Uptake of C14-atrazine by prairie grasses in a phytoremediation setting.

Agrochemicals significantly contribute to environmental pollution. In the USA, atrazine is a widely used pesticide and commonly found in rivers, water systems, and rural wells. Phytoremediation can be a cost-effective means of removing pesticides from soil. The objective of this project was to investigate the ability of prairie grasses to remove atrazine. 14C-labeled atrazine was added to sterilized sand and water/nutrient cultures, and the analysis was performed after 21 days. Switchgrass and big bluestem were promising species for phytoremediation, taking up about 40% of the applied [14C] in liquid hydroponic cultures, and between 20% and 33% in sand cultures. Yellow Indiangrass showed low resistance to atrazine toxicity and low uptake of [14C] atrazine in liquid hydroponic cultures. Atrazine degradation increased progressively from sand to roots and leaves. Most atrazine taken up by prairie grasses from sand culture was degraded to metabolites, which accounted for 60-80% of [14C] detected in leaves. Deisopropylatrazine (DIA) was the main metabolite detected in sand and roots, whereas in leaves further metabolism took place, forming increased amounts of didealkylatrazine (DDA) and an unidentified metabolite. In conclusion, prairie grasses achieved high atrazine removal and degradation, showing a high potential for phytoremediation.

Metabolism and metabolites of polychlorinated biphenyls.

Abstract The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity, and thereby on the assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general, and in humans in particular. The aim of this document is to provide an overview of PCB metabolism, and to identify the metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed.

Microbial community analysis of switchgrass planted and unplanted soil microcosms displaying PCB dechlorination.

Polychlorinated biphenyls (PCBs) pose potential risks to human and environmental health because they are carcinogenic, persistent, and bioaccumulative. In this study, we investigated bacterial communities in soil microcosms spiked with PCB 52, 77, and 153. Switchgrass (Panicum virgatum) was employed to improve overall PCB removal, and redox cycling (i.e., sequential periods of flooding followed by periods of no flooding) was performed in an effort to promote PCB dechlorination. Lesser chlorinated PCB transformation products were detected in all microcosms, indicating the occurrence of PCB dechlorination. Terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis showed that PCB spiking, switchgrass planting, and redox cycling affected the microbial community structure. Putative organohalide-respiring Chloroflexi populations, which were not found in unflooded microcosms, were enriched after 2 weeks of flooding in the redox-cycled microcosms. Sequences classified as Geobacter sp. were detected in all microcosms and were most abundant in the switchgrass-planted microcosm spiked with PCB congeners. The presence of possible organohalide-respiring bacteria in these soil microcosms suggests that they play a role in PCB dechlorination therein.

Atmospheric dispersion of PCB from a contaminated Lake Michigan harbor.

Indiana Harbor and Ship Canal (IHSC) in East Chicago is an industrial waterway on Lake Michigan and a source of PCBs to Lake Michigan and the overlying air. We hypothesized that IHSC is an important source of airborne PCBs to surrounding communities. We used AERMOD to model hourly PCB concentrations, utilizing emission fluxes from a prior study and hourly meteorology provided by the State of Indiana. We also assessed dispersion using hourly observed meteorology from a local airport and high resolution profiles simulated by the Weather Research and Forecasting model. We found that emissions from IHSC waters contributed about 15% of the observed ΣPCB concentrations close to IHSC when compared on an hourly basis and about 10% of observed annual concentrations at a nearby school. Concentrations at the school due to emissions from IHSC ranged from 0 to 18,000 pg m-3, up to 20 times higher than observed background levels, with an annual geometric mean (GSD) of 19 (31) pg m-3. Our findings indicate that IHSC is an important source of PCBs to East Chicago, but not the only source. Four observed enriched PCB3 samples suggest a nearby non-Aroclor source.

Enhanced Polychlorinated Biphenyl Removal in a Switchgrass Rhizosphere by Bioaugmentation with Burkholderia xenovorans LB400.

Phytoremediation makes use of plants and associated microorganisms to clean up soils and sediments contaminated with inorganic and organic pollutants. In this study, switchgrass (Panicum virgatum) was used to test for its efficiency in improving the removal of three specific polychlorinated biphenyl (PCB) congeners (PCB 52, 77 and 153) in soil microcosms. The congeners were chosen for their ubiquity, toxicity, and recalcitrance. After 24 weeks of incubation, loss of 39.9 ± 0.41% of total PCB molar mass was observed in switchgrass treated soil, significantly higher than in unplanted soil (29.5 ± 3.4%) (p<0.05). The improved PCB removal in switchgrass treated soils could be explained by phytoextraction processes and enhanced microbial activity in the rhizosphere. Bioaugmentation with Burkholderia xenovorans LB400 was performed to further enhance aerobic PCB degradation. The presence of LB400 was associated with improved degradation of PCB 52, but not PCB 77 or PCB 153. Increased abundances of bphA (a functional gene that codes for a subunit of PCB-degrading biphenyl dioxygenase in bacteria) and its transcript were observed after bioaugmentation. The highest total PCB removal was observed in switchgrass treated soil with LB400 bioaugmentation (47.3 ± 1.22 %), and the presence of switchgrass facilitated LB400 survival in the soil. Overall, our results suggest the combined use of phytoremediation and bioaugmentation could be an efficient and sustainable strategy to eliminate recalcitrant PCB congeners and remediate PCB-contaminated soil.

Effective alleviation of aluminum phytotoxicity by manure-derived biochar.

The alleviation of aluminum phytotoxicity to wheat plants in a hydroponic system through the amendment of biochar is investigated to explore the possibility of applying biochar in acidic soil amelioration. Biochar derived from cattle manure pyrolyzed at 400 °C (CM400) and the CM400 biochar washed with distilled-deionized water to remove alkalinity (WCM400) were prepared to determine the roles of the liming effect and adsorption during the alleviation of Al toxicity. Upon addition of 0.02% (W/V) CM400 to the exposure solution, the inhibition of plant growth by Al was significantly reduced while the toxic threshold was extended from 3 to 95 µmol/L Al(3+). Due to the biochar liming effect, the aluminum species were converted to Al(OH)(2+) and Al(OH)2(+) monomers, which were strongly adsorbed by biochar; furthermore, the highly toxic Al(3+) evolved to less toxic Al(OH)3 and Al(OH)4(-) species. Adsorption of Al by the biochar is dominated by surface complexation of the carboxyl groups with Al(OH)(2+)/Al(OH)2(+) rather than through electrostatic attraction of Al(3+) with negatively charged sites. Compared to the liming effect, the adsorption by biochar exhibited a sustainable effect on the alleviation of Al toxicity. Therefore, the biochar amendment appears to be a novel approach for aluminum detoxification in acidic soils.

Simulating and explaining passive air sampling rates for semivolatile compounds on polyurethane foam passive samplers.

Passive air samplers (PAS) including polyurethane foam (PUF) are widely deployed as an inexpensive and practical way to sample semivolatile pollutants. However, concentration estimates from PAS rely on constant empirical mass transfer rates, which add unquantified uncertainties to concentrations. Here we present a method for modeling hourly sampling rates for semivolatile compounds from hourly meteorology using first-principle chemistry, physics, and fluid dynamics, calibrated from depuration experiments. This approach quantifies and explains observed effects of meteorology on variability in compound-specific sampling rates and analyte concentrations, simulates nonlinear PUF uptake, and recovers synthetic hourly concentrations at a reference temperature. Sampling rates are evaluated for polychlorinated biphenyl congeners at a network of Harner model samplers in Chicago, IL, during 2008, finding simulated average sampling rates within analytical uncertainty of those determined from loss of depuration compounds and confirming quasilinear uptake. Results indicate hourly, daily, and interannual variability in sampling rates, sensitivity to temporal resolution in meteorology, and predictable volatility-based relationships between congeners. We quantify the importance of each simulated process to sampling rates and mass transfer and assess uncertainty contributed by advection, molecular diffusion, volatilization, and flow regime within the PAS, finding that PAS chamber temperature contributes the greatest variability to total process uncertainty (7.3%).

Subchronic inhalation exposure study of an airborne polychlorinated biphenyl mixture resembling the Chicago ambient air congener profile.

Although inhalation of atmospheric polychlorinated biphenyls (PCBs) is the most universal exposure route and has become a substantial concern in urban areas, research is lacking to determine the body burden of inhaled PCBs and consequent health effects. To reflect the Chicago airshed environment and mimic the PCB profile in Chicago air, we generated vapors from a Chicago air mixture (CAM). Sprague-Dawley rats were exposed to the CAM vapor for 1.6 h/day via nose-only inhalation for 4 weeks, 520 ± 10 µg/m(3). Congener-specific quantification in tissue and air samples was performed by gas chromatography-tandem mass spectrometry (GC/MS/MS). In contrast to the lower-chlorinated congener-enriched vapor, body tissues mainly contained tri- to hexachlorobiphenyls. Congener profiles varied between vapor and tissues and among different organs. The toxic equivalence (TEQ) and neurotoxic equivalence (NEQ) were also investigated for tissue distribution. We evaluated a variety of end points to catalogue the effects of long-term inhalation exposure, including immune responses, enzyme induction, cellular toxicity, and histopathologic abnormalities. Glutathione oxidized/reduced ratio (GSSG/GSH) was increased in the blood of exposed animals, accompanied by elevation of hematocrit. This study demonstrated that inhalation contributed to the body burden of mostly tri- to hexachlorobiphenyls and produced a distinct profile of congeners in tissue, yet minimal toxicity was found at this exposure dose, estimated at 134 µg/rat.

Enantioselective biotransformation of chiral PCBs in whole poplar plants.

Chiral PCBs have been used as molecular probes of biological metabolic processes due to their special physical, chemical, and biological properties. Many animal studies showed the enantioselective biotransformation of chiral PCBs, but it is unclear whether plants can enantioselectively biotransform chiral PCBs. In order to explore the enantioselectivity of chiral PCBs in whole plants, poplars (Populus deltoides × nigra, DN34), a model plant with complete genomic sequence, were hydroponically exposed to 2,2',3,5',6-pentachlorobiphenyl (PCB95) and 2,2',3,3',6,6'-hexachlorobiphenyl (PCB136) for 20 days. PCB95 and PCB136 were shown to be absorbed, taken-up and translocated in whole poplars, and they were detected in various tissues of whole poplars. However, the enantioselectivity of poplar for PCB95 and PCB136 proved to be quite different. The first eluting enantiomer of PCB95 was enantioselectively removed in whole poplar, especially in the middle and bottom xylem. It was likely enantioselectively metabolized inside poplar tissues, in contrast to racemic mixtures of PCB95 remaining in hydroponic solutions in contact with plant roots of whole and dead poplars. Unlike PCB95, PCB136 remained nearly racemic in most parts of whole poplars after 20 days exposure. These results suggest that PCB136 is more difficult to be enantioslectively biotransformed than PCB95 in whole poplars. This is the first evidence of enantioselectivity of chiral PCBs in whole plants, and suggests that poplars can enantioselectively biotransform at least one chiral PCB.

Sedimentary Records of Non-Aroclor and Aroclor PCB mixtures in the Great Lakes.

Three sediment cores from Lake Ontario, Lake Erie and Indiana Harbor Ship Canal were collected, segmented and analyzed for Aroclor and non-Aroclor polychlorinated biphenyl congeners (PCBs). PCBs associated with the commercially produced Aroclor mixtures 1248 and 1254 dominate the sediment signal and the sum of all congeners (ΣPCB) peaks in concentration and accumulation around 1970 in the Great Lakes. This trend is very similar to Aroclor production history. In the Indiana Harbor Ship Canal, PCBs appear around 1935 and remain at very high levels between 1940 and 1980, probably reflecting the history of use at the nearby steel mill. In contrast, the non-Aroclor PCBs in the Lake Ontario and IHSC sediment cores, including PCB11 and heavily chlorinated congeners PCB206, 207, 208 and 209 reach a peak in the 1950s, decline and peak again in the 1970s or in the early 1980s. All five congeners have been previously measured in commercial paint pigment. PCB11 was found to peak about 5 years later than ΣPCBs, and is probably associated with the production or use history of diarylide yellow pigments. The temporal distribution profiles of these non-Aroclor PCBs are well correlated with the production history of paint pigments and dyes. Although it is well known that the production of Aroclor PCBs is preserved in Great Lakes sediments, this study is the first to show that production of non-Aroclors are also preserved in the sediments as a record of long term trends in environmental exposure.

Time course of congener uptake and elimination in rats after short-term inhalation exposure to an airborne polychlorinated biphenyl (PCB) mixture.

Despite the continued presence of PCBs in indoor and ambient air, few studies have investigated the inhalation route of exposure. While dietary exposure has declined, inhalation of the semivolatile, lower-chlorinated PCBs has risen in importance. We measured the uptake, distribution, and time course of elimination of inhaled PCB congeners to characterize the pulmonary route after short-term exposure. Vapor-phase PCBs were generated from Aroclor 1242 to a nose-only exposure system and characterized for congener levels and profiles. Rats were exposed via inhalation acutely (2.4 mg/m3 for 2 h) or subacutely (8.2 mg/m3, 2 hx10 days), after which pulmonary immune responses and PCB tissue levels were measured. Animals acutely exposed were euthanized at 0, 1, 3, 6, and 12 h post exposure to assess the time course of PCB uptake and elimination. Following rapid absorption and distribution, PCBs accumulated in adipose tissue but decayed in other tissues with half-lives increasing in liver (5.6 h)

Atmospheric PCB congeners across Chicago.

We have measured PCBs in 184 air samples collected at 37 sites in the city of Chicago using an innovative system of high-volume air samplers mounted on two health clinic vans. Here we describe results of sampling conducted from November 2006 to November 2007. The samples were analyzed for all 209 PCB congeners using a gas chromatograph with tandem mass spectrometry (GC-MS/MS). The ΣPCBs (sum of 169 peaks) in Chicago ranged from 75 pg m(-3) to 5500 pg m(-3) and primarily varied as a function of temperature. The congener patterns are surprisingly similar throughout the city even though the temperature-corrected concentrations vary by more than an order of magnitude. The average profile resembles a mixture of Aroclor 1242 and Aroclor 1254, and includes many congeners that have been identified as being aryl hydrocarbon receptor (AhR) agonists (dioxin-like) and/or neurotoxins. The toxic equivalence (TEQ) and neurotoxic equivalence (NEQ) in air were calculated and investigated for their spatial distribution throughout the urban-industrial complex of Chicago. The NEQ concentrations are linearly correlated with ΣPCBs while the TEQ concentrations are not predictable. The findings of this study suggest that airborne PCBs in Chicago are widely present and elevated in residential communities; there are multiple sources rather than one or a few locations of very high emissions; the emission includes congeners associated with dioxin-like and neurotoxic effects and congeners associated with unidentified sources.

Laboratory evaluation of mobility and sorption for the veterinary antibiotic, tylosin, in agricultural soils.

Veterinary medicines, including antibiotics, are utilized in large quantities in intensive livestock farming. It is evidenced that tylosin, one of the most frequently used antibiotics, is only partially metabolized in animals and not completely degraded in the manure storage stage before application to the farmland. In order to assess the mobility of tylosin in soil, a soil-column leaching study and a simple batch sorption experiment were conducted in the laboratory. Tylosin had strong sorption to various soils, with sorption distribution coefficients ranging from 42 to 65 ml/g. The range of concentrations in leachate was detected from non-detectable to 0.27 ng/mL after four simulated rainfall events in one month, and leachability of tylosin is dependent on soil properties and manure amendment. Percentage of clay, organic matter, cation exchange capacity, and manure amendment were positively correlated with sorption, and negatively correlated with mobility of tylosin in soil. The majority of tylosin was not recovered in the testing system, indicating that tylosin was most likely mineralized, or irreversibly bound to solid particles since no major degradation products were detected. Some trace level tylosin residues from manure-applied farmlands may be the major source to surface water systems through soil erosion and preferential flow processes.

Evaluation of the environmental fate of thymol and phenethyl propionate in the laboratory.

BACKGROUND: The natural monoterpenoid pesticides thymol and phenethyl propionate (PEP) are used indoors and outdoors, but their fate in the environment has not been reported. In order better to understand their impact on the environment, water metabolism and soil metabolism studies were conducted with thymol and PEP at a concentration of 10 microg g(-1) in water and in soil under laboratory conditions. RESULTS: Dissipation half-lives of thymol and PEP were 16 and 5 days in water and 5 and 4 days in soil. 2-Phenylethanol and 2-(4-hydroxyphenyl)ethanol were detected as primary degradation products of PEP. Over time, a considerable volatilization loss of thymol, but not of phenethyl propionate, was found in the 1 month study under the experimental conditions used. Less than 6% of thymol and PEP were detected as bound residues, and less than 3% were mineralized during the 30 day study. CONCLUSION: In order to maximize the pesticidal effect, more attention should be paid to the temperature for thymol than for PEP when they are being applied, owing to the high volatility of the former. Thymol and PEP pose low risks to the ecosystem because of their rapid dissipation and low bound residues in the environment.

Aerobic degradation and photolysis of tylosin in water and soil.

Veterinary antibiotics enter the environment through the application of organic fertilizers to cropland. In this study, the aerobic degradation of tylosin, a widely used antibiotic in the production of livestock and poultry, was conducted in water and in soil in an effort to further investigate its environmental fate. Tylosin is a macrolide antibiotic, which consists of four factors (A, B, C, D). Water and soil were sampled at selected times and analyzed for tylosin and its degradation products by high-performance liquid chromatography (HPLC), with product identification confirmed by HPLC-mass spectrometry. Tylosin A is degraded with a half-life of 200 d in the light in water, and the total loss of tylosin A in the dark is 6% of the initial spiked amount during the experimental period. Tylosin C and D are relatively stable except in ultrapure water in the light. Slight increases of tylosin B after two months and formation of two photoreaction isomers of tylosin A were observed under exposure to light. However, tylosin probably would degrade faster if the experimental containers did not prevent ultraviolet transmission. In soil, tylosin A has a dissipation half-life of 7 d, and tylosin D is slightly more stable, with a dissipation half-life of 8 d in unsterilized and sterilized soil. Sorption and abiotic degradation are the major factors influencing the loss of tylosin in the environment, and no biotic degradation was observed at the test concentration either in pond water or in an agronomic soil, as determined by comparing dissipation profiles in sterilized and unsterilized conditions.

Pharmacokinetics of florfenicol in healthy and Escherichia coli-infected broiler chickens.

The pharmacokinetics of florfenicol were studied in both healthy (n=9) and Escherichia coli-infected (n=27) broiler chickens following intravenous (i.v.), intramuscular (i.m.) and oral administration at a single dose of 30 mg/kgbw. Infection was induced artificially in broiler chickens (35 d) by an intraperitoneal injection with 1.3 x 10(9) colony-forming units of E. coli. After i.v. administration in healthy and infected broiler chickens, the disposition kinetics of florfenicol were described by a bi-exponential equation. In diseased broiler chickens, a decrease in the elimination half-life and the apparent volume of distribution were found. The pharmacokinetic parameters such as total body clearance and the areas under curves of plasma concentrations were comparable in healthy and E. coli infected chickens. Following i.m. and oral administration in infected chickens, the plasma concentration-time data for florfenicol were found to fit a single-compartment open model. The elimination half-lives (t(1/2 el)) of florfenicol were 129(13) and 104(15)min, the systemic bioavailability 87% and 71% after i.m. and oral administration.

In vitro and in vivo metabolite profiling of valnemulin using ultraperformance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry.

Valnemulin, a semisynthetic pleuromutilin derivative related to tiamulin, is broadly used to treat bacterial diseases of animals. Despite its widespread use, metabolism in animals has not yet been fully investigated. To better understand valnemulin biotransformation, in this study, metabolites of valnemulinin in in vitro and in vivo rats, chickens, swines, goats, and cows were identified and elucidated using ultraperformance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry (UPLC-Q/TOF-MS). As a result, there were totally 7 metabolites of valnemulin identified in vitro and 75, 61, and 74 metabolites detected in in vivo rats, chickens, and swines, respectively, and the majority of metabolites were reported for the first time. The main metabolic pathways of valnemulin were found to be hydroxylation in the mutilin part (the ring system) and the side chain, oxidization on the sulfur of the side chain to form S-oxides, hydrolysis of the amido bond, and acetylization in the amido of the side chain. In addition, hydroxylation in the mutilin part was proposed to be the primary metabolic route. Furthermore, the results revealed that 2ß-hydroxyvalnemulin (V1) and 8α-hydroxyvalnemulin (V2) were the major metabolites for rats and swines and S-oxides (V6) in chickens.

Bioaccumulation and elimination kinetics of hydroxylated polybrominated diphenyl ethers (2'-OH-BDE68 and 4-OH-BDE90) and their distribution pattern in common carp (Cyprinus carpio).

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have attracted wide concerns due to their toxicities and universal presence in wildlife and humans. The relatively high Kow values of OH-PBDEs imply these compounds may have a significant bioaccumulation potential, but so far, the existing data provide little information regarding the kinetics of uptake and depuration in any organisms. Here we exposed common carps separately to two OH-PBDEs, 2'-OH-BDE68 and 4-OH-BDE90, for 30 days (d) in a flow-through system, followed by a 60-d depuration period in clean water to investigate compound-specific bioaccumulation and tissue distribution. Two OH-PBDEs could accumulate in common carp, and the high concentration was observed in liver or kidney. The uptake rates (k1) of two OH-PBDEs ranged from 0.15 to 21.3 d(-1) in fish, and the elimination rates (k2) ranged from 0.027 to 0.075 d(-1), which leaded to their BCF values in 4.8-299.2 ranges. Half-lives ranged from 9.2 d to 25.6 d. The exposure concentration significantly affected BCF values but didn't change their relative compositions in liver, kidney and muscle after a long exposure time. To our knowledge, this is the first study to systematically assess uptake, depuration kinetics and tissue distribution for OH-PBDEs via a controlled experimental animal model.

Dechlorination of PCBs in the rhizosphere of switchgrass and poplar.

Polychlorinated biphenyl (PCB) congeners (PCB 52, 77, and 153) singly and in mixture were spiked and aged in soil microcosms and subsequently planted with switchgrass (Panicum virgatum) or poplar (Populus deltoids x nigra DN34). The planted reactors showed significantly greater reductions in PCB parent compounds when compared to unplanted systems after 32 weeks. There was evidence of reductive dechlorination in both planted and unplanted systems, but the planted microcosms with fully developed roots and rhizospheres showed greater biotransformation than the unplanted reactors. These dechlorination products accounted for approximately all of the molar mass of parent compound lost. Based on the transformation products, reductive dechlorination pathways are proposed for rhizospheric biotransformation of PCB 52, 77, and 153. This is the first report of rhizosphere biotransformation pathways for reductive dechlorination in marginally aerobic, intermittently flooded soil as evidenced by a mass balance on transformation products.

Metabolic pathways of T-2 toxin in in vivo and in vitro systems of Wistar rats.

In the present study, metabolites of T-2 toxin in in vivo and in vitro systems of Wistar rats were identified and elucidated by ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS). Expected and unexpected metabolites were detected by Metabolynx(XS) software, which could automatically compare MS(E) data from the sample and control. A total of 19 metabolites of T-2 toxin were identified in this research, 9 of them being novel, which were 15-deacetyl-T-2, 3'-OH-15-deacetyl-T-2, 3',7-dihydroxy-T-2, isomer of 3',7-dihydroxy-T-2, 7-OH-HT-2, isomer of 7-OH-HT-2, de-epoxy-3',7-dihydroxy-HT-2, 9-OH-T-2, and 3',9-dihydroxy-T-2. The results showed that the main metabolic pathways of T-2 toxin were hydrolysis, hydroxylation, and de-epoxidation. In addition, the results also revealed one novel metabolic pathway of T-2 toxin, hydroxylation at C-9 position, which was demonstrated by the metabolites 9-OH-T-2 and 3',9-dihydroxy-T-2. In addition, hydroxylation at C-9 of T-2 toxin was also generated in in vitro of liver systems. Interestingly, several metabolites of hydroxylation at C-7 of T-2 toxin were also detected in in vivo male Wistar rats, but they were not found in in vivo female rats and in in vitro systems of Wistar rats.