Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities

Microbial communities in water from Baltimore Harbor and from the mainstem of Chesapeake Bay were examined for sensitivity to mercuric chloride, monomethyl mercury, stannic chloride, and tributyltin chloride. Acute toxicity was determined by measuring the effects of [3H]thymidine incorporation, [14C]glutamate incorporation and respiration, and viability as compared with those of controls. Minimum inhibitory concentrations were low for all metals (monomethyl mercury, less than 0.05 microgram liter-1; mercuric chloride, less than 1 microgram liter-1; tributyltin chloride, less than 5 micrograms liter-1) except stannic chloride (5 mg liter-1). In some cases, mercuric chloride and monomethyl mercury were equally toxic at comparable concentrations. The Chesapeake Bay community appeared to be slightly more sensitive to metal stress than the Baltimore Harbor community, but this was not true for all treatments or assays. For culturable bacteria the opposite result was found. Thymidine incorporation and glutamate metabolism were much more sensitive indicators of metal toxicity than was viability. To our knowledge, this is the first use of the thymidine incorporation method for ecotoxicology studies. We found it the easiest and fastest of the three methods; it is at least equal in sensitivity to metabolic measurements, and it likely measures the effects on greater portion of the natural community.     

Mercury-Resistant Bacteria and Petroleum Degradation

The concentration of mercury in water and sediment and in the oil extracted from water and sediment was determined for samples collected in Colgate Creek, located in Baltimore Harbor of the Chesapeake Bay. The concentration of mercury in the oil was 4,000 times higher than in sediment and 300,000 times higher than in water samples. The mercury-resistant bacterial populations of the samples studied have been shown to degrade oil, suggesting these bacteria to be a significant factor in the degradation of oil in Colgate Creek.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment.

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Cytotoxicity of tin on human peripheral lymphocytes in vitro.

The comparative effects of inorganic and organic tin compounds on chromosomes were assessed in human peripheral blood lymphocytes of healthy donors 20-40 years of age. The endpoints observed were chromosomal abnormalities, sister-chromatid exchanges (SCEs) and cell cycle kinetics. The maximum concentrations which reduced the replicative index by about 50%, of stannic chloride and trimethyltin chloride were 40 micrograms and 2 micrograms per culture respectively. The tested doses were 20 micrograms and 10 micrograms of stannic chloride and 1 microgram and 0.5 microgram of trimethyltin chloride. Both doses of stannic chloride induced a much higher frequency of chromosomal abnormalities (P less than 0.05-P less than 0.001) and a greater reduction of cell cycle kinetics than the corresponding relative doses of trimethyltin chloride. The frequencies of SCEs/cell induced by the latter were, however, slightly higher than those induced by the former.     

Heterotrophic utilization of glucose and glutamate in an estuary: effect of season and nutrient load.

Uptake of 14C-labeled glucose and glutamate was studied at several sites in Baltimore Harbor, Eastern Bay, and the Rhode River. Levels of uptake of 14C-labeled glutamate, measured over a period of 1 year during September, December, April, May, and June to estimate seasonal effects on heterotrophic utilization of selected nutrients, were highest in May and lowest in December. In a comparison of visibly polluted and unpolluted sites, the greatest amount of incorporation of glucose or glutamate and the highest Vmax values were observed at those sites where the microorganisms were exposed to varied and higher levels of pollutants, suggesting that Vmax may function as an indicator of relative pollution. Mineralization values ranged from 30 to 43%. A range of 0.44 to 2.32 mumol of C/liter per day was calculated for glutamate uptake.     

Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.     

Estimating Bacterial Production in Marine Waters from the Simultaneous Incorporation of Thymidine and Leucine

We examined the simultaneous incorporation of [³H]thymidine and [¹⁴C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 ± 0.2 [mean ± standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic acid-insoluble fraction. There were no significant differences in thymidine incorporation between dual- and single-label incubations (dual/ single = 1.03 ± 0.13). Addition of the two substrates in relatively large amounts (25 nM) did not apparently increase bacterial activity during short incubations (<5 h). With the dual-label method we found that thymidine and leucine incorporation rates covaried over depth profiles of the Chesapeake Bay. Estimates of bacterial production based on thymidine and leucine differed by less than 25%. Although the need for appropriate conversion factors has not been eliminated, the dual-label approach can be used to examine the variation in bacterial production while ensuring that the observed variation in incorporation rates is due to real changes in bacterial production rather than changes in conversion factors or introduction of other artifacts.     

Bioaccessibility of Mercury in Soils

The initial risk assessment for the East Fork Poplar Creek (EFPC) floodplain in Oak Ridge, Tennessee, a superfund site heavily contaminated with mercury, was based on a reference dose for mercuric chloride. Mercuric chloride, however, is a soluble mercury compound not expected to be present in the floodplain, which is frequently saturated with water. Previous investigations had suggested mercury in the EFPC floodplain was less soluble and therefore potentially less bioavailable than mercuric chloride, possibly making the results of the risk assessment unduly conservative. A bioaccessibility study, designed to measure the amount of mercury available for absorption in a child's digestive tract (the most critical risk pathway endpoint), was performed on 20 soils from the EFPC floodplain. The average bioac-cessible mercury for the 20 soils was 5.3%, compared with 100% of the mercuric chloride subjected to the same conditions. The alteration of the procedure to more closely mimic conditions in the digestive tract did not significantly change the results. Therefore, the use of a reference dose for mercuric chloride at EFPC, and potentially at other mercury-contaminated sites, without incorporating a corresponding bioavailability adjustment factor may overestimate the risk posed by the site.     

The ecology of mercury-resistant bacteria in Chesapeake Bay

Total ambient mercury concentrations and numbers of mercury resistant, aerobic heterotrophic bacteria at six locations in Chesapeake Bay were monitored over a 17 month period. Mercury resistance expressed as the proportion of the total, viable, aerobic, heterotrophic bacterial population reached a reproducible maximum in spring and was positively correlated with dissolved oxygen concentration and sediment mercury concentration and negatively correlated with water turbidity. A relationship between mercury resistance and metabolic capability for reduction of mercuric ion to the metallic state was established by surveying a number of HgCl₂-resistant cultures. The reaction was also observed in microrganisms isolated by differential centrifugation of water and sediment samples. Mercuric ion exhibited an average half-life of 12.5 days in the presence of approximately 10⁵ organisms/ml. Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmercuric acetate (PMA) were classified into eight generic categories.Pseudomonas spp. were the most numerous of those bacteria capable of metabolizing both compounds; however, PMA was more toxic and was more selective forPseudomonas. The mercury-resistant generic distribution was distinct from that of the total bacterial generic distribution and differed significantly between water and sediment, positionally and seasonally. The proportion of nonglucose-utilizing mercury-resistantPsuedomonas spp. was found to be positively correlated with total bacterial mercury resistance. It is concluded from this study that numbers of mercury-resistant bacteria as established by plate count can serve as a valid index ofin situ Hg²⁺ metabolism.     

Mercury inhibition of fatty acid synthesis in chicks.

Male chicks were fed a commercial ration and were given drinking water which contained 0, 50, 100, 150, 200 or 300 mug of mercury/ml as mercuric chloride from hatching to 3 weeks of age. In one experiment, the mercuric chloride was administered by injection into the abdominal cavity rather than in the drinking water. At 3 weeks the chicks were killed, and the livers were removed and weighed. The activity of fatty acid synthetase in the 800 X gav supernatant fractions of the liver homogenates and in vivo incorporation of [14C]acetate into liver and carcass fatty acids and respiratory 14CO2 was determined as indicated. Administration of mercury at a treatment level of 300 mug/ml of drinking water depressed growth, feed and water consumption, liver weight, hepatic fatty acid synthetase activity, and in vivo incorporation of [14C]acetate into liver and carcass fatty acids, and increased the production of respiratory 14CO2 as compared with controls. In experiments in which graded doses of mercury were administered, body weights, liver weights, and feed and water intakes of the chicks receiving 0, 50 and 100 mug of mercury/ml of drinking water were similar to each other, but these parameters were severely depressed by 200 mug of mercury/ml of drinking water. Mercury caused a dose-related decrease of fatty acid synthetase activity. Incorporation of [14C]acetate into carcass fatty acid was depressed by 50 and 200 mug of mercury/ml of drinking water; incorporation into liver fatty acids and production of respiratory 14CO2 was not affected by mercury. Intra-abdominal injection of 6 mg of mercury/100 g body weight (as mercuric chloride) into well alimented chicks depressed hepatic fatty acid synthetase activity at 1 h post-injection. The data are consistent with the hypothesis that a portion of the effects of mercury on fatty acid synthesis are direct rather than a secondary effect of inanition.     

Diseases of the gladiolus: I. Control of hard rot, due to Septoria gladioli Passer., by fungicidal treatment of the corms

Losses from hard rot, measured by an arbitrary disease index, were reduced by treating the dehusked corms before planting with mercuric chloride (with or without the addition of 10% hydrochloric acid), mercurous chloride (calomel), three proprietary mercury compounds (Aretan, Uspulun and Ceresan) and one proprietary non-mercury compound (Folosan). Calomel was the least effective. All the treatments were relatively less effective when corms with definite lesions were treated.
The weight of clean corms produced per old corm planted (weight index) was usually increased by all the fungicides tried, but calomel and Ceresan were less satisfactory than the others.
Mercuric chloride (3 hr. steep in a 0.1% solution) was not rendered more effective by the addition of hydrochloric acid nor by a preliminary dip in methylated spirits to facilitate wetting, while the addition of a proprietary wetting compound (Agral) was definitely harmful to the corms and usually less effective than mercuric chloride alone. Increase in time of steeping or concentration of mercuric chloride was not beneficial and was sometimes harmful. Reduction in time of steeping to 1 hr. gave promising results.
Treatment in November had some advantages over treatment in March.
All the mercury compounds tended to delay flowering, this being most marked in the presence of the wetting compound. Stunted foliage and poor quality flowers resulted from the use of Ceresan.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates.

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Metachromasia of basic dyes induced by mercuric chloride. I

Summary Interactions of the cationic dye methylene blue with mercuric chloride have been studied conductometrically, analytically and spectrophotometrically. Methylene blue produces red colored precipitate with mercuric chloride; in presence of large excess of mercuric chloride a strong metachromasia is induced in the dye. Metachromasia induced by mercuric chloride is more hypsochromic as well as hypochromic than that induced by chromotopes like heparin. The complexes formed between methylene blue and mercuric chloride have variable compositions, the complex responsible for the red metachromatic color of the dye has the composition 2 dye: 1 HgCl₂. A model has been proposed for the metachromatic complex consisting hexa-coordinated mercury, dye is coordinated to the mercury by donating the lone pair electrons of terminal nitrogen. The non-metachromatic dye capri blue also interacts with mercuric chloride but without any change in the visible spectrum. Potassium iodide also gives metachromatic reddish blue colored precipitate with methylene blue.University Research Scholar.     

Mercury incorporation by mature and immature red blood cells

Intact and ghost erythrocytes and reticulocytes were incubated with 0.1 ppm 203-Hg as either mercuric chloride or methyl mercury chloride. Both mature and immature cells accumulated alkyl mercury more avidly than inorganic mercury. Methyl mercury chloride, but not mercuric chloride, readily penetrated the membrane and became incorporated into the intracellular compartment of intact cells. Although uptake of alkyl mercury was approximately the same for intact erythrocytes and reticulocytes, developing cells accumulated inorganic mercury more avidly than did mature cells. Increased uptake of inorganic mercury represented predominantly an increase in stromal binding, illustrating differences in the surface membrane or reticulocytes and erythrocytes.     

Mercury Resistance Is Encoded by Transferable Giant Linear Plasmids in Two Chesapeake Bay Streptomyces Strains

The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28.     

A Probabilistic Ecological Risk Assessment of Tributyltin in Surface Waters of the Chesapeake Bay Watershed

The goal of this study was to conduct a probabilistic ecological risk assessment for tributyltin (TBT) in surface waters of the Chesapeake Bay watershed. Ecological risk was characterized by comparing the probability distributions of environmental exposure concentrations with the probability distributions of species response data determined from laboratory studies. The overlap of these distributions was a measure of risk to aquatic life. Tributyltin exposure data from the Chesapeake Bay watershed were available from over 3600 water column samples from 41 stations in nine basins from 1985 through 1996. Most of the stations were located in the Virginia waters of Chesapeake Bay, primarily the James, Elizabeth and York Rivers. In Maryland waters of the Bay, various marina, harbor and river systems were also sampled. As expected, the highest environmental concentrations of tributyltin (based on 90th percentiles) were reported in and near marina areas. The sources of TBT causing these high concentrations were primarily boat hulls and painting/depainting operations. Lower concentrations of TBT were reported in open water areas, such as the Potomac River, Choptank River and C and D Canal, where the density of boats was minimal. Temporal data from a ten year data base (1986-1996) from two areas in Virginia showed that TBT water column concentrations have declined since 1987 legislation prohibited the use of TBT paints on recreation boats (<25?m). Acute saltwater and freshwater TBT toxicity data were available for 43 and 23 species, respectively. Acute effects for saltwater species were reported for concentrations exceeding 420?ng/L; the lowest acute value for a freshwater species was 1110?ng/L. The acute 10th percentiles for all saltwater and freshwater species were 320 and 103?ng/L, respectively. The order of sensitivity from most to least sensitive for saltwater trophic groups and corresponding acute 10th percentiles were as follows: zooplankton (5?ng/L), phytoplankton (124?ng/L), benthos (312?ng/L) and fish (1009?ng/L). For freshwater species, the order of sensitivity from most to least sensitive trophic groups and corresponding acute 10th percentiles were: benthos (44?ng/L), zooplankton (400?ng/L), and fish (849?ng/L). Chronic data for both saltwater and freshwater species were limited to a few species in each water type. Based on these limited data, the saltwater and freshwater chronic 10th percentiles were 5 and 102?ng/L, respectively. Limited mesocosm and microcosm studies in saltwater suggested that TBT concentrations less than 50?ng/L did not impact the structure and function of biological communities. The saltwater acute (320?ng/L) and chronic (5?ng/L) 10th percentiles were used to determine ecological risk because all exposure data were from saltwater areas of the Chesapeake Bay watershed. Highest ecological risk was reported for marina areas in Maryland waters of Chesapeake Bay and for areas in Virginia such as the Elizabeth River, Hampton Creek and Sarah Creek. Low ecological risk was reported for areas such as the Potomac River, Choptank River, C and D Canal and Norfolk Harbor. Regulation of TBT on recreational watercraft in 1987 has successfully reduced water column concentrations of this organometallic compound. However, various studies have showed that TBT may remain in the sediment for years and continue to be source for water column exposures.     

The ecology of mercury-resistant bacteria in Chesapeake Bay

Total ambient mercury concentrations and numbers of mercury resistant, aerobic heterotrophic bacteria at six locations in Chesapeake Bay were monitored over a 17 month period. Mercury resistance expressed as the proportion of the total, viable, aerobic, heterotrophic bacterial population reached a reproducible maximum in spring and was positively correlated with dissolved oxygen concentration and sediment mercury concentration and negatively correlated with water turbidity. A relationship between mercury resistance and metabolic capability for reduction of mercuric ion to the metallic state was established by surveying a number of HgCl₂-resistant cultures. The reaction was also observed in microrganisms isolated by differential centrifugation of water and sediment samples. Mercuric ion exhibited an average half-life of 12.5 days in the presence of approximately 10⁵ organisms/ml. Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmercuric acetate (PMA) were classified into eight generic categories.Pseudomonas spp. were the most numerous of those bacteria capable of metabolizing both compounds; however, PMA was more toxic and was more selective forPseudomonas. The mercury-resistant generic distribution was distinct from that of the total bacterial generic distribution and differed significantly between water and sediment, positionally and seasonally. The proportion of nonglucose-utilizing mercury-resistantPsuedomonas spp. was found to be positively correlated with total bacterial mercury resistance. It is concluded from this study that numbers of mercury-resistant bacteria as established by plate count can serve as a valid index ofin situ Hg²⁺ metabolism.     

Inhibition of bacterial and phytoplanktonic metabolic activity in the lower River Rhine by ditallowdimethylammonium chloride.

The effects of a quaternary ammonium compound, ditallowdimethylammonium chloride (DTDMAC), on natural populations of bacteria and phytoplankton from the lower River Rhine were examined to estimate their sensitivity to the discharges of cationic surfactants in the river basin. In short-term experiments, significant decreases in the growth rate of bacterioplankton and in the photosynthetic rate of phytoplankton were observed at a nominal concentration of 0.03 to 0.1 mg of DTDMAC liter-1. Nitrification was measured with an ion-selective electrode and by the rate of acid production in ammonium-spiked river water and was found to be only sensitive to the addition of concentrations higher than 1 mg of DTDMAC liter-1. This does not support an earlier suggestion that ammonium-oxidizing bacteria are specifically sensitive to quaternary ammonium compounds. The effect of DTDMAC on thymidine incorporation was shown to depend strongly on the concentration of suspended material, which varied with the sampling date. This effect was also quantified in experimental manipulations with Rhine water. Calculations on the partitioning of DTDMAC between water and suspended matter confirmed the role of suspended solids and showed that an increase of the dissolved DTDMAC concentration in Rhine water by circa 0.01 mg liter-1 leads to a slight inhibition of the growth of heterotrophic bacteria. It is concluded that a total concentration of circa 0.01 mg of DTDMAC liter-1 measured in the River Rhine is likely to have biological consequences.