90

14.80 18.50

HgL4

HgLOH

15.70 19.30

3.20

HgHL

Hg(HL)L

Hg(HL)2

20.10

-1.00 14.50

11.90 20.00 11.40 17.90 13.00 12.40 18.10 20.60 17.30 12.10 19.60 17.70 18.90 17.80 17.60 20.00 45.20

6.90

15.30

28.20

22.40 33.60

7.10 6.80

a p values (20, 22, 23, 27, 29; B. Podanyi and R. S. Reid, Inorg. Chem., in press) are provided for equilibrium reactions of the form:

Hg2+ + n H20= Hg(OH)2 -n+ n.H+

b

Hg2+ + m Lz = HgL2 -m Hg2+ + m LZ + n.H20 = HgLm(OH)2 -,m,z -n Hg2+ + m L + q H+ = Hg(HL)Lm_ q2mz Ac, Acetate.

nitrate consisted of one trial with three replicates for each treatment. Mercury(II) speciation. The computer program GEO CHEM-PC (26, 32) was used to calculate the aqueous speciation of Hg(II) in the synthetic growth medium (MIIY). The effects of chloride, citrate, and cysteine on Hg(II) speciation were also computed. The variables used to calculate Hg(II) speciation were pH, total metal concentration, total ligand concentration, and ionic strength. The data base (GEODATA) was modified by the addition of stability constants for selected combinations of the metals and ligands listed in Table 1. Stability constants for the Hg(II)-ligand complexes are listed in Table 2. RESULTS Bacterial isolates. Isolates were obtained from a composite sample of the sediments collected at Buffalo Pound Lake, Saskatchewan. The sample had a total viable count of 2.0 x 106 CFU/g (dry weight) of sediment and a population of Hgr heterotrophs of 3.1 x 101 CFU/g (dry weight) of sediment. Three Hgr pseudomonads (tentatively identified as P. fluorescens) were isolated from the serial dilution plates. One isolate capable of methylating Hg(II), BPL85-47 (R. E. Farrell, P. M. Huang, J. J. Germida, U. T. Hammer, and W. K. Liaw, Verh. Int. Ver. Limnol., in press), was chosen for use in the toxicity assessment studies. Toxicity assessment. Selection of the growth medium used in the toxicity assessment experiments was based on three

criteria: (i) the medium provided adequate nutrition for sustained bacterial growth, (ii) the medium was chemically well characterized, hence, thermodynamic modelling was feasible, and (iii) medium components did not mask the speciation effects of selected ligands added to the medium. Criteria i and ii were validated experimentally. Criterion iii was validated by using computer simulations in which the input data included the concentrations of all medium components for which valid stability constant data were available plus the concentrations of the ligands in question (i.e., chloride, cysteine, and citrate). Based on the results of these validations, the M-IIY medium (Table 1) was chosen for use in this study. Typical growth curves for isolate BPL85-47 in the M-IIY medium and at different pH levels are presented in Fig. 1. The isolate exhibited good growth in the pH range from 6 to 8. Because of the poor growth of the isolate at pH 5, toxicity assessment studies at this pH level were omitted. Bioassays were conducted to determine the effects of pH on the acute toxicity of different Hg salts (i.e., mercuric nitrate, mercuric acetate, and mercuric chloride) to P. fluorescens isolate BPL85-47. Results of the bioassays, reported in terms of the IC50s, are summarized in Table 3. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s (i.e., no significant difference at P = 0.05 or P = 0.01); mercuric chloride, on the other hand, always produced lower IC50s (significant at P = 0.05 for pH 6.0 and 7.0). For each Hg salt, the IC50 was lowest at pH 6.0 and increased significantly (P = 0.05) at pH 7.0. Increasing

VOL. 56, 1990

BIOTOXICITY OF Hg(II) SPECIES

*

0.4

E

3009

pH=8 =7

O pHz *

pH=6

O

pH=5

O

0.3

0

co

0.2

co

*~~1 °

0~~ cn .0

0.1

0.0 '

48

24

o

72

96

120

Time (h) FIG. 1. Growth of P. fluorescens isolate BPL85-47 in the synthetic growth medium M-IIY in the pH

the pH to 8.0 had no effect on the IC50s obtained for mercuric acetate or mercuric nitrate but did produce a significant (P = 0.05) increase in the IC50 for mercuric chloride. Bioassays were also conducted to determine the effects of chloride (as potassium chloride), citrate (as citric acid), and cysteine (as L-cysteine) on the acute toxicity of mercuric nitrate at pH 8.0. Results of the bioassays are summarized in Table 4. Addition of a low concentration (1.54 x 10-4 M) of chloride had no significant effect on the IC50. Increasing the chloride concentration to 1.05 x 10-3 M, however, produced a marked decrease (55%) in the IC50, and at a chloride concentration of 1.00 x 10-2 M, the IC50 was less than 16% that of the control. The effect of chloride on the toxicity of mercuric nitrate is more clearly illustrated in Fig. 2. Bacterial growth, as measured by the optical density (i.e., absorbance) of the test cultures, was inhibited by the addition of chloride to the Hg-amended medium (M-IIY, total Hg = 40 ,uM). Moreover, the magnitude of the inhibitory response increased as the concentration of chloride increased. Experiments with a set of chloride controls (i.e., M-IIY plus chloride, 5.36 x 10-5 to 1.00 x 10-2 M, but without Hg) demonstrated that chloride alone had no deleterious effect on the growth of the bacterial culture. Additions of cysteine (at cysteine:Hg molar ratios greater than 1:10) to the M-IIY medium produced significant (P = toxicity (IC50) of Hg(II), added as different mercury salts to cultures of P. fluorescens Hgr (isolate BPL85-47) grown in solutions of M-IIY medium on acute

IC50 (pLg of Hg ml-') Hg(11)

HSD

salt

pH 6.0

Hg(CH3COO)2 Hg(NO3)2 HgCl2 HSD (0.05)b

pH 7.0

7.65 0.10 25.18 8.03 + 0.24 25.32 4.85 0.74 19.03 2.48

4.47

1.28 2.05 2.05

pH 8.0

25.37 24.83 20.01

3.02 1.90 2.54

(.5, (0.05)a

5.84 2.19 0.95

6.14

Tukey's honest significant difference, at the 5% level of probability, between mean values within rows (i.e., pH effect). b Tukey's honest significant difference, at the 5% level of probability, between mean values within columns (i.e., salt effect). a

from 5 to 8.

0.01) increases in the IC50 (Table 4) to the extent that Hg toxicity was virtually eliminated at a cysteine:Hg molar ratio of 2:1. The effects of cysteine on the growth of the P. fluorescens isolate in a Hg-amended system (M-IIY, total Hg = 200 ,uM) are illustrated in Fig. 3. In the absence of cysteine, bacterial growth was completely inhibited during a 16-h incubation. However, growth inhibition decreased significantly when the cysteine concentration of the medium was >25 p,M (i.e., at cysteine:Hg molar ratios of >0.125). In general, bacterial growth increased in a sigmoidal fashion as the cysteine concentration of the medium was increased. That is, the antagonistic effect of cysteine on mercury toxicity was negligible at cysteine concentrations of 2). Cysteine alone had no significant (P = 0.01) effect on bacterial growth (data not shown). Citrate additions had no significant (P = 0.01) effect on either the IC50 (Table 4) or bacterial growth (data not shown). Mercury(II) speciation. The computer program GEO CHEM-PC (26, 32) was used to calculate the aqueous speciation of the different Hg salts in the M-IIY medium and the effects of pH on the distribution (i.e., activities) of the various Hg-ligand species. (Throughout the text, activity 0.6 200

pMHg(II)

p

refers to the effective concentration of the various Hg complexes in solution. It is defined mathematically as follows: ai = fci, where ai is the activity of an aqueous species, ci is the actual concentration of that species, and f is the activity coefficient.) Free Hg2" ion and 56 Hg-ligand species (Table 2) were considered in the calculations. For a given pH, mercuric nitrate and mercuric acetate produced essentially the same distribution of Hg species. Mercuric chloride, on the other hand, produced distribution patterns that were substantially different, especially at pH 6.0. The most noteworthy difference in the speciation pattern of mercuric chloride was a significant increase in the amount of Hg(II) bound in the form of Hg-chloro complexes. The primary Hg-ligand species present in the M-IIY 1.0

8.0

0.5 E

0.8

O 0

0.6

CD

C

o

0.4

VI

o c 0 .0

0.3 0

Mole fraction

0.4

0 Absorbance

0.2

C,'

0.2

/ 1

10

CIJ

1 00

Cysteine concentration

O

>~ ~ ~ ~ ~0

1 000

(gM)

FIG. 3. Effects of cysteine concentration on the growth of P. fluorescens isolate BPL85-47 during a 16-h incubation and the formation of the Hg(CYS)22- complex in the synthetic growth medium M-IIY. The mole fraction of the Hg(CYS)22- complex was calculated from the results of the computer simulation study. Each point is the average of duplicate determinations.

BIOTOXICITY OF Hg(II) SPECIES

VOL. 56, 1990

medium (defined as those species present at concentrations of -0.01 mol% of the total Hg) are listed in Table 5. Because mercuric nitrate and mercuric acetate yielded essentially the same results, only the data for mercuric nitrate and mercuric chloride are reported. The number of primary species as well as the distribution of the total Hg among these species was pH dependent, yet, in all cases, they accounted for more than 99.9% of the total Hg. Based on pH effects, the primary Hg-ligand species were classified into two groups: group I complexes, those species whose activity increased as the pH of the medium increased, and group II complexes, those species whose activity increased as the pH decreased. For all group I complexes, there was a strong positive correlation (linear, with r 2 0.984) between the calculated IC50s and the activities of the Hg-ligand species (Fig. 4). That is, the acute toxicity of the Hg salts decreased as the amount of Hg(II) bound in group I complexes increased. For the group II complexes, the relationship between the IC50 and the activities of the Hg-ligand species was more ambiguous (Fig. 5A to D). For the group II complexes in general, the IC50 decreased as an exponential function of the activity of the Hg-ligand species (Fig. SA). Yet in several instances, no clear relationship between IC50 and activity could be established [e.g., Hg(H-HIS)HIS+, Fig. SC]. It was observed, however, that in these cases the IC50s decreased as an exponential function of the mole fraction of the Hg-ligand species (Fig. SD). Indeed, for all group II complexes, the IC50s were more highly correlated with the mole fraction of the Hg-ligand species than with the activity of the species (Table 6). This reflects the fact that toxicity depends not only on the activity of individual Hg-ligand species but also on the combined activities of all Hg-ligand species in the growth medium. Mole fraction, therefore, is a relative intensity factor which describes the effects of species interactions on the overall toxicity of the Hg salts. The effects of chloride, citrate, and cysteine on the aqueous speciation of Hg(II), added to the M-IIY medium (pH = 8.0) as mercuric nitrate, were also computed. As expected, the fraction of the total Hg(II) bound in the form of chloro complexes increased as the chloride concentration of the medium was increased (Fig. 2). Although the total activity of the Hg-chloro complexes increased as a near-linear function

TABLE 5. Primarya Hg-ligand species present in M-IIY medium at total Hg(II) concentrations equivalent to the IC50s obtained at the different pH levels mol% total Hg(II)

Hg(NO3)2

Hg species

HgCl2

pH 7 pH 8 pH 6 pH 7 pH 8 (8.03)b (25.32) (24.83) (4.85) (19.03) (20.01) pH 6

Group I complexes (activity increases as the pH increases)

Hg(THR)2' Hg(LEU)20 Hg(NH3)22+ Hg(GLY)2' Hg(GLY)OH°

Hg(SER)2' Hg(ALA)20 Hg(ALA)OH°

Hg(VAL)20

Hg(VAL)OH°

Hg(OH)20 Group II complexes (activity increases as the pH decreases) HgCl+

HgCl20

HgCl3 HgCIOH°

HgNH32+ HgHPO40 HgGLY+ HgALA+ Hg(H-HIS)HIS+

Hg(H-HIS)22+

HgOH+

0.07 0.10 0.09 0.06 0.01 0.02 0.02 0.01 3.53 4.73 4.41 2.84 1.55 1.83 1.82 1.28 5.65 7.24 7.57 4.60 0.15 0.21 0.21 0.12 21.60 21.48 21.80 18.55 16.80 19.73 20.75 13.95 1.31 1.62 1.63 1.07 6.55 8.64 9.04 5.30 21.23 29.40 31.48 17.02

0.03 5.40

2.11 0.01 0.64 0.05 0.03 9.30 3.93 0.02

0.09 0.08 0.02 0.02 4.51 4.26 1.84 1.86 7.05 7.46 0.20 0.20 22.60 23.24 19.64 21.03 1.61 1.64 8.31 8.81 27.92 30.01

0.04 16.10 1.86 0.01 0.03 3.27 1.41 0.01 0.50 0.04 0.04 0.02 1.16 10.64 4.92 0.01 4.47 0.21 0.01

0.09

0.32 0.04 4.37 0.19

0.02 0.15

1.17 0.01

a Defined as those species present at -0.01 mol% of the total Hg(II). The number of primary species is dependent on the pH of the growth medium. b Values in parentheses are the IC50s in micrograms of Hg per milliliter.

1.4_ O Hg(GLY)OH * Hg(VAL)OH 0 Hg(ALA)OH .t r 1UnAl Al_ ngt#L^) 2/ A Hg(OH)2

12 0

1.0 0

0

0 0.8

pH

=

8.0

0

0 0.6

-6.0

-5.7

-5.4

3011

-5.1

-4.8

-4.5

-4.2

log Activity of Hg-Species FIG. 4. Relationship between the toxicity index, log IC50, and logarithm of the activity of selected group I complexes.

3012

APPL. ENVIRON. MICROBIOL.

FARRELL ET AL.

1.6

I

1.6

I

I

1.4

U

1.4 U

0

I

I

I

I

3

I

-3

-2

B

A

.

*

.

U

.

.

0

1.2

C.)uw

1.2

0

1.0

10

0

1.0

0.8

0.8

.

0.6 .9 1.0

-8.0

*

I

.

-7.0

-6.0

n-A -.W .6g

-5.0

I

-4

-

-1

0

1.6

C

1.4

1.4 0

0

U)

-5

.

log Mole Fraction of HgCI2

log Activity of HgCl2 1.6

I

.

1.2

1.2

0) 0

0) 0

.-

1.0

0.8

0.8 .

I

0.6 -6.1

1.0

.

I

0.6 L.. -5.7

-5.9

log Activity of

-5.3

-5.5

Hg(H-HIS)HIS'

-2.2

-1.8

-1.4

-1.0

log Mole Fraction of Hg(H-HIS)HlS+

FIG. 5. Relationship between the toxicity index, log IC_O, and logarithm of the activity (A and C) or the mole fraction (B and D) of selected II complexes.

group

TABLE 6. Coefficients of multiple determination (R2) computed for selected group II complexes; determined using the regression equationa y = alO-bx Hg species

Hg2+ HgALA+ HgCl+

HgCl20 HgCI3HgClOH° HgGLY+ Hg(H-HIS)(HIS)+

Hg(H-HIS)22+ HgHPO40 HgNH32+ HgOH+

R2

R2

(activity)

(mole fraction)

0.537 0.450 0.889 0.887 0.541 0.231 0.415 0.147 0.745 0.538 0.392 0.384

0.803 0.804 0.986 0.927 0.770 0.910 0.793 0.817 0.935 0.783 0.782 0.780

a In the regression equation, y is the predicted IC50 when the activity or mole fraction of the Hg-ligand species is x; a and b are the regression coefficients.

of the total chloride concentration, there was no significant increase in the mole fraction until the total chloride concentration exceeded 10-3 M. This corresponded to the chloride concentration at which significant decreases in bacterial growth were first observed. Similar results were observed when the mole fractions of the individual Hg-chloro complexes (i.e., HgCl+, HgCl20, HgCl3-, HgC142-7 and HgClOH°) were plotted versus total chloride concentration. Additions of cysteine to the M-IIY medium resulted in significant amounts of Hg(II) being complexed as Hg(CYS)22- (Fig. 3). Because of the extreme affinity of mercury for sulfhydryl groups (the formation constant for the 1:2 Hg-cysteine complex is 1045-2), Hg(II) is strongly bound to the added cysteine. Indeed, each twofold increase in the cysteine concentration was accompanied by a doubling of the activity of Hg(CYS)22-; however, no significant increase in the mole fraction of Hg(CYS)22- occurred until the cysteine concentration exceeded 25 ,uM (i.e., at cysteine:Hg molar ratios of >0.125). This corresponded to the cysteine concentration at which significant increases in bacterial growth were first observed. The addition of citric acid to the Hg-amended M-IIY medium had no significant effect on the distribution of the Hg

VOL. 56, 1990

species and no appreciable Hg-citrate was formed (data not shown). DISCUSSION In a companion study (Farrell et al., in press), the meth-

ylation of mercuric nitrate by P. fluorescens isolate BPL85-47 in the M-IIY medium was investigated and found to be inversely related to the acute toxicity of mercury. The present study was conducted to identify the Hg-ligand species responsible for toxicity in the M-IIY medium and to evaluate the effects of chloride, citrate, and cysteine on Hg(II) speciation and acute toxicity. It was demonstrated that the toxicities of different mercury salts were influenced by the nature of the co-ion. Distribution of the Hg-ligand species in the M-IIY medium was essentially the same whether Hg(II) was added as mercuric nitrate or mercuric acetate; consequently, it is not surprising that there were no significant differences in the toxicities of these two salts (Table 3). When Hg(II) was added as mercuric chloride, however, the distribution of Hg-ligand species was quite different, i.e., Hg(II) was bound preferentially in the Hg-chloro complexes with concomitant decreases in the activities of all other Hg-ligand species. This was especially true at pH .7.0. It was anticipated, therefore, that the toxicity of mercuric chloride would be different from that of mercuric nitrate or mercuric acetate, especially at pH s7.0. Indeed, it was observed that mercuric chloride was generally more toxic than the other mercury salts and that significant differences in the acute toxicity occurred at pH 6.0 and 7.0 (Table 3). These results indicate that chloride enhances the toxicity of Hg(II) and also suggest that the Hg-chloro complexes are among the more toxic Hg species. The influence of the co-ion on the toxicity of mercury salts has also been reported by Ribo et al. (30). The acute toxicity of mercury increased as the pH of the M-IIY medium decreased (Table 3). This was especially evident when the pH was decreased from 7.0 to 6.0. How-

ever, because P. fluorescens isolate BPL85-47 grew slower and produced decreased yields at pH 6.0 (Fig. 1), it is possible that pH-induced physiological stress contributed to the increased toxicity of the mercury salts at pH 6.0. In general, the mechanisms involved in pH-metal toxicity relations are poorly understood (5). Consequently, the contribution of pH-induced changes in Hg speciation to the toxic response of the bacteria cannot easily be separated from that of the increased susceptibility of the pH-stressed bacteria to the various Hg species. Nevertheless, decreasing the pH from 8.0 to 7.0 also resulted in a significant increase in the toxicity of mercuric chloride (Table 3), although it had no effect on bacterial growth (Fig. 1). These results, together with those obtained from the computer simulations of the effects of pH on Hg(II) speciation, indicated that pH-induced changes in Hg(II) speciation were most likely responsible for the increased toxicity of mercuric chloride at pH 7.0. Intuitively, these results suggest that the effects of pH on the chemical speciation of Hg(II) must also have contributed to the increased toxicity of the mercury salts at pH 6.0. Because the computed activities of the Hg-ligand species were highly correlated to each other (i.e., very collinear), it was difficult to make definite conclusions regarding the relative toxicities of individual Hg-ligand species. Based on trends in the speciation data and toxicity tests, however, it was possible to identify a number of Hg species that were positively correlated with toxicity. Twenty-two primary Hg-ligand species were identified in

BIOTOXICITY OF Hg(Il) SPECIES

3013

the M-IIY medium (Table 5). Of these, 11 (the group I complexes) were negatively correlated with acute toxicity, i.e., the P. fluorescens isolate was more tolerant of Hg(II) under conditions which promoted the formation of these complexes (high pH and/or low chloride concentration). Regardless of pH or the nature of the co-ion, complexes of Hg(II) with alanine, Hg(ALA)2' and Hg(ALA)OH°, and hydroxyl, Hg(OH)20, were the predominant group I species in the M-IIY medium and together accounted for between 49.52 and 74.28 mol% of the total mercury. The remaining 11 primary Hg-ligand species (the group II complexes) (Table 5) were implicated as the main causative agents of toxicity, i.e., bacterial growth was inhibited under the conditions which favored the formation of these complexes (low pH and/or high chloride concentration). In the mercuric nitrate and mercuric acetate systems, the Hghistidine complex, Hg(H-HIS)HIS+, was the predominant group II species under all pH conditions. In the mercuric chloride system, however, Hg(H-HIS)HIS+ was the predominant species only under neutral and alkaline pH conditions; the HgCl20 complex was the predominant group II species at pH 6.0. Based on calculated concentrations, the 30 remaining Hg-ligand species were classified as either secondary (CHg2+ S CHg-L < 10-4 HgT [CHg2+ = concentration of free Hg2+ ion, CHg-L = concentration of a given Hg-ligand complex, and HgT = total concentration of Hg(II)]) or tertiary (CHg-L < CHg2+) species. This classification was highly dependent on pH, e.g., the number of tertiary Hg-ligand species at pH 6.0, 7.0, and 8.0 was 20, 15, and 7, respectively. Because the tertiary species were present at concentrations less than that of the free Hg2+, it was assumed that their contribution to the overall toxicity of the Hg salts was not significant. On the other hand, the concentrations of the secondary species were generally 102 to 105 times that of the free Hg2+; thus, it was assumed that the secondary group II complexes did have a significant impact on the overall toxicity of the Hg salts. Cationic metal-ligand species are generally considered to be more toxic than anionic or neutral species because they are better able to compete for sites on cell surfaces (6). Thus, although the free Hg2+ ion is generally considered to be the most toxic form of the metal, cationic 1-coordinate Hgligand species (e.g., HgCl and HgALA+), in which the R-Hg+ ion is free to form complexes with other ligands, presumably would also be highly toxic. Indeed, our results demonstrated a positive correlation between the cationic HgL2-ZL species and the acute toxicity of the Hg salts. It is to be expected, however, that the relative toxicities of these complexes would be less than that of the free Hg2+ because of the reduced charge (net charge = 2 - ZL) and the effects of steric hindrance. Nevertheless, the calculated activities of these complexes were generally 103 to 107 times that of the free Hg2+, which suggests that the 1-coordinate complexes contribute significantly to the overall toxicity of the Hg salts. Several 1-coordinate species that were positively correlated with acute toxicity but which had a net charge of .0 were also identified (i.e., HgSO40, HgHPO40, HgGLU°, HgASP°, and HgPO4-). The toxicity of the HgGLU° and HgASP° complexes is assumed to be a consequence of the polar nature of the molecules (i.e., the Hg2+ ion is bound through the a-amino and a-carboxyl groups and carries a charge of +1 while the terminal carboxyl group is negatively charged, resulting in a net charge of zero). The toxicity of the HgSO4 , HgHPO40, and HgPO4 complexes, on the other hand, is presumably related to the weakness of the Hg-O bond.

3014

FARRELL ET AL.

The binding preference of Hg2" for sulfhydryl, thioether, and imidazole groups at catalytically active centers in enzymes provides the biochemical basis for much of its toxicity (24). Consequently, molecular arrangements which inhibit the ability of Hg2+ to combine with these enzymes will result in reduced toxicity. The group I complexes were predominantly 2-coordinate complexes with a net charge of -0 [e.g., HgALA20, Hg(OH)20, Hg(ALA)OH°, and Hg(GLU)22-]. Because these complexes are neutral or anionic they have a low affinity for the binding sites on the bacterial cells. Moreover, because the Hg2+ ion is bound at the center of these molecules, it is presumably shielded from direct interaction with the bacterial cells. Presumably, it is this combination of charge and steric factors which accounts for the low toxicity of these complexes. Not all the 2-coordinate complexes were classified as group I complexes. Unlike most of the amino acids, which bind Hg2+ through the a-amino and ot-carboxyl groups, histidine and arginine bind Hg2+ through the imidazole and guanidine groups, respectively. Thus, in addition to forming group I complexes of the type HgL20, histidine and arginine also form group II complexes of the type Hg(HL)L+ and Hg(HL)22+. The increased toxicity of the latter species is presumably a result of the cationic nature of the com-

plexes.

Our results also demonstrated that the toxicity of the Hg salts was highly correlated to the mole fraction of the Hg-chloro complexes, regardless of the charge of the complex. Although it was not possible to quantitatively determine the relative toxicities of the various Hg-chloro complexes, it was postulated that the toxicity of these complexes should decrease in the order HgCl+ > HgC20 2 HgCIOH° > HgCl3- 2 HgCl42-. The HgCl+ complex is expected to exert the greatest toxicity because of its cationic nature and because the Hg2+ ion is still free to interact directly with other ligands. On charge considerations, we would expect HgCl20 to be a low-toxicity species; however, there is evidence to suggest that the toxicity of the HgCl2' complex arises from its high permeability through lipid bilayer membranes (18). Likewise, enhanced permeability due to chloride may also account for the higher than expected toxicity of the HgCIOH° complex. The anionic complexes (HgCI3- and HgC142-) would be expected to exert the lowest toxicities because of their net negative charge, low permeability through the membrane (18), and the increased shielding of the Hg2+ ion provided by the additional Cl ions. In summary, it was found that the Hg-chloro complexes, the 1:1 Hg-ligand species (i.e., complexes of the type HgL and HgHL), and the protonated 1:2 Hg-ligand species [i.e., complexes of the type Hg(HL)L and Hg(HL)2] were positively correlated with the acute toxicity of Hg(II) in the M-IIY medium. Conversely, with the exception of HgCl20 and HgCIOH°, the 1:2 Hg-ligand species of the type HgL2 and HgLOH were negatively correlated with acute

toxicity. Although chloride concentrations in natural freshwater

systems are generally low (16), additions of chloride as a result of the application of deicing salts to roadways (3), the use of potash (KCl) and nitrogen (NH4Cl) fertilizers, and the discharge of chlorinated wastewater can raise chloride levels significantly. For example, chloride concentrations ranging from 0.28 to 7.7 mM have been measured in the interstitial waters of sediments collected from the upper Qu'Appelle River basin, including Buffalo Pound Lake (unpublished data). Consequently, the effect of chloride on the bioavail-

APPL. ENVIRON. MICROBIOL.

ability of Hg(II) in freshwater systems remains an important issue. When chloride was added to Hg-amended media, the toxicity of mercuric nitrate was enhanced (Table 4). Moreover, the toxic response (i.e., growth inhibition) to mercury of P. fluorescens isolate BPL85-47 increased as the chloride concentration increased (Fig. 2). When the total Hg(II) concentration was 40 ,uM, the critical chloride concentration was 1.05 mM (i.e., the critical chloride:Hg molar ratio was 26:1). This was somewhat higher than critical molar ratio of 20:1 which was observed for a system with a total Hg concentration of 75 ,uM (Farrell et al., in press). Additional experiments demonstrated that even relatively small amounts of mercury became toxic when the chloride concentration was high enough (e.g., 5 ,uM mercuric nitrate was toxic only when the chloride concentration exceeded 5 mM, i.e., at chloride:Hg molar ratios greater than 1,000:1). These results clearly demonstrate that chloride enhances the toxicity of mercury and that the critical chloride:Hg molar ratio increases as the mercury concentration decreases. Toxicity enhancement was attributed to an increase in the activities of the Hg-chloro complexes, formation of which was accomplished primarily by a redistribution of Hg(II) from the group I complexes. Redistribution of Hg(II) from the group II complexes was negligible until the total chloride concentration exceeded 5.0 mM. Low-molecular-weight organic ligands such as cysteine and citrate are common in aquatic ecosystems (16) and can affect the dispersion of mercury from sediments. The influence of these ligands on the speciation and bioavailability of mercury, however, has not been extensively studied. Additions of cysteine to Hg-amended media reduced the toxicity of mercuric nitrate, and this effect was enhanced as the total cysteine concentration was increased. In the presence of 200 p,M Hg, bacterial growth was inhibited by more than 95% at cysteine concentrations of