Comparison of the effects of seleno-L-methionine, seleno-DL-methionine, and selenized yeast on reproduction of mallards

The toxicities of seleno-L-methionine, seleno-DL-methionine, and selenized yeast were compared. Ten pairs of mallards were fed a control diet and 15 pairs were fed diets containing 10 ppm selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. Hatching of fertile eggs was significantly lower for females fed 10 ppm selenium as seleno-DL-methionine (7.6%) and seleno-L-methionine (6.4%) than for controls (41.3%). Survival of ducklings was lower when their parents had been fed 10 ppm selenium as seleno-L-methionine (20.0%) than for controls (98.4%). The number of 6-day-old ducklings produced per female was significantly lower for mallards fed 10 ppm selenium as seleno-DL-methionine (0.47) or selenized yeast (2.67) than for controls (6.10), and was significantly lower for mallards fed seleno-L-methionine (0.13) than for mallards fed selenized yeast. The eighth eggs of females fed the DL or L forms of selenomethionine contained means of 9.2 and 8.9 ppm selenium, wet weight; these means were higher than the mean (6.6 ppm) for females fed selenized yeast. Among embryos that died at 7 days of age or older, the percentage of embryos that were deformed was 1.3% for controls, 24.6% for seleno-DL-methionine, 28.2% for seleno-L-methionine, and 11.0% for selenized yeast. The results suggested that seleno-DL-methionine and seleno-L-methionine were of similar toxicity and were both more toxic than selenium from selenized yeast.     

Fluoride effects on the mulberry-silkworm system

Absorption and accumulation of fluoride (F) from ambient air by mulberry leaves, and its transfer and effects on silkworm development, were examined. When the concentration of F in air exceeded 1.5 microg dm(-2). day (-1), using lime filter papers as static monitors, the F content of mulbery leaves was more than 30 ppm, the threshold for injury to silkworm larvae. Fluoride-polluted mulberry leaves inhibited growth and development of silkworms. Leaves containing more than 80 ppm F severely inhibited cocoon production. Fluoride was absorbed from the atmosphere by the mulberry leaf and was transferred to the silkworm, soil, water, and back to the atmosphere, forming a cycle.     

Effects of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in zebrafish: general and reproductive toxicity

Mixed-sex populations of young adult zebrafish (~2-month-old) were exposed to measured RDX concentrations of 0, 1 or 9.6 ppm for up to 12 weeks followed by a 15-day rearing period in untreated water. RDX caused high mortality at 9.6 ppm, with most deaths occurring within the first 8 weeks of exposure. RDX at 9.6 ppm caused lower body weights at 4 and 8 weeks of exposure; and at 1 ppm, lower body weight was observed only at 4 weeks. Fish length was not affected by treatment at any time during the exposure period. The bioconcentration factor for RDX seemed to be influenced by time of exposure but not by water RDX concentration; its overall values were 1.01+/-0.13, 0.91+/-0.06 and 2.23+/-0.04 at 4, 8 weeks and 12 weeks, respectively. RDX was not detected in fish collected after the 15-day recovery period. In a separate experiment, adult females and males were separately exposed to RDX at measured concentrations of 0, 0.5 and 3.2 ppm for a period of 6 weeks. Reproductive performance was evaluated by biweekly breeding of the fish and measuring packed-egg volume (PEV) as index of fecundity. At 0.5 ppm, RDX caused elevated PEV levels relative to the control value at 2 weeks but not at 4 or 6 weeks, whereas no significant effects were noted at 3.2 ppm. Egg fertilization and embryo hatching rates were not affected by RDX at any of the concentrations tested. In conclusion, RDX at sublethal concentrations causes short-term negative effects on growth and, at 0.5 ppm, positive effects on fecundity.     

Factors influencing liver PCB concentrations in sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus) and herons (Ardea cinerea) in Britain

Large scale temporal and spatial changes in the exposure of terrestrial vertebrates to PCBs have been monitored in the UK by measuring liver residues in sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus) and grey herons (Ardea cinerea) from throughout the country. Residues in the three species are typically characterised by large intra- and inter-specific variation. Data for 306 sparrowhawks, 186 kestrels and 47 herons collected between 1992 and 1997 as part of a national Predatory Bird Monitoring Scheme were examined to determine how much of this variation was explained by body condition, age and sex, rather than other factors. In all three species, body condition was the single most important factor and accounted for up to 49% of the variation in PCB liver residues; starved birds had the highest liver concentrations. Age and sex were also significant but of lesser importance. Adult sparrowhawks and kestrels had liver PCB residues that were 2 to 10-fold higher than in first-year birds. Sex did not affect residue magnitude in a consistent manner. PCB concentrations in the liver were higher in males than females in both first-year and adult kestrels and in first-year sparrowhawks, but adult female sparrowhawks had similar PCB residues to adult males. Liver residues also varied seasonally. PCB concentrations in first-year sparrowhawks increased during the first year following fledging and a similar pattern was detected in adult female sparrowhawks following egg laying. When these physiological factors were taken into account, it was evident that while kestrels with high fat scores had significantly lower PCB concentrations than either sparrowhawks or herons, liver residues were similar in all three species when birds were in a starved condition. Overall during 1992-1997, the combined influence of body condition, age and sex explained more of the variation in liver PCB concentrations than species differences or other factors, such as geographical variation in exposure.     

Effects of dieldrin on operant behavior of bobwhites.

Bobwhites (Colinus virginianus) were trained to peck a green lighted key to receive food. Bird response performance was monitored for 14 days, then birds were dosed with five levels of dieldrin (50 to 300 micrograms every other day) for 42 days while their response performance was monitored. At the end of the 42-day dosage period, mean brain concentrations of dieldrin ranged from 2.6 ppm to 11.8 ppm; lower concentrations associated with lower dosage levels. All dieldrin dosage levels altered cage behavior of bobwhites, dieldrin dosage levels greater than 100 micrograms resulted in slower and less accurate responses by dosed birds during the 42-day trial period.     

Acute and chronic activity of perchlorate and hexavalent chromium contamination on the survival and development of Culex quinquefasciatus Say (Diptera: Culicidae)

Effects of water contamination with perchlorate and hexavalent chromium [Cr (VI)] on the mosquito Culex quinquefasciatus were assessed. The chronic (10-day) LC50s values for perchlorate and chromium were 74+/-8.0 mg/L and 0.41+/-0.15 mg/L, respectively. Relative Growth Index, a measure of growth and mortality rates in a population, was significantly reduced within 5 days for levels of perchlorate as low as 25 mg/L and for levels of chromium as low as 0.16 mg/L. Neither compound altered wing length of surviving adults. In combination, contaminants were synergistic, causing 14% more mortality than predicted. Acute (24-h) LC50 values for perchlorate and Cr (VI) were 17,000+/-3200 and 38+/-1.3 mg/L, respectively. Effects on mosquito larvae in contaminated environments are likely to be observed for Cr (VI) but not for perchlorate, which generally does not occur at levels as high as those shown here to affect larval mosquitoes.     

Evidence of immunomodulation in nestling American kestrels (Falco sparverius) exposed to environmentally relevant PBDEs

We investigated whether exposure to environmentally relevant polybrominated diphenyl ethers (PBDEs) causes immunomodulation in captive nestling American kestrels (Falco sparverius). Eggs within each clutch, divided by laying sequence, were injected with safflower oil or penta-BDE congeners-47, -99, -100, and -153 dissolved in safflower oil (18.7 microg sigmaPBDEs/egg) approximating Great Lakes birds. For 29 days, nestlings consumed the same PBDE mixture (15.6+/-0.3 ng/g body weight per day), reaching sigmaPBDE body burden concentrations that were 120x higher in the treatment birds (86.1+/-29.1 ng/g ww) than controls (0.73+/-0.5 ng/g ww). PBDE-exposed birds had a greater PHA response (T-cell-mediated immunity), which was negatively associated with increasing BDE-47 concentrations, but a reduced antibody-mediated response that was positively associated with increasing BDE-183 concentrations. There were also structural changes in the spleen (fewer germinal centers), bursa (reduced apoptosis) and thymus (increased macrophages), and negative associations between the spleen somatic index and sigmaPBDEs, and the bursa somatic index and BDE-47. Immunomodulation from PBDE exposure may be exacerbated in wild birds experiencing greater environmental stresses.     

Effects of chlornitrofen, a herbicide, on reproduction of Brachionus urceolaris (Rotatoria) through water and food (Chlorella)

Chronic effects of chlornitrofen (CNP) on the reproduction of Brachionus urceolaris (Rotatoria) were investigated by exposure of individuals to CNP from the egg stage, which had been attached to the adult. The survivors of 12 neonates, which had been exposed to CNP100, 70 or 40 microg liter(-1) decreased to 50% at the age of c. 2, 4 and 6 days, respectively, compared to c. 6.5 days for those exposed to 0, 10 and 20 microg liter(-1) CNP. Release of offspring (mostly two individuals per day) started at two days old. At the peak, four days old, a control female produced 8.1+/-0.9 offspring per day compared with 4.5+/-1.3 (mean+/-SD, n=12) at 40 microg liter(-1). The cumulative numbers of offspring produced by a female were 25.8+/-1.2, 24.2+/-2.9, 22.3+/-3.6 and 13.6+/-3.1 (mean+/-SD, n=12) at control, 10, 20 and 40 microg liter(-1) CNP exposure, respectively. The 50% reproductive impairment concentration was calculated to be 37 microg liter(-1). Growth of neonates was barely detectable at 70 microg liter(-1), and the rapid increase in the effect of CNP from 40 to 70 microg liter(-1) was attributed to an increase in tolerance with growth of the neonates. The effects of CNP on reproduction were also tested by CNP exposure through food (CNP-accumulated Chlorella). The 50% reproductive impairment concentration of CNP in the alga was calculated to be c.60 microg g(-1) (wet weight) by the same method used to assess the dissolved CNP, although the effect of CNP which may have been released from the alga to the water could not be estimated precisely.     

Fluoride and aluminium concentrations of tea plants and tea products from Sichuan Province,PR China

Some Tibetans in Sichuan Province in southwestern China have been suffering from fluorosis, due to drinking and eating tea with high fluoride (F) and aluminium (Al) contents. Tea plants, soils of tea plantations and tea products from Yaan, Gaoxian and Yibin Cities in Sichuan Province were investigated to evaluate the factors affecting F and Al contents in tea products. The F and Al concentrations of four commercial brands of brick teas were significantly higher than those of 11 brands of green teas. Chemical analysis indicated that total and available F and Al concentrations in tea plantation soils in Yaan and Gaoxian were within the normal range compared with acid soils in South China and tea soils in Fujian Province. Edaphic conditions did not contribute to the high F and Al concentrations in brick tea. Analysis of raw materials of brick tea indicated that old leaves were the major contributors to the high F and Al contents contained in brick tea. There were also great variations among different tea varieties in accumulating F and Al, and concentrations of F and Al in tea variety of Qianmei 303 were about 2-3-fold higher than the other three varieties. Selection of appropriate varieties would be important to lower F and Al contents in tea products.     

Competition between polyphosphate- and glycogen-accumulating organisms in enhanced-biological-phosphorus-removal systems: effect of temperature and sludge age.

Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.     

Residues of chlorpyrifos-methyl in balsam fir foliage, forest litter, soil, stream water, sediment and fish tissue after double aerial applications of Reldan

Chlorpyrifos-methyl was applied twice at 70 g A.I./ha by means of a fixed-wing aircraft to a mixed coniferous forest near Allardville, New Brunswick. Residue in balsam fir foliage was highest (1 ppm wet wt) 1 hr after spraying and rapidly declined to about 30% within 1 day, but persisted at a very low level (0.03 ppm wet wt) for 125 days. Current year's foliage contained a higher level of residue than old foliage. Chlorpyrifos-methyl persisted longer in forest litter than in soil. After 125 days, trace amounts (less than 6 ppb wet wt) were still found in litter but were not detected in soil. In stream water the residue dissipated very rapidly; more than 90% disappeared 3 hours after the second application and were not detected after 4 days. Low-level residue (less than 0.1 ppm wet wt) was present in the sediment and persisted for 10 days. Although brook trout and slimy sculpin captured in the stream within 3 days of the second application contained residues (less than 0.05 ppm fresh wt) none were detected in any fish captured, 9 and 47 days later.     

Factors Affecting Ozone Sensitivity and Susceptibility Of Cotton Plants

Cotton plants were investigated to ascertain the growth period of maximum sensitivity to ozone. Plants were grown from seed in the greenhouse to obtain a two-day interval age series to about three months old. Before fumigation with 0.2 to 1.0 ppm ozone for 1 hr, plants were preconditioned under standard growth chamber conditions with day temperatures of 32°, night temperatures of 21°, relative humidity of 50%, and a light intensity of 6 X 10⁴ ergs/cm²/sec. Also, stomatal opening was monitored with a resistance hygrometer. Ozone damage to leaves was assessed by rating per cent visible damage with an arbitrary scale. Approximately 0.6-0.7 ppm ozone exposure for 1 hr was necessary for visible damage. Leaves were susceptible to ozone when about three-quarters fully expanded. Leaves appearing on older plants became progressively less sensitive. Diurnally, susceptible leaves were only sensitive after several hours in the light and then lost sensitivity toward the end of a normal day. The two most critical questions concerning the nature of ozone sensitivity and susceptibility are: (7) what is the nature of susceptibility, and (2) what is the nature of sensitivity of susceptible leaves?     

Effects of Inorganic Fluorides on Animals

Damage to animals by atmospheric fluoride pollution involves only those animals who subsequently graze on contaminated forages. The chronic disease which results has been most extensively studied in cattle, where the general symptoms are: A n interference with normal dentition, development of osseous lesions, and a diminished appetite which may lead to weight loss or decreased milk production. Studies completed to date would indicate that depending on the conditions of exposure the tolerance level for dairy cattle is about 1.3 mg F/kg (40 ppm F in diet). Because the route of exposure is via the forage, it would seem unreasonable to base any control standard on atmospheric fluoride levels. The forage fluoride levels, analytical evidence from the animal, or even some early sign of fluoride ingestion on the animal might form a more logical basis for a standard.     

Air Quality Standards for the Protection of Farm Animals from Fluorides

The ingestion of forages contaminated with inorganic fluorides from industrial sources constitutes an air pollution problem for domestic livestock. In cattle, which have been studied most extensively, the syndrome is characterized by dental and skeletal lesions, lameness, chemical evidence of increased fluoride ingestion, and in severe cases by effects on appetite and milk production. The only practical basis for a standard appears to be one based on forage fluoride concentrations. It is concluded that a standard should protect cattle from loss of milk production and from severe dental fluorosis, but need not be set so low that the animals will be protected against any discernible deviations from normal which do not influence their general health, productive ability, or the soundness and wearing quality of their dentition. As there may be wide seasonal variations in forage fluoride concentrations, the basic standard should be expressed as a yearly average of the forage fluoride concentration. However, as the developing teeth may be adversely influenced by short periods of high exposure, the standard should contain a provision which limits both the extent and duration of time that high concentrations may be tolerated even though they are balanced by lower values at other months. Based on these criteria, a tentative standard which limits forage fluoride to an average of 40 ppm, and limits the time that forage concentrations may exceed 60 or 80 ppm F is proposed.     

Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta)

Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-1) azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F=2.75, P=0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F=15.33, P<0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species.     

Toxicity of trichlorotoluene isomers: a 28-day feeding study in the rat

Groups of 10 male and 10 female rats were fed alpha,alpha,alpha-, alpha,2,6- or 2,3,6- trichlorotoluene (TCT) in their diet at 0, 0.5, 5.0, 50 or 500 ppm for 28 days. Growth rate and food consumption were not affected by treatment. No deaths occurred. Significant increases in liver weights were observed in male rats fed 5.0 and 500 ppm 2,3,6-TCT. Mild serum biochemical changes occurred in male rats. These included increased SDH activities in the groups fed 5.0 and 50 ppm alpha, alpha, alpha-TCT, and 5.0 ppm 2,3,6-TCT. Alpha, alpha, alpha-TCT at 500 ppm caused elevated LDH activities in male rats. Hepatic microsomal aminopyrine N-demethylase activities were increased in male rats fed 500 ppm alpha,2,6-TCT. Hematological parameters were not affected by treatment. Mild histological changes were seen in the liver, kidney and thyroid of treated rats. Data presented here suggest that alpha, alpha, alpha-, alpha,2,6- and 2,3,6-TCT possess a low order of oral toxicity in the rat.     

Size- and density-distributions and sources of polycyclic aromatic hydrocarbons in urban road dust

Polycyclic aromatic hydrocarbons (PAHs) present in size- and density-fractionated road dust were measured to identify the important fractions in urban runoff and to analyse their sources. Road dust was collected from a residential area (Shakujii) and a heavy traffic area (Hongo Street). The sampling of road dust from the residential area was conducted twice in different seasons (autumn and winter). The collected road dust was separated into three or four size-fractions and further fractionated into light (<1.7 g/cm3) and heavy (>1.7 g/cm3) fractions by using cesium chloride solution. Light particles constituted only 4.0+/-1.4%, 0.69+/-0.03% and 3.4+/-1.0% of the road dust by weight for Shakujii (November), Shakujii (February) and Hongo Street, respectively but contained 28+/-10%, 33+/-3% and 44+/-8% of the total PAHs, respectively. The PAH contents in the light fractions were 1-2 orders of magnitude higher than those in the heavy fractions. In the light fractions, the 12PAH contents in February were significantly higher than the 12PAH contents in November (P<0.01), whereas in the heavy fractions, no significant difference was found (P>0.05). Cluster analysis revealed that there was a significant difference in the PAH profiles between locations rather than between size-fractions, density-fractions and sampling times. Multiple regression analysis indicated that asphalt/pavement was the major source of Shakujii road dust, and that tyre and diesel vehicle exhaust were the major sources of finer and coarser fractions collected from Hongo Street road dust, respectively.     

Rodenticides in British barn owls

Out of 145 Barn Owls found dead through accidents (66%), starvation (32%), shooting (2%) and poisoning (<1%), 10% contained residues of rodenticides, difenacoum or brodifacoum, in their livers. Difenacoum was in the range 0.005-0.106 microg g(-1) fresh weight, and brodifacoum was in the range 0.019-0.515 microg g(-1). Minimum levels of detection were about 0.005 microg g(-1) for both chemicals. Mice fed for 1 day on food containing difenacoum and brodifacoum died after 2-11 days. Within these mice residues were present at greater concentration in the liver than in the rest of the carcass. The mean mass of residue in a whole 35g mouse was estimated at 10.17 microg (range 4.73-20.65 microg) for difenacoum and 15.36 microg (range 8.07-26.55) for brodifacoum. Such poisoned mice were fed to Barn Owls for successive periods of 1, 3 and 6 days. All six owls fed on difenacoum-dosed mice survived all three treatments, in which up to an estimated 101.7 microg of difenacoum was consumed, and the coagulation times of their blood returned to near normal in less than 5-23 days. Four of the six owls fed on brodifacoum-dosed mice died 6-17 days after the 1-day treatment, but the survivors also survived the 3-day and 6-day treatments. Those that died had each eaten 3 mice, with a combined weight of about 105g and a total brodifacoum content of about 46.07 microg, which was equivalent to a dose of 0.150-0.182 mg kg(-1) of owl body weight. After death these owls had 0.63-1.25 micro g(-1) of brodifacoum in their livers. Blood from the survivors would not coagulate at 9 days post-treatment, but did so at 16 days in one bird and between 38 and 78 days in the other. It is concluded that: (1) Barn Owls in Britain are now widely exposed to second-generation rodenticides; (2) not all owls exposed to these chemicals are likely to receive a lethal dose; (3) brodifacoum is more toxic to owls than difenacoum; and (4) while there is yet no evidence that rodenticides have had any appreciable effect on Barn Owl populations in Britain, further monitoring of residue levels and population trends in desirable.     

Residues of chlorpyrifos‐methyl in balsam fir foliage,forest litter,soil, stream water,sediment and fish tissue after double aerial applications of Reldan®

Abstract

Chlorpyrifos‐methyl was applied twice at 70 g A.I./ha by means of a fixed‐wing aircraft to a mixed coniferous forest near Allardville, New Brunswick. Residue in balsam fir foliage was highest (1 ppm wet wt) 1 hr after spraying and rapidly declined to about 30% within 1 day, but persisted at a very low level (0.03 ppm wet wt) for 125 days. Current year's foliage contained a higher level of residue than old foliage. Chlorpyrifos‐methyl persisted longer in forest litter than in soil. After 125 days, trace amounts (< 6 ppb wet wt) were still found in litter but were not detected in soil. In stream water the residue dissipated very rapidly; more than 90% disappeared 3 hours after the second application and were not detected after 4 days. Low‐level residue (< 0.1 ppm wet wt) was present in the sediment and persisted for 10 days. Although brook trout and slimy sculpin captured in the stream within 3 days of the second application contained residues (< 0.05 ppm fresh wt) none were detected in any fish captured, 9 and 47 days later.     

Developmental exposure to diuron causes splenotoxicity in male Sprague-Dawley rat pups

This study investigated whether perinatal exposure to diuron [3-(3,4-dichlorophenyl)-1-1-dimethylurea] might exert adverse effects on rat lymphoid organs. Pregnant Sprague-Dawley (SD) rats were exposed to diuron at 500, 750 or 1250 ppm in the diet from gestational days (GD) 12-21 and during lactation. At postnatal day (PND) 42, male pups were euthanized and thymus, spleen, mesenteric lymph node and femur were collected for histopathological analysis. Food consumption and body weight gain were significantly reduced in dams exposed to 1250 ppm during gestation period. Also, Diuron at 750 and 1250 ppm produced: (1) increased relative spleen weight associated histologically with severe congestion in red pulp, (2) enhanced extramedullary hematopoiesis and hemosiderosis as well as (3) depletion of lymphoid follicles in white pulp. Flow cytometric analysis revealed a significant reduction in B lymphocytes (CD45RA+) in male pups but T lymphocytes (CD4+, CD8+ and CD4+/CD8+) were not markedly affected. Thus, data suggest that Diuron-induced maternal toxicity in dams exposed to high dose and perinatal exposure to this herbicide produced spleen toxicity as evidenced by a reduction in B lymphocyte number in male SD pups.