Internal Cd Research Articles

Cd 2+ transport and storage in the chloroplast of Euglena gracilis

Euglena gracilis lacks a plant-like vacuole and, when grown in Cd 2+-containing medium, 60% of the accumulated Cd 2+ is located inside the chloroplast. Hence, the biochemical mechanisms involved in Cd 2+ accumulation in chloroplast were examined. Percoll-purified chloroplasts showed a temperature-sensitive uptake of the free 109Cd 2+ ion. Kinetics of the uptake initial rate was resolved in two components, one hyperbolic and saturable ( V max 11 nmol 109Cd 2+ min −1 mg protein −1, K m 13 μM) and the other, linear and non-saturable. 109Cd 2+ uptake was not affected by metabolic inhibitors or illumination. Zn 2+ competitively inhibited 109Cd 2+ uptake ( K i 8.2 μM); internal Cd 2+ slightly inhibited 109Cd 2+ uptake. Cadmium was partially and rapidly released from chloroplasts. These data suggested the involvement of a cation diffusion facilitator-like protein. Chloroplasts isolated from cells grown with 50 μM CdCl 2 (ZCd 50 chloroplasts) showed a 1.6 times increase in the uptake V max, whereas the K m and the non-saturable component did not change. In addition, Cd 2+ retention in chloroplasts correlated with the amount of internal sulfur compounds. ZCd 50 chloroplasts, which contained 4.4 times more thiol-compounds and sulfide than control chloroplasts, retained six times more Cd 2+. The Cd 2+ storage-inactivation mechanism was specific for Cd 2+, since Zn 2+ and Fe 3+ were not preferentially accumulated into chloroplasts.

Bioconcentration of cadmium and toxic effects on life-history traits of pond snails (Lymnaea palustris and Lymnaea stagnalis) in laboratory bioassays.

We studied the bioconcentration and the individual effects of Cd on life-history traits of Lymnaea stagnalis and Lymnaea palustris exposed to increasing Cd concentrations for 4 weeks in controlled conditions. Internal Cd concentrations were linearly correlated to Cd concentrations of exposure in both foot and viscera of L. stagnalis and in the foot of L. palustris, and they were linked by a logistic relationship with environmental contamination in the viscera of L. palustris. If LC50S were higher than the highest Cd concentrations of exposure (L. stagnalis: 160 microg L(-1); L. palustris: 320 microg L(-1)), other dose-dependent responses affecting life-history traits were noted in both species. In L. stagnalis, EC50 for growth was evaluated at 142 microg L(-1) and growth inhibition was correlated with internal Cd concentrations by a linear relationship. L. palustris was more sensitive to Cd than L. stagnalis because its EC50-growth was three times lower (58 microg L(-1) after 28 days). Dose-dependent responses were obtained for several parameters of fecundity of L. palustris. EC50 for the mean number of egg masses or mean number of eggs per individual were close to 60 microg L(-1), whereas for the mean number of eggs per egg mass, the EC50 was higher, with a value of 124 microg L(-1). The percentage of hatching versus the total number of eggs was 60% in controls, and no embryos were able to hatch at the lowest tested Cd concentration, 40 microg L(-1). The high sensitivity of fertility was due to Cd blocking embryo development, particularly for the latest embryonic stages just before hatching. The present results constitute useful data to develop laboratory tests using pond snails for freshwater risk assessment.

Oral sealing using glue: a new method to distinguish between intestinal and dermal uptake of metals in earthworms

Earthworms may take up chemicals from soil and pore water, both through their skin (dermal) and by ingestion (oral). It remains unclear, however, what the relative importance of these pathways is. To assess bioavailability of pollutants in soil to earthworms, it is necessary that the contribution of each pathway is known. Lumbricus rubellus were sealed by means of medical histoacryl glue, to block ingestion of soil particles and pore water. For 6 d, these earthworms showed good survival and vitality and no soil ingestion was found. Equal metal uptake was found by sealed and unsealed earthworms exposed to an inert sand matrix continuously flushed with contaminated water. Therefore, pore water uptake via ingestion contributes little to metal accumulation. Uptake rates of Cd, Cu and Pb in sealed and unsealed earthworms exposed to two contaminated field soils were similar. Uptake and elimination kinetics of Zn were significantly lower in sealed earthworms exposed to one of the two field soils. Body concentrations of Cu and Pb could be completely attributed to the dermal route. For internal Cd and Zn concentrations, however, 0–17 and 21–30%, respectively, were derived from ingestion. It is concluded that for metals the dermal route is the uptake route of importance. The sealing method described here may be useful in a variety of earthworm nutrition and contamination-effect studies.

Adaptive reproductive variation along a pollution gradient in a wolf spider.

When populations are exposed to environmental pollutants, growth and reproduction might be strongly reduced due to an increased detoxification effort. Sublethal metal pollution is therefore to be expected to cause the same selection pressure as a low resource habitat and might alter the reproductive strategy. Optimality models of life history theory predict that when resource availability is reduced, growth and reproductive output are reduced and that the release of fewer but larger propagules will be favoured. This was tested by applying a life history model to reproductive trait measurements in six populations of the wolf spider Pirata piraticus in which the assumptions of the model are satisfied. Internal Cd, Cu and Zn body burden were strongly correlated with each other, and differed strongly between the populations, indicating consistently differing metal exposure at the different sites. Pb levels were extremely variable within each population and did not differ between the populations. Females from populations with high concentrations of the first three heavy metals showed a strongly reduced reproductive output and fecundity, indicating a high reduction in resource availability due to detoxification processes. Egg size in contrast was negatively correlated with fecundity and reproductive output and as a consequence positively related with internal metal burden. Our results are thus in strong agreement with the predictions of the optimality models and confirm the benefits of a larger propagule size when resource availability is reduced.

Is the cadmium uptake from soil important in bioaccumulation and toxic effects for snails?

To evaluate the contribution of digestive and epithelial transfer of Cd from soil to snail, Helix aspersa was exposed to an artificial substrate contaminated with 0,100,500, and 1000 μg Cd · g −1 for 4 weeks under laboratory conditions. Two modes of exposure were used: (1) the snails were in direct contact with the substrate (DC) or (2) were separated from substrate with a perforated plate (no contact (NC)) which allowed ingestion of substrate but avoided epithelial contact. Cd concentrations in DC snails were twice as high as in NC snails. The bioaccumulation factors were 0.51±0.13 in DC snails and 0.26±0.04 in NC snails. Dose-dependent growth inhibition was noted in DC snails (500< EC 50<1000 μg· g −1 and EC 10 around 100 μg· g −1) while only slight effects were noted in NC snails. Internal Cd concentrations in DC and NC snails were linearly correlated with growth effect whatever the contamination route.

EFFECTS OF CHRONIC Cd EXPOSURE VIA THE DIET OR WATER ON INTERNAL ORGAN-SPECIFIC DISTRIBUTION AND SUBSEQUENT GILL Cd UPTAKE KINETICS IN JUVENILE RAINBOW TROUT (ONCORHYNCHUS MYKISS)

New regulatory approaches to metal toxicity (e.g., biotic ligand model [BLM]) focus on gill metal binding and tissue-specific accumulation of waterborne metals; the dietary route of exposure and dietary/waterborne interactions are not considered, nor are the consequences of chronic exposure by either route. Therefore, we studied the effect of the same gill Cd load (approximately 2.5 microg/g), achieved by a chronic, 30-d exposure to Cd either via the diet (1,500 mg/kg) or the water (2 microg/L), on tissue-specific Cd distribution and subsequent uptake of waterborne Cd in juvenile rainbow trout (Oncorhynchus mykiss). These two exposure regimes resulted in a branchial Cd load that had been taken up across either apical gill membranes (waterborne Cd) or basolateral gill membranes (through the bloodstream for dietary Cd). The BLM characteristics of the gills (i.e., short-term Cd uptake kinetics) were altered: affinity (log K(Cd Gill) [95% confidence level]) decreased from 7.05 (6.75-8.76) for control to 6.54 (6.32-7.03) for waterborne Cd and 5.92 (5.83-6.51) for dietary Cd, whereas binding capacity (Bmax) increased from 3.12 (2.14-4.09) to 4.80 (3.16-6.43) and 5.50 (2.86-8.17) nmol x g(-1) for control, waterborne, and dietary Cd, respectively. Fish exposed to dietary Cd accumulated a much greater overall chronic Cd body burden relative to fish exposed to waterborne Cd or control fish. The carcass accumulated the greatest percentage of total body Cd in control and waterborne-exposed fish, whereas the intestinal tissue accumulated the greatest percentage in dietary-exposed fish. Tissue-specific Cd burdens were highest in the kidney in both dietary and waterborne treatments. We conclude that chronic Cd exposure alters Cd uptake dynamics, and that the route of Cd exposure, whether waterborne or dietary, results in differences of internal Cd accumulation and branchial Cd uptake characteristics. These factors should be considered in future BLM development.

Quantification of metal bioavailability for lettuce (Lactuca sativa L.) in field soils.

Understanding metal bioavailability of plants in soils requires, apart from physiological processes and symbiosis with arbuscular mycorrhizal fungi, the consideration of the chemical availability in the soil solution (the intensity of the toxic exposure) and the soil's capacity to supply the metal (capacity). In this contribution we report on the time-dependent accumulation of As, Cd, Cr, Cu, Ni, Pb, and Zn in lettuce (Lactuca sativa L.). Bioassays with 17 Dutch field soils and two artificially metal-contaminated soils were carried out. Phytotoxicity was observed in soils with pH (pore water) <4.8. Metal uptake is shown to be both metal- and soil-dependent and strongly depends on the amount of water the plant transpired and the available concentration in the water. No net accumulation of As, Pb, Ni, and especially Cr was observed in most soils tested. The latter observation is in agreement with findings of Zayed et al. (Planta, 1998 206:293-299), who reported that translocation of Cr from roots to shoots is extremely limited. Internal Cd levels in the plants varied greatly among soils, whereas plant tissue concentrations of Zn and especially Cu appear to be regulated at more or less fixed levels. The 0.01 M CaCl(2)-extractable metal pool provides the best descriptor for the capacity of the soil to supply Cd and Zn. This enabled the development of models that are suited to predict Zn and Cd uptake by lettuce in both field soils (weathered soils) and soils to which metal salts were added, which is common practice in toxicity testing of chemicals. It is concluded that of all metals included in this study, Cd is the metal of most concern due to bioaccumulation through the soil-plant-animal food chain as Cd is the only metal that might pose human or animal health risks at plant tissue concentrations that are not directly phytotoxic. Finally, application of the models for risk assessment purposes is discussed.

Bioaccumulation and toxicity of four dissolved metals in Paracentrotus lividus sea-urchin embryo

The bioaccumulation of four metals (Cd, Cu, Pb, Zn) dissolved in water was assessed measuring the concentrations recorded within sea-urchin larvae ( Paracentrotus lividus, Lmk.) after a 48-h exposure period. Concurrently, the frequencies of abnormalities were evaluated at the 48-h pluteus stage to check the actual toxicity of such contaminants with regards to larval development. Maximum metal concentrations in the larvae reached 43.8±6.7 μg Cd g −1 dry weight (d.w.), 1269±392 μg Cu g −1 d.w., 3106±679 μg Pb g −1 d.w., 534±62 μg Zn g −1 d.w. Bioconcentration factors (BCFs) reached very high values for Pb (maximum value: 16,282±973) and indicated that the concentrations of Pb and Cd were directly related to the water concentration at least in the range of concentrations tested. Zn appeared to be partially regulated above a certain threshold concentration (between 5 and 25 μg l −1). Cu was accumulated with an active uptake at least below lethal threshold level (100 μg l −1). Nevertheless, above these thresholds, high mortality of embryos prevent any interpretation of Zn and Cu bioconcentration factors. Concurrent studies carried out on embryotoxicity confirmed the high toxicity of Cu towards sea-urchin pluteus larvae and highlighted the different behaviour of Cd. The internal Cd concentrations were directly related to the external ones, comparable to other non-essential metals, but Cd had little toxic effect on the initial larval development.

Those Ubiquitous Archimedean Circles

(1999). Those Ubiquitous Archimedean Circles. Mathematics Magazine: Vol. 72, No. 3, pp. 202-213.

Prediction of Metal Bioavailability in Dutch Field Soils for the Oligochaete Enchytraeus crypticus

Current risk assessment procedures ignore that variation in soil properties results in substantial differences for uptake and effects in organisms in different soils. In this contribution is presented the results of a study on the soil-related factors that modulate metal uptake and elimination by the oligochaete worm Enchytraeus crypticus. Uptake of Cd, Cu, Pb, and Zn was quantified in 20 Dutch field soils as a function of time. Uptake rate constants and equilibrium concentrations were estimated using compartment modeling. Internal metal concentrations varied less than the corresponding external levels. Zn and especially Cu provided the most extreme examples of this general behavior, which suggests regulation by the organism. Body residues by Cd increased linearly over time in 11 of the 20 soils studied, whereas in the remaining 9 soils equilibration of internal Cd levels was observed. CaCl2 extraction could be used to discriminate the 9 soils in which there is Pb accumulation from the 11 soils in which bioavailable Pb levels were too low to allow for uptake. Multivariate expressions that describe uptake rate constants and bioaccumulation factors as a function of soil characteristics were derived. pH and cation exchange capacity were the most important parameters. The formulae were very similar to those describing partitioning of metals over the solid and liquid phase of the soils, which suggests pore water-mediated uptake. A semi-mechanistic approach yielded further evidence of pore water-related uptake, modulated by competition between H+ and metal ions at the active sites of the membranes.

A record of estuarine water contamination from the Cd content of foraminiferal tests in San Francisco Bay, California

A five-year dissolved Cd time series from San Francisco Bay and adjacent coastal water shows that the composition of surface water towards the mouth of the estuary is determined largely by the effect of coastal upwelling. Cd concentrations inside and outside the estuary Ž0.2–1.0 nmolrkg. increase as Cd-rich deep water is advected to the surface near the coast during spring and summer. On average, the mean Cd concentrations inside San Francisco Bay Ž0.54 nmolrkg. during 1991–1995 was significantly higher than outside Ž0.35 nmolrkg , however. Surface samples collected throughout San . Francisco Bay confirm an internal Cd source unrelated to river discharge. The Cd content of the test of a benthic foraminifer Ž Elphidiella hannai . in a dated sediment core from San Francisco Bay was measured to determine if the water column Cd enrichments in San Francisco Bay could be related to the rapid development of the watershed. The method is based on the observation that the CdrCa ratio of carefully cleaned tests of foraminifera is determined by the dissolved Cd content of overlying water at the time of test formation. Pre-industrial foraminiferal CdrCa ratios in the sediment core average 274 15 nmolrmol Ž n s 19. nmolrmol. Foraminiferal CdrCa ratios increased to 386 33 nmolrmol Ž n s 19. over the past several decades indicating a 40% increase in the mean Cd content of surface water in Central San Francisco Bay. We suggest that, in addition to Cd discharges into the estuary, indirect consequences of agricultural development in the Central Valley of California could have contributed to this increase. This new method to reconstruct estuarine contamination is not affected by some of the processes that complicate the interpretation of changes in bulk sediment metal concentrations. q 1999 Elsevier Science B.V. All rights reserved.

Cadmium Decreases SGLT1 Messenger RNA in Mouse Kidney Cells

Mouse renal cortical tubule cells in primary culture exposed to cadmium (Cd2+) develop decreased Na+-glucose cotransport activity as measured by uptake of the glucose analogue α-methylglucoside. RNA was isolated from kidney cell cultures, and after reversed transcription, the DNA was amplified with primers to rat SGLT1 (the high affinity isoform of the sodium glucose cotransporter) and mouse β-actin. Only one product was identified after amplification with the rat SGLT1 primers, which on sequencing was 96% identical to rat SGLT1. Compared to β-actin, the intensity of the SGLT1 message declined progressively as CdCl2concentration in the medium increased from 0 to 10 μM. Similar decreases in SGLT1 mRNA were also observed as media zinc (Zn2+) concentrations rose from 0 to 75 μM or as copper (Cu) concentrations increased from 0 to 150 μM. Exposure to 8 μM Cd as Cd-metallothionein (Cd7-MT) also caused a fall in relative SGLT1 mRNA abundance, and at nearly identical internal Cd concentrations of 40-43 pmol/μg DNA, both Cd7-MT and CdCl2reduced SGLT1 mRNA to 33% of control. In general, the fall in SGLT1 mRNA was more rapid than the decline in Na+-dependent glucose uptake after cells were exposed to Cd2+. These findings suggest that the effects of Cd2+and other metals on renal glucose transport are related to decreased expression of SGLT1 message.

Cadmium in the blood and seminal fluid of nonoccupationally exposed adult male subjects with regard to smoking habits

Blood cadmium (B-Cd) and seminal fluid cadmium (Sf-Cd) were measured in 120 adult male subjects not occupationally exposed to cadmium (Cd), comprising 42 nonsmokers (including nine former smokers) and 78 smokers. The respective median and range values were: 0.46 (0.19-1.49) microgram/l of B-Cd and 0.54 (0.17-1.67) microgram/l of Sf-Cd in nonsmokers, and 4.33 (0.49-13.33) micrograms/l of B-Cd and 0.85 (0.29-3.56) microgram/l of Sf-Cd in smokers. Both indicators showed a highly significant difference in Cd exposure between the groups (P < 0.0001), although the increase in B-Cd was considerably more pronounced than that of Sf-Cd in smokers compared with nonsmokers. The results suggest a nonlinear relationship (log Sf-Cd/log B-Cd: r = 0.501, P < 0.0001), rather than linear relationship (Sf-Cd/B-Cd: r = 0.430, P < 0.0001), between the indicators. Significant correlations were found between smoking habits, i.e., the number of cigarettes per day, and an increase in B-Cd in smokers (r = 0.296, P < 0.01) and in all 120 subjects (r = 0.685, P < 0.0001), as well as between smoking habits and an increase in Sf-Cd in smokers (r = 0.378, P < 0.001) and in all 120 subjects (r = 0.488, P < 0.0001). Both indicators are necessary for evaluation of individual internal Cd dose, since they appear to differ in reflecting recent and long-term cumulative Cd exposure and/or the amount of Cd at the site(s) of its effect(s) in the body.

Reversibility of microproteinuria in cadmium workers with incipient tubular dysfunction after reduction of exposure.

The study aimed at assessing the evolution of cadmium (Cd)-induced renal tubular dysfunction in Cd workers according to the severity of the microproteinuria observed at the time the exposure was substantially decreased. Male workers employed in the Cd production industry for whom formerly high exposure had markedly decreased by 1984 and for whom standardized medical data were available during two observation periods (1980-1984 and 1990-1992) were eligible for the study. A total of 32 Cd workers fulfilling this profile were divided into two groups on the basis of historical records of urinary Cd concentration (Cd-U) covering the period until 1984. The workers with Cd-U values of > 10 micrograms Cd/g creatinine were subdivided further on the basis of the urinary concentration of beta 2-microglobulin (beta 2 MG-U) measured during the first observation period (1980-1984). In each group, the tubular microproteinuria as reflected by beta 2 MG-U and the concentration of retinol-binding protein in urine as well as the internal Cd dose as reflected by the concentration of Cd in blood and urine were compared between the first and second (1990-1992) observation periods. Increased microproteinuria was often diagnosed in cases with Cd-U values of > 10 micrograms Cd/g creatinine. The evolution of tubular renal function has been found to depend on the extent of the body burden of Cd (as reflected by Cd-U) and the severity of the initial microproteinuria at the time high Cd exposure was reduced or ceased. When reduction of Cd exposure took place while beta 2 MG-U did not exceed the upper reference limit of 300 micrograms/g creatinine, the risk of developing tubular dysfunction at a later stage was likely to be low, even in cases with historical Cd-U values occasionally > 10 but always < 20 micrograms Cd/g creatinine. When the microproteinuria was mild (beta 2 MG-U > 300 and < or = 1,500 micrograms/g creatinine) at the time exposure was reduced, and the historical Cd-U values had never exceeded 20 micrograms Cd/g creatinine, there was indication of a reversible tubulotoxic effect of Cd. When severe microproteinuria (beta 2 MG-U > 1,500 micrograms/g creatinine) was diagnosed in combination with historical Cd-U values exceeding 20 micrograms Cd/g creatinine, Cd-induced tubular dysfunction was progressive in spite of reduction or cessation of Cd exposure.

Cadmium distribution in maize inbred lines: Effects of pH and level of Cd supply

In order to investigate the physiological basis of the differential Cd distribution and the degree of variation of this Cd distribution among maize inbred lines, six inbreds designated earlier as ‘shoot Cd excluders’ (B73, H99, and H96) and ‘non-shoot Cd excluders’ (B37, H98, and N28) were grown in nutrient solution culture at different external Cd levels or at different pH. The characterization of the inbreds according to their shoot/root partitioning of Cd was consistent, independent of pH or level of Cd supply. The Cd concentrations in the plants were highest at the highest pH of the solution cultures. Generally, there was a positive correlation between the Cd concentrations in shoots and xylem exudates. It was shown that the Cd concentration in the roots is particularly important in the Cd distribution process. Above a ‘critical’ internal Cd concentration in the roots, specific for each inbred, the ability to retain Cd is strongly diminished. It is concluded that structural and/or physiological characteristics of the roots are involved in Cd partitioning.

The response of Lemna trisulca L. to cadmium

Lemna trisulca was grown, using aseptic culture techniques in a filter-sterilized medium, a portion of which was replaced regularly during experiments. L. trisulca responded to the addition of 0·64 μM Cd with a reduction in multiplication rate (MR) 2 ± 1 days after exposure. The internal Cd content reached 1000 ± 140 μg Cd/g (dry wt) within 2 days exposure to 0·64 μM Cd. The final yield was reduced by an average of c.8% for each day of exposure to 0·64 μM Cd in a 14 day experiment. This implies that an equilibration period should be used for short-term bioassay tests before the effect of a toxicant is determined. Pretreating L. trisulca with 0·08 or 0·32 μM Cd for 6 weeks had no significant effect on MR or Cd uptake when plants were subsequently exposed to a range of Cd concentrations or grown in a control medium. This suggests that L. trisulca does not become acclimated to elevated Cd concentrations. The MR of L. trisulca fluctuated over a period of almost 600 days and the doubling time ranged from 1·6 to 2·4 days. This produced more than a fivefold difference in final yield in experiments of 14 days duration. The reduction in MR in response to 0·32 μM Cd during this same 600 days period averaged 24% with a coefficient of variation of 38%, and varied with the MR of control cultures. Fluctuations in the intrinsic growth rate and the effect of a toxicant on L. trisulca could potentially confound the assessment of toxicity and must be carefully considered when designing test protocols for aquatic plants.

Influence of the stage of the hair cycle on Cd deposition in hair

Shortly after a single injection of Cd a much higher deposition of Cd was observed in growing hair (anaphase) than in resting hair (telophase). Shifting the time of the administration between 7 d before the onset of matrix production and full hair growth did not appreciably alter the initial deposition in spite of the rapid decline of Cd in blood plasma between the injection and the start of hair growth. After the initial deposition the concentration decreased in growing as well as in resting hair. In growing hair this is attributed to the addition of new matrix containing less Cd due to its declining supply via blood. In the resting hair it may reflect the decline of Cd in the follicular tissue adhering to the hair roots. The study demonstrates that the deposition of internal Cd in hair occurs mainly in those sections of hair growing at the time of the actual intake of Cd into the organism. The quantity of Cd found in a particular section of hair in the absence of external contamination, therefore, indicates first and foremost the quantity of Cd taken up into the blood stream—after ingestion or inhalation—at the time of the actual formation of this section notwithstanding the actual body burden at that time.

Cadmium and calcium interactions in the freshwater amphipod Gammarus pulex

SUMMARY. The accumulation of cadmium from an experimental medium by the freshwater amphipod Gammarus pulex is described.Much of the uptake is internal as opposed to adsorption on the body surface, and after an apparent saturation of the exoskeleton the hepatopancreas becomes an increasingly important site of cadmium storage. The haemolymph cadmium concentration reaches a high level compared with marine crustaceans, achieving a concentration factor (ratio of internal Cd/ external Cd) of 100 after about 60 h uptake.The cadmium uptake process is severely inhibited after exposure of experimental animals to 0.5 mM 2:4 Dinitrophenol, indicating the mediation of an active process. This fact together with the negative relationship between cadmium uptake rate and the calcium concentration of the animal suggests that cadmium accumulation by this species may be at least partially accounted for by a process of ‘accidental’ active cadmium uptake, with cadmium substituting for calcium on a calcium regulatory mechanism.As yet it has not been possible to establish a true stoichiometric relationship between the two metals. Although calcium influx and cadmium uptake (influx) rates are similar over a wide range of external concentrations, calcium influx is clearly inhibited by a low external ratio of cadmium to calcium. This indicates that the relationship between the two metals is far from an equimolar one and the possibility of non‐competitive inhibition of calcium influx by cadmium cannot be eliminated.

The microwave spectra of ortho-fluorotoluene with the asymmetric internal rotors CH 2D and CD 2H

The rotational spectra of α d 1- and α d 2-ortho-fluorotoluene in the ground state of the methyl group torsion have been measured. The evaluation of the spectra has been based on the theory for the internal rotation of an asymmetric internal top formulated earlier by several authors. The barrier potential being threefold symmetric ( V 3), each torsional level consists of three nondegenerate substates, designated as sy and ±asy. The sy-state is assigned to the conformation with the unique methyl hydrogen isotope within the molecular heavy-atom plane (sy-rotamer), while the ±asy-states belong to the respective out-of-plane conformation (asy-rotamer). In the torsional ground state the level spacing between the ±asy substates is very small and numerous accidental close degeneracies are present between the rotational level systems based on these torsional substates. The rotational levels involved are strongly perturbed by the coupling between molecular overall rotation and internal rotation. Large deviations from a rigid rotor spectrum and (+) ↔ (−) intersystem (“tunneling”) transitions are observed. The spectrum of the asy-rotamer can be well reproduced by a “two-dimensional” Hamiltonian containing 11 “rotational constants,” 9 of which are determined by a fit to the spectrum. Several are sufficiently barrier-dependent to derive V 3. We obtain (in cal/mole) 567 ± 48 for α d 1-ortho-fluorotoluene, 711 ± 40 for the α d 2-isotope. The deviations from 649 cal/mole for the normal isotope are appreciable, probably indicating shortcomings of the semirigid model. The sy-rotamer presents a rigid rotor spectrum.