Evaluation of post-Katrina flooded soils for contaminants and toxicity to the soil invertebrates Eisenia fetida and Caenorhabditis elegans.

This research evaluated soil samples from a New Orleans neighborhood in the Chalmette, Saint Bernard Parish, that had been inundated by flooding associated with Hurricane Katrina. The goal was to determine if ecological risks persisted from flood waters that had come in contact with hazardous surface chemicals before inundating this low-lying neighborhood for a prolonged period. Research objectives were to establish the presence or absence of volatile organic and heavy metal contaminants, and then asses the toxicity of this soil to Eisenia fetida in a soil exposure assay and Caenorhabditis elegans in a simulated porewater exposure assay. Soil analysis revealed detectable levels of metals and organics in the surface soil at each location. No contaminant was detected in concentrations above human health screening values. Chromium and mercury were detected at levels in excess of typical ecological risk values. Soil extracts revealed concentrations of nitrate, sulfate, and chloride above those from an unflooded background sample. Toxicity testing resulted in no acute effects to either test species, but did show bioaccumulation of arsenic, cadmium, and lead in E. fetida exposed to several samples. The combination of mercury and sulfate provide the potential for mercury methylation should flooding and prolonged inundation occur again.

Using sulfate-amended sediment slurry batch reactors to evaluate mercury methylation.

In the methylated form, mercury represents a concern to public health primarily through the consumption of contaminated fish tissue. Research conducted on the methylation of mercury strongly suggests that the process is microbial in nature and facilitated principally by sulfate-reducing bacteria. This study addressed the potential for mercury methylation by varying sulfate treatments and wetland-based soil in microbial slurry reactors with available inorganic mercury. Under anoxic laboratory conditions conducive to the growth of naturally occurring sulfate-reducing bacteria in the soil, it was possible to evaluate how various sulfate additions influenced the methylation of inorganic mercury added to overlying water as well as the sequestration of dissolved copper. Treatments included sulfate amendments ranging from 25 to 500 mg/L (0.26 to 5.2 mM) above the soil's natural sulfate level. Mercury methylation in sulfate treatments did not exceed that of the nonamended control during a 35-day incubation period. However, increases in methylmercury concentration were linked to bacterial growth and sulfate reduction. A time lag in methylation in the highest treatment correlated with an equivalent lag in bacterial growth. The decrease in dissolved copper ranged from 72.7% in the control to 99.7% in the highest sulfate treatment. It was determined that experimental systems such as these can provide some useful information but that they also have severe limitations once sulfate is depleted or if sulfate is used in excess.

Mercury body burdens in Gambusia holbrooki and Erimyzon sucetta in a wetland mesocosm amended with sulfate.

This study used an experimental model of a constructed wetland to evaluate the risk of mercury methylation when the soil is amended with sulfate. The model was planted with Schoenoplectus californicus and designed to reduce copper, mercury, and metal-related toxicity in a wastestream. The sediments of the model were varied during construction to provide a control and two levels of sulfate treatment, thus allowing characterization of sulfate's effect on mercury methylation and bioaccumulation in periphyton and two species of fish--eastern mosquitofish (Gambusia holbrooki) and lake chubsucker (Erimyzon sucetta). After one year in the experimental model, mean dry-weight normalized total mercury concentrations in mosquitofish from the non-sulfate treated controls (374+/-77 ng/g) and the reference location (233+/-17 ng/g) were significantly lower than those from the low and high sulfate treatments (520+/-73 and 613+/-80 ng/g, respectively). For lake chubsucker, mean total mercury concentration in fish from the high sulfate treatment (276+/-63 ng/g) was significantly elevated over that observed in the control (109+/-47 ng/g), the low sulfate treatment (122+/-42 ng/g), and the reference population (41+/-2 ng/g). Mercury in periphyton was mostly inorganic as methylmercury ranged from 6.6 ng/g (dry weight) in the control to 9.8 ng/g in the high sulfate treatment, while total mercury concentrations ranged from 1147 ng/g in the control to a high of 1297 ng/g in the low sulfate treatment. Fish methylmercury bioaccumulation factors from sediment ranged from 52 to 390 and from 495 to 3059 for water. These results suggest that sulfate treatments add a factor of risk due to elevated production of methylmercury in sediment and porewater which biomagnified into small fish, and may potentially increase through the food web.

Methylmercury formation in a wetland mesocosm amended with sulfate.

This study used an experimental model to evaluate methylmercury accumulation when the soil of a constructed wetland is amended with sulfate. The model was planted with Schoenoplectus californicus and designed to reduce wastestream metals and metal-related toxicity. The soil was varied during construction to provide a control and two sulfate treatments which were equally efficient at overall mercury and copper removal. After an initial stabilization period, methylmercury concentrations in porewater were up to three times higher in the sulfate-treated porewater (0.5-1.6 ng/L) than in the control (<0.02-0.5 ng/L). Mean percent methylmercury was 9.0% in the control with 18.5 and 16.6% in the low- and high-sulfate treatments, respectively. Methylmercury concentrations measured in mesocosm surface water did not reflect the differences between the control and the sulfate treatments that were noted in porewater. The mean bulk sediment methylmercury concentration in the top 6 cm of the low-sulfate treatment (2.33 ng/g) was significantly higher than other treatment means which ranged from 0.96 to 1.57 ng/g. Total mercury in sediment ranged from 20.8 to 33.4 ng/g, with no differences between treatments. Results suggest that the non-sulfate-amended control was equally effective in removing metals while keeping mercury methylation low.

A comparison of the daphnids Ceriodaphnia dubia and Daphnia ambigua for their utilization in routine toxicity testing in the Southeastern United States.

U.S. regulatory agencies commonly require effluent toxicity testing with Ceriodaphnia dubia--a practice that has led to the criticism that this species and test protocol often does not reflect local taxa or site-specific conditions. Using an indigenous test species may produce a more realistic model of local effects and may minimize test endpoint variance due to regional differences in water quality. This study addressed the substitution of C. dubia with Daphnia ambigua for toxicity testing in the southeastern United States. This investigation determined that D. ambigua could be laboratory cultured with only minimal changes to established regulatory protocol and that the life-cycle characteristics of this species were conducive to traditional acute and chronic aquatic toxicity test methods used with other daphnids. Acute toxicity tests showed that D. ambigua was less sensitive to some toxicants (sodium chloride, copper sulfate, and sodium lauryl sulfate) but more sensitive to others (chlorpyrifos). Chronic tests with copper sulfate and sodium chloride resulted in lower EC50S for D. ambigua reproduction with both compounds. When exposed to low-alkalinity, low-pH stream waters typical of many southeastern United States watersheds, C. dubia demonstrated a significant reproductive depression in two of three streams tested, whereas D. ambigua experienced no chronic effect. These results suggest that D. ambigua may serve as a suitable surrogate for C. dubia as an toxicity indicator species in these types of receiving streams.

Large vessel occlusion with vasculitis in systemic lupus erythematosus.

Patients with SLE may have acute large vessel occlusion due to vasculitis and/or circulating antiphospholipid antibodies, as illustrated by the case we have reported. Unfortunately, delayed medical attention led to gangrene of the foot and amputation. Early recognition and appropriate treatment may significantly decrease morbidity and mortality. Medical treatment may include corticosteroids, thrombolysis, anticoagulation, or immunosuppression.

Lipid globule staining to aid in differentiating Bacillus species.

The use of the lipid globule stain to aid in differentiating the Bacillus cereus group (i.e., B. cereus, B. cereus var. mycoides, and B. thuringiensis) from other Bacillus species was investigated. Smears from colonies grown on suitable agar were made on precleaned slides, stained, and examined microscopically for characteristic deep blue lipid globules. The study included a total of 649 cultures of Bacillus species plus 143 incompletely characterized Bacillus isolates from food. Only B. cereus, B. cereus var. mycoides, B. thuringiensis, B. megaterium, and B. sphaericus were consistently positive for lipid globules, although at times, a few cells of B. aneurinolyticus and B. thiaminolyticus were also positive. The lipid globule stain procedure is of value in differentiating Bacillus species, especially when performed by an experienced analyst and used in conjunction with tests for cell and spore morphology.

Clostridium perfringens food poisoning: use of serotyping in an outbreak setting.

An outbreak of Clostridium perfringens food poisoning occurred among attendees of a firehouse luncheon. The predominant symptoms of diarrhea (100%) and abdominal pain (81%) among case-patients, the mean incubation period (13.4 h), and the mean duration of illness (21.2 h) were all characteristic of C. perfringens enteritis. Roast beef, although not epidemiologically implicated, was the most likely vehicle of transmission. Fecal specimens from case-patients contained a median C. perfringens spore count of greater than 10(6) and yielded isolates that were heat sensitive and predominantly nonhemolytic, produced C. perfringens enterotoxin A, and, in the majority of specimens (four of five), were identical in serotype. Food samples were negative. This outbreak demonstrates that following enumeration of C. perfringens from a suitable number of fecal specimens from case-patients, serotyping of the isolates may be helpful in implicating C. perfringens as the cause of foodborne illness. This is especially true when implicated food items test negative or are no longer available for testing.

Enumeration of Clostridium perfringens spores in human feces: comparison of four culture media.

Enumeration of Clostridium perfringens spores was compared using 4 culture media. Duplicate 1 g portions of 35 stools (25 from C. perfringens food poisoning outbreaks and 10 from normal stools) were heat treated 20 min at 75 degrees C and tested on tryptose-sulfite-cycloserine (TSC) agar, trypticase-soy-blood (TSB) agar, lactose-sulfite (LS) medium, and iron milk (IM) medium. Dilutions were plated directly onto TSB and TSC, and a 3-tube most probable number determination was made with each specimen in LS and IM incubated at 45 degrees C. TSB was easiest to use and nonhemolytic food poisoning strains were readily differentiated from the normal hemolytic biotype on this medium. Confirmed counts on TSC and TSB were similar for all specimens, but counts of 8 of 25 outbreak specimens were 2-4 log units lower in LS and IM than on plating media; spores in specimens associated with 2 of 5 outbreaks were intolerant of the elevated temperatures. Results showed that elevated temperature MPN methods in LS and IM are inappropriate for the examination of outbreak stools.

Detection of Clostridium perfringens enterotoxin in stool specimens and culture supernatants by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to detect and quantitate Clostridium perfringens enterotoxin A in culture supernatants and in stool specimens from cases of diarrhea in which high numbers of enterotoxin-producing Clostridium perfringens were isolated. To analyze for enterotoxin A, polyvinyl chloride microtiter plates were coated with dilute immune whole rabbit serum. Enterotoxin A standards and samples were allowed to react with sensitized wells. The presence of the immobilized antigen in the wells was detected by the binding of immune rabbit immunoglobulin conjugated with peroxidase. Nanograms of enterotoxin were detectable. Four enterotoxin-positive and seven enterotoxin-negative cultures grown in Duncan-Strong medium gave expected results. Eighteen of 23 diarrheal stool specimens obtained after a food-poisoning outbreak at a state hospital were found to contain microgram quantities of enterotoxin per gram of stool, whereas five control diarrheal specimens contained less than 0.6 ng enterotoxin per gram of stool. These results indicate that the enzyme-linked immunosorbent assay technique is useful for differentiating enterotoxigenic strains and for diagnosing diarrhea caused by enterotoxigenic Clostridium perfringens.

Two successive outbreaks of Clostridium perfringens at a state correctional institution.

An outbreak of acute gastrointestinal illness of short duration involving 100 inmates at a correctional institution followed a similar outbreak among the same population by eight days. Clostridium perfringens was the specific etiology in both outbreaks; the vehicle was roast beef in the first outbreak, ham in the second. Direct observation of food handling practices revealed that the meats were not cooled quickly enough following cooking; not reheated adequately prior to serving, and; held at improper temperatures prior to serving.

Comparison of Selective Plating Media for Enumeration of Bacillus cereus in Foods.

Enumeration of Bacillus cereus on raw sprouts of mung beans and wheat was compared in three agars: mannitol-egg yolk-polymyxin (MYP), polymyxin pyruvate-egg yolk-mannitol-bromthymol blue, and trypticase-soy-polymyxin blood. Ten different strains of B. cereus were used to seed the sprouts. Rates of recovery for the three media were not significantly different. However, with MYP agar, B. cereus could be differentiated more easily from other microorganisms and required fewer confirmatory tests.

New method for differentiating members of the Bacillus cereus group: collaborative study.

A collaborative study was conducted of a new method for differentiating members of the Bacillus cereus group. Using the new method, each of 14 collaborators identified 8 Bacillus cultures, which represented 3 biotypes of the B. cereus group. Each culture was tested for motility, hemolytic activity on trypticase-soy-sheep blood agar, and rhizoid growth on nutrient agar; carbol-fuchsin stain was used to determine the presence of protein toxin crystals. Cultures were identified as B. cereus (biotype 1), B. cereus var. mycoides (biotype 2), or B. thuringiensis (biotype 3). All collaborators correctly identified the unknown cultures and classified them correctly as to biotype. There were no statistically significant differences in the identification rates among the different laboratories. Additional tests by one participant on 5 strains of Bacillus anthracis showed that the new method is also adequate for differentiating B. anthracis from typically reacting strains of B. cereus. The method has been adopted interim official first action.

Enumeration and confirmation of Bacillus cereus in foods: collaborative study.

A collaborative study was conducted in 15 laboratories to evaluate 2 different techniques for enumerating Bacillus cereus in foods. A direct plating technique using mannitol-egg yolk-poly-myxin agar and a most probable number (MPN) technique using trypticase-soy-polymyxin broth were compared for the enumeration of high and low populations of B. cereus in mashed potatoes. The collaborative results showed that the overall mean recovery obtained with the low population level was essentially the same by both techniques. However, the overall mean recovery was significantly higher by the direct plating technique at the high population level. A statistical evaluation of the data also showed that the direct plating technique had better repeatability and reproducibility than did the MPN technique at both the high and low population levels. These results suggest that the MPN technique is suitable for examining foods containing low populations of B. cereus, but that the direct plating technique is preferable for foods that contain a high population of this organism. The confirmatory technique used in the proposed method is reliable for presumptive identification of isolates as B. cereus. The method has been adopted as official first action.

Comparison of Stomacher and Waring Blendor for homogenizing foods to be examined for Clostridium perfringens.

The Colworth Stomacher Model 400 homogenizer was compared with the Waring Blendor for preparing food homogenates to be examined for Clostridium perfringens. Forty-eight samples representing 6 different food types were inoculated with C. perfringens and examined by the AOAC official first action method for enumeration of C. perfringens in foods. Identical paired specimens of each food type were blended with the 2 devices, and plate counts were made as specified in the official first action method. The effects of frozen storage on plate counts were determined by examining 24 food samples that had been stored for 3 days at -68 degrees C and homogenized both devices. Results of a statistical analysis of the experimental data indicated no significant difference overall (P greater than 0.05) in the plate counts of homogenates prepared with the Waring Blendor or the Stomacher 400, either before or after frozen storage of the food samples. However, the overall plate count average of the 48 samples was slightly higher with the Waring Blendor than with the Stomacher 400 homogenizer.

ICMSF methods studies. XII. Comparative study for the enumeration of Clostridium perfringens in feces.

As the second phase of an international comparative study for the enumeration of Clostridium perfringens, four methods were compared for "total" and spore counts of C. perfringens in fecal specimens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserine) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods. In both the total and spore count procedures, the confirmed C. perfringens counts in method D were lower than in methods A, B, and C. Little differences among methods were found in the percentages of presumptive colonies confirmed as C. perfringens. The nonspecific counts in methods A and D were generally greater than in B and C, but nonspecific microorganisms did not interfere in the enumeration of C. perfringens spores by any of the four methods. In overall performance, methods B and C were superior to A and D. The mean C. perfringens spore count was only 0.17 log lower than the mean total count. Spore counts alone are, therefore, adequate in investigations of C. perfringens outbreaks.

Method for maintaining viability of Clostridium perfringens in foods during shipment and storage: collaborative study.

A collaborative study was conducted in 12 laboratories to determine the effectiveness of a new method for maintaining vegetative cells of Clostridium perfringens in viable condition during storage and transport of food specimens to the laboratory. The collaborative results showed that treatment of brown gravy and roast beef samples with an equal amount by weight of sterile buffered glycerol-sodium chloride solution to give a final 10% glycerol concentration and storage with Dry Ice for 10 days at -56 degrees C resulted in plate counts of C. perfringens which were 2-4 log cycles higher with 2 different strains than counts with untreated specimens stored by the usual method at -20 degrees C. Plate counts obtained with the treated specimens stored with Dry Ice were less than 1 log cycle lower than counts made with identical specimens before freezing for storage and shipment to the collaborators. The results with treated specimens were also more uniform among the different laboratories. Because the new method for storage and shipment of food samples was so effective for maintaining viability of the organism, the official first action method for C. perfringens (46.B01) was changed to incorporate these procedures as part of the method.

Media for Confirming Clostridium perfringens from Food and Feces.

Several media recommended for confirming isolates of Clostridium perfringens from selective plating media were evaluated. Media for testing motility, reduction of nitrate to nitrite, fermentation of lactose, and liquefaction of gelatin were found to be the most useful. A modified motility-nitrate medium, developed during the study, and lactose-gelatin medium were the most satisfactory for doing these tests. Fermentation of salicin and raffinose in peptone-yeast extract medium was also useful for differentiating atypically reacting strains of C. perfringens from a variety of culturally similar clostridia.