Optimization and validation of enzyme-linked immunosorbent assay for the determination of endosulfan residues in food samples

In this study, an enzyme-linked immunosorbent assay (ELISA) was optimized and applied to the determination of endosulfan residues in 20 different kinds of food commodities including vegetables, dry fruits, tea and meat. The limit of detection (IC₁₅) was 0.8 μg kg^?1 and the sensitivity (IC₅₀) was 5.3 μg kg^?1. Three simple extraction methods were developed, including shaking on the rotary shaker at 250 r min^?1 overnight, shaking on the rotary shaker for 1 h and thoroughly mixing for 2 min. Methanol was used as the extraction solvent in this study. The extracts were diluted in 0.5% fish skin gelatin (FG) in phosphate-buffered saline (PBS) at various dilutions in order to remove the matrix interference. For cabbage (purple and green), asparagus, Japanese green, Chinese cabbage, scallion, garland chrysanthemum, spinach and garlic, the extracts were diluted 10-fold; for carrots and tea, the extracts were diluted 15-fold and 900-fold, respectively. The extracts of celery, adzuki beans and chestnuts, were diluted 20-fold to avoid the matrix interference; ginger, vegetable soybean and peanut extracts were diluted 100-fold; mutton and chicken extracts were diluted 10-fold and for eel, the dilution was 40-fold. Average recoveries were 63.13–125.61%. Validation was conducted by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results of this study will be useful to the wide application of an ELISA for the rapid determination of pesticides in food samples.     

Elemental uptake by edible herbs and lettuce (Latuca sativa)

The total concentration of toxic elements (aluminum, cadmium, chromium and lead) and selected macro and micro elements (iron, manganese, copper and zinc) are reported in six leafy edible vegetation species, namely lettuce, spinach, cabbage, chards and green and red types of Amaranth herbs. Although spinach and chards had greater than 125 mv of iron, both the amaranthus herbs recorded > than 320 microg g(-1) dry weight. In both the spinach and chard species, the Mn and Zn levels were appreciable recording > 225 microg g(-1) and 150 microg g(-1) dry weight, respectively. Aluminum concentrations were (in microg g(-1) dry weight) lettuce (10), cabbage (11), spinach (167), chards (65), amaranthus green (293) and amaranthus red (233). All the micro and macro elements and the toxic elements (Ni, Cr, Cd and Pb) elements analyzed, were below the recommended maximum permitted levels (RMI) in vegetables. Further the elemental uptake and distribution of the nine elements, at three growth stages of the lettuce plant grown on soil bed under controlled conditions are detailed. In the soil, except for iron (16%), greater than 33% of the other cations were in exchangeable form. Generally in the lettuce plant, roots retained much of the iron (> 224 microg g(-1)) and aluminum (> 360 microg g(-1)), while leaves had less than 200 microg g(-1) of iron and 165 microg g(-1) of Al. Although the concentrations of elements marginally decreased with growth, the lettuce leaves had significant amounts of Mn (30 microg g(-1)), Zn (50 microg g(-1)) and Cu (3.6 microg g(-1)). Some presence of lead in leaves (2.0 microg g(-1)) was noticed, but all the toxic and other elements analyzed were well below the RMI values for the vegetables.     

Depuration of copper and zinc by green oysters and blue mussels of Taiwan

This paper describes depuration processes of copper and zinc in green oysters (Crassostrea gigas) and in blue mussels (Mytilus smarangdium) collected from an environment with heavy copper contamination, and then transferred to natural clean seawater. Results show that the total loss of copper content per oyster is an exponential function of exposure time for the first 6 days with a depuration rate of 351 microg g(-1) day(-1) and then levels off. During this exponential decrease period approximately 67% of the copper accumulated in green oysters was depurated. However, when the copper contents in the oysters decreased from 2225 +/- 111 microg g(-1) to 344 +/- 18.7 microg g(-1) the depuration rates decreased from 245 microg g(-1) day(-1) to 0.08 microg g(-1) day(-1). This means that green oysters had a 16-fold higher copper depuration rate (351 microg g(-1) day(-1)) than normal oysters (21.5 microg g(-1) day(-1)) for the first 6 days. However, the depuration of accumulated copper and zinc by the mussels was a fast process in natural clean seawater. About 91% of the accumulated copper was lost during the first 6-day period; copper contents declined from 20.2 +/- 3.41 microg g(-1) to 1.80 +/- 0.21 microg g(-1). Only 36% of the accumulated zinc was lost during a depuration period of 6 days. Calculations show that the biological half-lives of copper in green and normal oysters were 11.6 and 25.1 days, respectively. The biological half-lives of zinc in green and normal oysters were 16.7 and 30.1 days, respectively. In spite of the relatively low initial copper content in blue mussels being 20.2 +/- 3.41 microg g(-1), the biological half-life is only 6.40 days. From these results it is important to emphasise that the fastest turnover rate is for copper in blue mussels. However, zinc is more retentive in blue mussels than copper.     

Analysis of herbicide Krovar I by liquid chromatography with atmospheric pressure chemical ionization mass spectrometry

A simple, very efficient method is presented for routine analysis of herbicide Krovar I (active components bromacil and diuron) in water and soil samples. Water samples were extracted by liquid-liquid extraction with dichloromethane (DCM) as extraction solvent. For soil samples two different extraction techniques were compared: microwave-assisted solvent extraction and a shaking technique using a platform shaker. Extracts were analyzed by high performance liquid chromatography using a water:methanol gradient. Liquid chromatography was coupled with atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) for quantification of bromacil and diuron. Optimization of the APCI-MS was done by using standards in the flow injection analysis mode (FIA). Method detection limit for liquid samples for bromacil is 0.04 microg L(-1) and for diuron 0.03 microg L(-1). Method detection limit for soil samples is 0.01 microg g(-1) dry weight for both compounds. Results of analysis of field samples of water and soil are also presented.     

Tube-immunoassay for rapid detection of carbaryl residues in agricultural products

An antibody-based rapid, quantitative, and qualitative tube enzyme-linked immunosorbent assay (tube-ELISA) was developed and used to determine carbaryl (1-naphthyl methylcarbamate) residues in agricultural products (apple, Chinese cabbage, rice, and barley). The tube-ELISA is a competitive immunoassay in which the antibody is coated in the polystyrene tube, with a dynamic range between 0.7 and 46.3 microg kg(-1). Carbaryl was extracted from each agricultural sample by hand-shaking with methanol and examined for application to on-site analysis. After the liquid extraction, the sample extracts diluted with buffer were analyzed by rapid tube-ELISA directly. The overall test time was around 15-30 min, including sample preparation and assay performance. The results obtained from tube-ELISA correlated well with high-performance liquid chromatography (R2 > 0.9). The study shows that tube-ELISA is useful as a quality control tool and can be used to quantitatively detect carbaryl as well.     

Application of an ELISA for PCB 118 to the screening of dioxin-like PCBs in retail fish

A commercially available enzyme-linked immunosorbent assay (ELISA) kit was evaluated for the determination of toxic equivalents (TEQs) of dioxin-like polychlorinated biphenyls (PCBs) in retail fish. The ELISA was highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118), which is generally the most abundant dioxin-like PCB isomer found in fish. The quantitative limit of the ELISA (using 3,3',4'-trichloro-4-methoxybiphenyl as a surrogate standard for PCB 118) was 10 ng ml(-1) (125 pg assay(-1)) in the standard curve, corresponding to 50 pg PCB 118 g(-1) in the tested sample. Good recoveries of PCB 118 (78.7-112.3%) were obtained for spiked purified fish extracts according to the ELISA. Good linearity was also obtained in dilution tests using purified fish extracts. No significant interference of the matrix was observed in the ELISA when this purification procedure was used. Recovery tests in which PCB 118 was added to fish samples also resulted in acceptable recoveries (60.2-82.3%) in the ELISA following purification. The ELISA results for fish samples correlated well with the TEQ concentrations of dioxin-like PCBs obtained by high-resolution gas chromatography/high-resolution mass spectrometry (r = 0.92, n = 26). These data indicate that the ELISA kit is suitable for screening retail fish for the TEQs of dioxin-like PCBs.     

Assessment of heavy metals (Cd, Cr and Pb) in water, sediment and seaweed (Ulva lactuca) in the Pulicat Lake, South East India

The concentrations of three heavy metals chromium (Cr), cadmium (Cd) and lead (Pb) were examined in water, sediment and green algae (Ulva lactuca); collected from six different stations at Pulicat Lake, which receives effluents from industries located in North Chennai Coastal region. Concentrations of Cd (64.21 microg g(-1)) and Cr (28.51 microg g(-1)) were found to be high in sediment, whereas in green algae concentration of Pb (8.32 microg g(-1)) was higher than water and sediment samples. The relative abundance of these heavy metals in U. lactuca and sediment were found to be in the order Cd>Cr>Pb, whereas in water the ratio was found to be Cr>Pb>Cd. The seasonal variations in Cd and Pb followed a similar pattern in both seaweeds and sediments, but not in water samples. Spearman correlation coefficient study showed no significant correlation in the concentration of metals in U. lactuca, water and sediment samples.     

Comparison of gas chromatography and immunoassay methods for analysis of total DDT in calcareous soils

Three different calcareous soil samples from Lebanon were analyzed for total DDT pesticide residue using GC and ELISA methods. Two experiments were conducted on three different calcareous soil samples. In each experiment, triplicates of 5 gm soil samples were each fortified with standard solutions of DDE to reach concentrations of 0, 5, 25, 50, 100 and 200 ng g(-1) and allowed to equilibrate at room temperature for 6 hours. Each sample was then extracted with 25 mL of 90% methanol by shaking in glass bottles on a mechanical shaker for 16 hours. The bottles were allowed to stand for 30 minutes and aliquots were taken from the clear supernatant for analyses without further cleanup. The total DDT in the extract was measured in triplicate by GC and ELISA. The results indicated that the two methods were highly correlated (R = 0.955-0.994). Differences in soil properties did not affect the accuracy of the detection limits of ELISA. Immunoassay technique can be used for rapid and accurate measurement of total DDT residues in mineral calcareous soils in Lebanon.     

Biodegradation of nonylphenol in river sediment

We investigated the biodegradation of nonylphenol monoethoxylate (NP1EO) and nonylphenol (NP) by aerobic microbes in sediment samples collected at four sites along the Erren River in southern Taiwan. Aerobic degradation rate constants (k1) and half-lives (t1/2) for NP (2 microg g(-1)) ranged from 0.007 to 0.051 day(-1) and 13.6 to 99.0 days, respectively; for NP1EO (2 microg g(-1)) the ranges were 0.006 to 0.010 day(-1) and 69.3 to 115.5 days. Aerobic degradation rates for NP and NP1EO were enhanced by shaking and increased temperature, and delayed by the addition of Pb, Cd, Cu, Zn, phthalic acid esters (PAEs), and NaCl, as well as by reduced levels of ammonium, phosphate, and sulfate. Of the microorganism strains isolated from the sediment samples, we found that strain JC1 (identified as Pseudomonas sp.) expressed the best biodegrading ability. Also noted was the presence of 4'-amino-acetophenone, an intermediate product resulting from the aerobic degradation of NP by Pseudomonas sp.     

Assessing the critical level of SO2 for Scots pine in situ

A field survey was performed in eastern Finland, where measured ambient SO2 concentrations were 1.4-3.8 microg m(-3) a(-1) and bulk S deposition 0.17-0.32 g m(-2) a(-1) in 1991-1993. The accumulation of sulphur (S) in needles of Scots pine (Pinus sylvestris L.) was studied with XRF, IC and FESEM analyses and the needle damage examined under a light microscope and by SEM. Foliar N concentrations were also measured. Foliar total S concentrations were observed to be above the normal S level (500-700 microg g(-1)) over almost the whole area. Slight chlorosis and/or necrosis of the needle tips and stomatal areas, changes in the needle surface waxes and localization of S into needle tips and mesophyll cells around the stomata suggested the impact of S deposition, as did the calculations of St/Nt, and 'predicted' and 'excess' S. A concentration of about 900 microg g(-1) may be considered a critical level for foliar St in areas with low N supply.     

Determination of ozone in outdoor and indoor environments using nitrite-impregnated passive samplers followed by ion chromatography

An improved ion chromatographic (IC) method has been developed for the separation of nitrate in filter extracts in the presence of high concentrations of nitrite. This analytical method was successfully used for an indirect measurement of ozone (O3) in outdoor and indoor air, following its collection using a nitrite-impregnated passive sampler. The limit of detection and the limit of quantification, using the modified IC method, were 6 microg l(-1) (3sigma) and 20 microg l(-1) (10sigma), respectively. Improved detection limits and low baseline noise were obtained with the use of eluent generator and high-capacity ion exchange column. The optimized method was used for assessing O3 concentration in both indoor and outdoor environments of 28 child care centers (CCCs) located in different parts of Singapore. The O3 concentrations ranged from 0.1 to 11.95 parts per billion (ppb) in indoor and from 3.2 to 21.7 ppb in outdoor environments during the study period. It was found that, among the CCCs investigated in this study, air-conditioned CCCs and those located in close proximity to traffic emissions had significantly lower O3 concentrations indoors.     

High performance liquid chromatographic method for residue determination of sulfosulfuron

A method was developed for sulfosulfuron [(1-(2-ethylsulfonylimidazo [1,2-a] pyridin-3-ylsulfonyl)-3-(4,6-dimethoxy pyrimidin-2yl)] and its three major metabolites by HPLC utilizing photodiode array detector. The method makes use of Lichrosphere RP-8 column and acetonitrile:water:orthophosphoric acid (80:20:0.1 v/v/v) as mobile phase at a flow rate of 1 ml min(-1). Using these condition sulfosulfuron, and compounds II, III and IV were resolved with distinct Rt of 2.088, 2.216, 2.302 and 2.476 minutes, respectively. Sulfosulfuron residues were analysed in soil, wheat grain and straw samples by extracting with a mixture of acetonitrile and 2 M ammonium carbonate (100 ml, 9:1, v/v) using horizontal shaker for soil and Soxhlet apparatus for wheat grain and straw samples. The extracts were cleaned up by partitioning with dichloromethane in case of soil and hexane followed by dichloromethane for plant samples. The percent recovery ranged between 71 to 75.2 for soil and 70.8 to 74.7 for plant samples. The limit of determination of sulfosulfuron was 0.25 microg g(-1).     

Age- and sex-based comparative distribution of selected metals in the scalp hair of an urban population from two cities in Pakistan

Concentrations of Cu, Zn, Pb and Ni in the scalp hair of male and female donors, with an age range of 6-60 years, were determined by ICP atomic emission spectroscopy. The donors were drawn from the densely populated city of Lahore and the relatively less-populated capital city of Islamabad for comparative evaluation of the metal levels in relation to age, sex and location. Linear regression analyses and correlation between paired metals indicated a positive correlation between Cu and Zn for both sexes ( [Formula: see text] ) and between Pb and Ni ( [Formula: see text] ) for males and females of Lahore. Metal concentrations varied between the two cities and the two sexes. The highest mean concentration was found for Zn at 180.5 microg g(-1) for males and 202.4 microg g(-1) for females from Lahore, while for counterparts from Islamabad the values were 105.2 microg g(-1) and 206.6 microg g(-1). Copper showed an identical mean concentration (20.8 microg g(-1)) in the hair of both males and females from Lahore; however, relatively lower mean concentrations (7.7 and 10.8 microg g(-1)) were observed for donors from Islamabad. Mean Pb concentrations in hair of male donors from Lahore and Islamabad were 9.4 and 7.0 microg g(-1), respectively; in female groups the concentrations were 14.3 and 5.7 microg g(-1), respectively. Ni showed the lowest concentration range (4.3-4.5 microg g(-1)) of all the four metals for subjects from Lahore, and this was higher than the corresponding range (2.0-3.2 microg g(-1)) for subjects from Islamabad. The findings are discussed in relation to the available data from the literature.     

Procedures of trophic chain samples preparation for determination of triazines by HPLC and metals by ICP-AES methods

The aim of this research was monitoring the distribution of atrazine and simazine as well as metals Pb, Cd, Zn, Al, Co, Ni, and V along with trophic chains: soil-vegetables and soil, carrot or grass and meat. Different techniques of herbicides extraction by means of many solvents were examined. Triazines were analysed by means of HPLC, metals by means of ICP-AES. Detection limits: LOD=0.2 microg ml(-1), determination limits: LOQ=0.73 microg ml(-1) for atrazine and LOD=0.3 microg ml(-1), LOQ=1.12 microg ml(-1) for simazine were obtained. The content (microg g(-1)) of simazine in soil was in range: 3.45-8.60, in vegetable roots: 6.62-38.15, in vegetable leaves: 2.45-31.71, in rabbit fat: 0.13-49.90. The content (microg g(-1)) of atrazine in soils was in range: 11.9-13.03, in vegetable roots: 13.61-92.90. In analysed material the particular metals after microwave or dry digestion were determined in range (microg g(-1)): Pb: 6.48-43.18; Cd: 0.11-0.57; Zn: 8.79-51.90; Al: 10.22-24.48; Co: 0.18-3.89; Ni: 0.37-6.36; V: 0.29-1.48.     

The long-term environmental behaviour of strontium and barium released from former mine workings in the granites of the Sunart region of Scotland, UK

The concentrations of strontium and barium have been measured in water, sediment and the shells of mussels (Mytilus edulis) from a river system in the Sunart region of Scotland, UK. The aim was to establish the fate and mobility of these elements, which are slowly being released from old mine workings on the Strontian granites. Enhanced strontium (1500-2000 microg l(-1) and 250-290 microg l(-1)) and barium concentrations (316 microg l(-1) and 83 microg l(-1)) were found in the waters originating from the two mine drains studied. Both element were also found at significant levels in the river sediments taken from the vicinity of each drainage site (Sr: 225 microg g(-1) and 120-125 microg g(-1); Ba: 1380 microg g(-1) and 126-170 microg g(-1)). The data suggests that the sediments are acting as a reservoir for these group II cations from where they become distributed throughout the river system. Strontium is found to be incorporated into the shells (3.16-3.46 microg g(-1)) and pearls (3.57 microg g(-1)) of the blue mussel, located at the estuarine margin some 10 km downstream, at values close to the maximum expected (3.3% by weight of the calcium content). The study presents a view of the fate of barium and strontium in a river system over a prolonged period of time. As such it provides valuable information for studies that seek to model the impact of the accidental release of barium and strontium (including the important radionuclide 90Sr) into the environment.     

Copper regulation and homeostasis of Daphnia magna and Pseudokirchneriella subcapitata: influence of acclimation

This study aimed to evaluate (1) the capacity of the green alga Pseudokirchneriella subcapitata and the waterflea Daphnia magna to regulate copper when exposed to environmentally realistic copper concentrations and (2) the influence of multi-generation acclimation to these copper concentrations on copper bioaccumulation and homeostasis. Based on bioconcentration factors, active copper regulation was observed in algae up to 5 microg Cu L(-1) and in daphnids up to 35 mug Cu L(-1). Constant body copper concentrations (13+/-4 microg Cu g DW(-1)) were observed in algae exposed to 1 through 5 microg Cu L(-1) and in daphnids exposed to 1 through 12 microg Cu L(-1). At higher exposure concentrations, there was an increase in internal body copper concentration, while no increase was observed in bioconcentration factors, suggesting the presence of a storage mechanism. At copper concentrations of 100 microg Cu L(-1) (P. subcapitata) and 150 microg Cu L(-1) (D. magna), the significant increases observed in body copper concentrations and in bioconcentration factors may be related to a failure of this regulation mechanism. For both organisms, internal body copper concentrations lower than 13 microg Cu g DW(-1) may result in copper deficiency. For P. subcapitata acclimated to 0.5 and 100 microg Cu L(-1), body copper concentrations ranged (mean+/-standard deviation) between 5+/-2 microg Cu g DW(-1) and 1300+/-197 microg Cu g DW(-1), respectively. For D. magna, this value ranged between 9+/-2 microg Cu g DW(-1) and 175+/-17 microg Cu g DW(-1) for daphnids acclimated to 0.5 and 150 microg Cu L(-1). Multi-generation acclimation to copper concentrations >or =12 microg Cu L(-1) resulted in a decrease (up to 40%) in body copper concentrations for both organisms compared to the body copper concentration of the first generation. It can be concluded that there is an indication that P. subcapitata and D. magna can regulate their whole body copper concentration to maintain copper homeostasis within their optimal copper range and acclimation enhances these mechanisms.     

Evaluation of the phytotoxicity of contaminated sediments deposited "on soil". I. Impact of water draining from the deposit on the germination of neighbouring plants

As part of a study of the phytotoxic risk of spreading of contaminated sediments "on soil", we carried out a laboratory experiment assessing the impact of water draining from sediments in a deposit scenario on the peripheral vegetation. The plant tested were the Chinese cabbage (Brassica campestris L. var. chinensis), maize (Zea mays L.) and ryegrass (Lolium perenne L.). The draining water samples (A1, B1 and C1) were obtained after decantation in laboratory of the sediments. The classification of the water sampled in decreasing order of cumulative contamination was C1 > A1 > B1. The B1 and C1 water samples inhibited the germination of seeds tested to various extents. The percentage of seeds that did not germinate was 1.3-fold times higher for Chinese cabbage with B1 than for the control and 2.3-fold times higher for ryegrass with C1 than for the control. Seeds watered with B1 had lower moisture contents than the control: 10% lower for ryegrass and maize and 50% lower for Chinese cabbage. An increase (about 1.5 microg/ml) in total soluble protein (versus the control) was observed for all three plant species tested in the presence of C1. Glutamine synthetase activity was significantly (1.35 times) higher in ryegrass seeds in the presence of C1 than in the control. We also observed changes in the specific activity of phosphoenolpyruvate carboxylase, which increased for ryegrass and decreased for maize as the concentration of contaminants in the water increased. The results show (i) the necessity to use different plant species to evaluate the toxic effect of sediment deposited on soil on the vegetation, and (ii) that soon as on germination an evaluation of an impact is possible.     

Effects of nitrogen dioxide pollution on the growth of three fern species

The effects of exposing plants of Dryopteris filix-mas (L.) Schott, Phyllitis scolopendrium (L.) Newman and Polypodium vulgare L. to 60 nl litre(-1) (122 microg m(-3)) NO(2) for 37 weeks were investigated in a closed chamber fumigation system. There was no effect of NO(2) on the numbers of fronds produced for any species at any time during the exposure period. However, at the end of the study, there was a lower dry weight yield of green shoots of D. filix-mas and P. scolopendrium and a higher yield of green shoots of P. vulgare for plants in the NO(2) treatment as compared to control plants. These differences in shoot dry weights were not accompanied by an effect of NO(2) exposure on total plant dry weights.     

Distribution of trace element pollutants in a contaminated ecosystem established on metalliferous fluorspar tailings. 3: fluoride

High total soil fluoride (10 000 microg g(-1)) in the metalliferous fluorspar tailings was reflected by elevated concentrations in standing live vegetation (300-1000 microg g(-1)); plant roots (c. 6000 microg g(-1)); plant litter (c. 4000 microg g(-1)); total body concentrations of invertebrates (400-4000 microg g(-1)) and the small mammals Microtus agrestis (120-360 microg g(-1)) and Sorex araneus (140-250 microg g(-1)). Seasonal changes in the standing live vegetation and the availability of soil fluoride to plants are discussed. Seasonal changes in total body concentrations of the small mammals were related to the age structure of the populations as well as dietary levels. In the small mammals, the concentration ratios were < 0.5 at the tailings dam and > 1.1 at the control site, indicating that both species were able to regulate fluoride accumulation at the higher levels of intake. Soft tissue concentrations were, as expected, very low compared to the hard tissues but, still, were generally significantly higher at the tailings dam compared to the control site. Evidence of dental fluorosis was found in Microtus agrestis, but not Sorex araneus.     

Comparison of spontaneous antibiotic resistance frequency of Salmonella Typhimurium growth in glucose amended continuous culture at slow and fast dilution rates

The objective of the study was to determine the frequency of spontaneous acquisition of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in glucose amended continuous flow culture at slow (D = 0.025 h(-1)) or fast (D = 0.27 h(-1)) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25) containing no antibiotics. Upon achieving steady state, samples were plated to tryptic soy agar (TSA) alone or supplemented (per ml) with 2 and 16 microg oxytetracycline, 4 and 16 microg tetracycline, 2 and 64 microg kanamycin, and 0.25 and 2 microg enrofloxacin. Regardless of growth rate, CFU of resistant ST from the TSA containing antibiotics was less than 2 x 10(1) except for 2 microg kanamycin and 0.25 microg enrofloxacin treatments (higher than 1 x 10(9) and 4 x 10(7) CFU of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST from the TSA containing the higher antibiotic concentrations was less than 1 in 10(9) for all antibiotics, but was higher on the media containing 2 microg kanamycin and 0.25 microg enrofloxacin at both slow and fast growth rates. In general, minimal susceptibility differences were detected for isolates from slow and fast dilution rates.