Metal bioaccumulation in the greater white-toothed shrew, Crocidura russula, inhabiting an abandoned pyrite mine site

Hepatic and renal concentrations of iron, magnesium, zinc, lead, copper, manganese, mercury, cadmium, molybdenum, chromium, and nickel were quantified in shrews (Crocidura russula) inhabiting a pyrite mine site in Portugal. Several morphological parameters (body weight, residual index, and relative weights) were also examined to clarify the physiological effects of pollution. Shrews from the mine showed increased bioavailability of Fe, Pb, Hg, Cd, Mo, and Ni in comparison with reference specimens. Adult shrews had the highest Cd levels while Cr and Ni concentrations diminished. Intersexual differences were found for Mo and Ni. As a consequence of metal pollution, the relative hepatic weight was higher in shrews from the mine site when compared with reference specimens. These data indicate that C. russula is a good bioindicator of metal pollution. We also evaluated the toxic effects of Pb, Hg, Cd, and Ni, because several shrews from the polluted site showed high concentrations of these metals. To approximate at the real biological impact of abandoned mines, after this first step it is necessary to associate the bioaccumulation levels and morphological effects with other physiological, ecological and genetical biomarkers.     

Haematological characteristics and bone fluoride content in Bufo melanostictus from an aluminium industrial site

Fluoride concentration in bones and differential haemotological characteristics (RBC, haemoglobin, haematocrit, mean corpusclar haemoglobin and mean corpuscular volume) were measured in amphibians, Bufo melanostictus, collected from fluoride-contaminated and -uncontaminated areas. The average haemoglobin content, total RBC count and haematocrit (%) in blood samples were found to be significantly reduced, while mean corpuscular concentration and volume were significantly elevated in individuals from the contaminated area in comparison to those from the uncontaminated area. Fluoride concentration was approximately 11 times greater in the bones of toads from the contaminated area.     

Do wood mice (Apodemus sylvaticus L.) use food selection as a means to reduce heavy metal intake?

Food preference of wood mice from two with heavy metals polluted sites and two unpolluted sites was tested under laboratory and field conditions with two-way choice experiments. In the laboratory, wood mice preferred to eat acorns from unpolluted sites over acorns from polluted sites. Previous experience with polluted food had no influence on food choice. Preference was negatively related to acorn metal content. Furthermore, the nutrient content of the acorn endosperm was consistently lower in polluted sites. We therefore conclude that wood mice used absolute metal concentration in the acorn, nutrient content, or both as a food selection cue. The results of the laboratory experiment could not be confirmed under field conditions. We hypothesized that search time constraints due to the presence of predators, competitors and/or other stress factors in the field have prevented the mice to forage selectively.     

Time to death response in carabid beetles exposed to multiple stressors along a gradient of heavy metal pollution

We investigated the responses of invertebrates inhabiting polluted environments to multiple stressors. Carabid beetles (Pterostichus oblongopunctatus F.) were subjected to food deprivation and insecticide treatment (dimethoate) to resolve trends associated with a gradient of heavy metal pollution. Metal concentrations along the gradient of five sites ranged from approximately 150 to 10,500 mg/kg Zn, 136 to 2600 mg/kg Pb, and 0.84 to 81.9 mg/kg Cd. There was no difference in body mass along the pollution gradient. However, the beetles originating from the most contaminated sites were significantly less tolerant to food deprivation than beetles from the reference site. Median survival time was 120 h for the two most polluted sites, compared with 168 h at the reference site. Beetles from the two most polluted sites were also significantly more susceptible to dimethoate at 0.1 microgram active ingredient/beetle. Median survival times were 12 and 123 h for beetles from the two most polluted sites and 359 h for the reference site. Carabid beetles exposed to chronic pollution, therefore, exhibit elevated susceptibility to additional stressors.     

Chlorpyrifos-induced changes in oxidant/antioxidant status and haematological parameters of Cyprinus carpio carpio: Ameliorative effect of lycopene

The present study was carried out to investigate the potential ameliorative effects of lycopene against chlorpyrifos (CPF) toxicity in carp. The fish were divided into 7 different experimental groups and received the following treatments: Group 1, control; Group 2, orally administered corn oil; Group 3, oral lycopene (10 mg kg^?1 body weight); Group 4, exposure to 0.040 mg L^?1 CPF; Group 5, exposure to 0.040 mg L^?1 CPF plus oral administration of 10 mg kg^?1 lycopene; Group 6, exposure to 0.080 mg L^?1 CPF; and Group 7, exposure to 0.040 mg L^?1 CPF plus oral administration of 10 mg kg^?1 lycopene. Treatment was continued for 14 d and samples of the blood and tissue (liver, kidney, and gill) were collected at the end of the experiment and analysed for their oxidant–antioxidant status, including the malondialdehyde (MDA) levels, and the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity. The samples were also measured for changes in the haematological parameters, such as the red blood cell (RBC) and white blood cell (WBC) counts, the haemoglobin concentration (Hb), the haematocrit (Ht) level, and the erythrocyte indices: the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC). The findings of this study demonstrated that CPF had a negative effect on the haematological parameters and the antioxidant enzyme activities of the fish; this toxic effect was neutralised by the administration of lycopene. The present results suggest that lycopene (10 mg kg^?1) can be effective in the protection of CPF-induced toxicity in fish.     

Effects of complex waste mixtures on hepatic monooxygenase activities in brown bullheads (Ictalurus nebulosus)

Hepatic MFO components (cytochrome P-450, cytochrome b(5), and ethoxyresorufin O-deethylase, EROD) were measured in brown bullheads (Ictalurus nebulosus) inhabiting a creek receiving a complex mixture of organics and trace metals. The activities of these same enzymes were also measured in bullheads from an uncontaminated reference site to assess the relative ability of MFO parameters to serve as a biomarker of aquatic pollution. Bullheads analyzed from the polluted site had lower hepatic microsomal P-450 (p < 0.01) concentrations and similar EROD activities per mg protein as compared to bullheads from the reference site. However, analysis of enzyme turnover ratios revealed greater EROD activity per mg cytochrome P-450 (p < 0.05) in the fish from the polluted site. No difference in cytochrome b(5) activities were observed between the two groups. As compared to the reference site, bullheads collected from the polluted creek had an increased occurrence of lip and lower jaw lesions and liver damage, including elevated liver/body weight ratios. Accordingly, the monooxygenase activities measured in this study were not reliable indicators of chemical pollution or contaminant stress in bullheads in the polluted creek. Further research is needed concerning contaminant interactions, particularly among organic pollutants and metals and their effects on monooxygenase activities.     

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus)

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mre?nica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.     

Transferrin, haematological indices and liver glycogen in field mice collected from a coal mining area and a control site in Poland

A polymorphism for serum transferrin in field mice from polluted and control sites was compared with body-morphological and haematological parameters, and with liver glycogen concentration. The distribution of genotypes in the polluted area was not panmictic, due to higher frequencies of the rare allele Trf(c) occurring mainly in males. In the polluted area, transferrin-c was accompanied by slowly migrating, Fe-binding protein fractions. Since the Trf(c)/Trf(c) homozygote possessed higher red blood cell parameters, it is suggested that these extra proteins have an Fe-transport function. The morpho-physiology of heterozygotes from the polluted area did not differ significantly from those from the control site. The animals from the polluted area possessed a high concentration of liver glycogen, indicative of poor environmental conditions. Also, a generally lower level of red blood cell parameters suggests that non-oxygenic processes dominate over oxygenic ones in the polluted area.     

Effects of heavy metal pollution on red wood ant (Formica s. str.) populations

We studied the species composition, mound population densities, relative abundance and colony sizes of red wood ants along a well known air pollution gradient of a copper smelter in Southwest Finland. The dominant species, Formica aquilonia, was further studied for heavy metal (Al, Cu, Cd, Ni, Zn, As, Pb, Hg) levels and morphological characters (body mass, head width, labial gland disease) of workers. We found five species belonging to Formica s. str., and two of them showed changes in their relative abundance, which could not be explained by natural habitat differences. Nest mound volumes were 34% smaller in the polluted area, suggesting that smaller colonies can be maintained there. The heavy metal levels in F. aquilonia workers were higher in the polluted area for all metals, except Hg. The largest relative differences between the study areas (polluted/unpolluted) were found for As (4.1), Ni (2.4), Cu (2.1) and Pb (1.8). Morphological characters of workers were not related to the heavy metal levels. Our data showed that red wood ants can tolerate relatively high amounts of heavy metals and maintain reproducing colonies even in a heavily polluted area, but on the basis of smaller colony sizes, pollution stress may also cause trade-offs in reproduction.     

Contamination of the River Ganga and its toxic implication in the blood parameters of the major carp Labeo rohita (Ham)

A field study was conducted to examine different physicochemical properties of water and various haematological and biochemical parametres of the fish Labeo rohita collected from the Ganga River (National river of India) at Varanasi district, India. The water was found to be greatly contaminated with a number of dissolved metals (Fe, Cr, Zn, Cu, Mn, Ni and Pb) whose concentrations were above the safe limits suggested by Bureau of Indian Standard (BIS 1991) for drinking water (Fe, 1,353.33 %; Cr, 456 %; Mn, 553.33 %; Ni, 4,490 % and Pb, 1,410 %). The metal accumulation in the fish blood was very high (Fe, 2,408 %; Cr, 956.57 %; Zn, 464.90 %; Cu, 310.57 %; Mn, 1,115.48 %) in comparison to the control fish maintained under strict quality control. Lower values of the various haematological parameters (total erythrocytes count, haemoglobin, haematocrit, mean corpuscular volume and O₂-carrying capacity) in the river fish in comparison to the control indicate toxic manifestation exerted by the contaminated river water on the fish. The higher level of total leucocytes count further illustrates stressed condition of the river fish. The toxic impact of the Ganga water is also expressed in the fish by the presence of higher levels of cholesterol, glucose, elevated activities of the enzymes aspartate amino transferase and alanine amino transferase, and lowered protein concentration.     

Metallothionein levels in Algerian mice (Mus spretus) exposed to elemental pollution: an ecophysiological approach

The potential use of metallothioneins (MTs) as biomarkers of trace metal contamination was evaluated for the first time in the Algerian mouse (Mus spretus). Mice were collected seasonally in an abandoned mining area (Aljustrel) and in a reference area, both located in southern Portugal. MT levels were quantified in liver and kidney by differential pulse polarography and hepatic elemental concentrations (Mn, Fe, Cu, Zn, Se) were determined by particle-induced X-ray emission. Hepatic iron and selenium concentrations were elevated in mice from Aljustrel mine when compared to reference animals. MTs levels were averagely higher in mice from Aljustrel than those originated from the reference area. A season-dependent significant effect was found on the hepatic and renal MT concentrations, characterized by higher levels in winter and lower in autumn. In contaminated mice positive relationship between liver elemental contents (Cu in autumn and Fe in winter) and MTs were found. The seasonal variation of MT suggests that probably physiological and environmental factors could influence hepatic and renal MT induction. Results seem to imply that some environmental disturbance occur in the vicinity of the Aljustrel mine. Therefore, for the management purposes MT levels should be followed in liver of M. spretus, especially in winter. Furthermore, other physiological factors that could influence MT expression and turnover in Algerian mouse should also be monitored.     

Limited effects of heavy metal pollution on foraging and breeding success in the curlew (Numenius arquata)

We examined the effect of heavy metal pollution on the foraging success and breeding performance of the curlew (Numenius arquata) along a documented pollution gradient from a point source, and also by comparing foraging and breeding parameters between the polluted site and two non-polluted areas. Prey biomass and abundance, and foraging success did not vary along the pollution gradient, and were no less at the polluted site than in the non-polluted areas. Furthermore, there was no difference in adult weight during incubation between the polluted and non-polluted areas. There were also no differences in measures of breeding success along the pollution gradient, or between areas, which could be attributed to pollution per se. Egg shells from the polluted area had higher concentrations of heavy metals than in the non-polluted areas, and egg shells close to the pollution source were contaminated more than those further from it. However, there was no difference in calcium concentrations of egg shells or egg-shell thickness between areas. We conclude that in this study there were minimal immediate effects of heavy metal pollution on foraging and breeding success in the curlew.     

A microcosm test of adaptation and species specific responses to polluted sediments applicable to indigenous chironomids (Diptera)

Chironomids may adapt to pollution stress but data are confined to species that can be reared in the laboratory. A microcosm approach was used to test for adaptation and species differences in heavy metal tolerance. In one experiment, microcosms containing different levels of contaminants were placed in polluted and reference locations. The response of Chironomus februarius to metal contaminants suggested local adaptation: relatively more flies emerged from clean sediment at the reference site and the reverse pattern occurred at the polluted site. However, maternal effects were not specifically ruled out. In another species, Kiefferulus intertinctus, there was no evidence for adaptation. In a second experiment, microcosms with different contaminant levels were placed at two polluted and two unpolluted sites. Species responded differently to contaminants, but there was no evidence for adaptation in the species where this could be tested. Adaptation to heavy metals may be uncommon and species specific, but more sensitive species need to be tested across a range of pollution levels. Factors influencing the likelihood of adaptation are briefly discussed.     

Sensitivity analyses of woody species exposed to air pollution based on ecophysiological measurements

GOAL, SCOPE AND BACKGROUND: Air pollution has been of a major problem in the Pearl River Delta of south China, particularly during the last two decades. Emissions of air pollutants from industries have already led to damages in natural communities and environments in a wide range of the Delta area. Leaf parameters such as chlorophyll fluorescence, leaf area (LA), dry weight (DW) and leaf mass per area (LMA) had once been used as specific indexes of environmental stress. This study aims to determine in situ if the daily variation of chlorophyll fluorescence and other ecophysiological parameters in five seedlings of three woody species, Ilex rotunda, Ficus microcarpa and Machilus chinensis, could be used alone or in combination with other measurements for sensitivity indexes to make diagnoses under air pollution stress and, hence, to choose the correct tree species for urban afforestation in the Delta area. METHODS: Five seedlings of each species were transplanted in pot containers after their acclimation under shadowing conditions. Chlorophyll fluorescence measurements were made in situ by a portable fluorometer (OS-30, Opti-sciences, U.S.A). Ten random samples of leaves were picked from each species for LA measurements by area-meter (CI-203, CID, Inc., U.S.A). DW was determined after the leaf samples were dried to a constant weight at 65 degrees C. LMA was calculated as the ratio of DW/LA. Leaf N content was analyzed according to the Kjeldhal method, and the extraction of pigments was carried out according Lin et al. RESULTS AND DISCUSSION: The daily mean Fv/Fm (Fv is the variable fluorescence and Fm is the maximum fluorescence) analysis showed that Ilex rotunda and Ficus microcarpa were more highly resistant to pollution stress, followed by Machilus chinensis, implying that the efficiency of photosystem II in I. rotunda was less affected by air pollutants than the other two species. Little difference in daily change of Fv/Fm in I. rotunda between the polluted and the clean site was also observed. However, a relatively large variation of Fv/Fm appeared in the other two species, particularly in M. chinensis, suggesting that they were more sensitive to air pollutants than I. rotunda. The mean LA was reduced for all species growing at the polluted site. The mean LMA for all species exceeded the sclerophylly threshold given by Cowling and Campbell and increased for those under pollution stress, which could be explained as one of the acclimation strategies for plants to air pollution stress. Little difference in leaf chlorophyll content was observed in F. microcarpa and M. chinensis, while remarkable differences were found in I. rotunda growing at the polluted and the clean site. Content of leaf carotenoids was largely reduced in I. rotunda growing at the polluted site, but increased in F. microcarpa and M. chinensis, compared with plants growing at the clean site. Plants growing at the clean site had a lower leaf N content than those growing at the polluted site. In addition, species with a higher resistance to pollution stress showed less difference in leaf N content than those sensitive species. CONCLUSION: Based on Fv/Fm measurements of the three woody species, I. rotunda showed the highest resistance to air pollutants from ceramic industries, followed by F. microcarpa. M. chinensis was the most sensitive species to air pollution, had lowest capacities to cope with the air pollution stress, which was consistent with visual injury symptoms observed in the crown profiles of plants at the polluted site. Fv/Fm, LAM, LA, leaf pigments and N content could be used alone or in combination to diagnose the extent of the physiological injury. The ratio of Fv/Fm, however, was the best and most effective parameter. RECOMMENDATION AND OUTLOOK: Tree species which have higher air-pollutant resistance, as diagnosed by such ecophysiological parameters, should be considered first and planted widely for urban afforestation or forest regeneration in areas where the forest was seriously degraded or forest health was markedly effected by the same kind of air pollutants.     

Field effects of pollutants in dynamic environments. A case study on earthworm populations in river floodplains contaminated with heavy metals

In industrialized countries river floodplains can be strongly polluted with heavy metals. Published studies on effects of heavy metal pollution on soil invertebrates in floodplains, however, are inconclusive. This is unexpected since studies in other less dynamic environments reported clear effects at even lower levels of pollution. Flooding induces extra variation in invertebrate biomass and abundance which may reduce the probability to detect heavy metal effects. In this paper we combine reported data from studies on river floodplains in The Netherlands and Belgium and statistically analyze the effect of heavy metals on species composition, biomass, density and individual weight of earthworms. Interaction effects of heavy metal stress and flooding are also considered. The results suggest clear effects of zinc and copper on all variables and interaction of heavy metals and flooding for individual weight.     

Impact of metals on histopathology and expression of HSP 70 in different tissues of Milk fish (Chanos chanos) of Kaattuppalli Island, South East Coast, India

Histological and Immunohistochemical studies were carried out to document the possible impact of heavy metal contamination in different tissues of Chanos chanos. Heavy metals such as Cu, Pb, Zn, Cd, Mn and Fe were predominant in water, sediment and biota of Kaattuppalli Island and varied significantly between two different sites. Histological changes such as swelling of muscle fiber and break down of muscle bundles were noted in the muscle. Similarly gill filament cell proliferation, increase in intercellular spaces and primary and secondary lamellar epithelium were evident in gills. The hepatocytes showed damage of central vein and rupture of irregular hepatic plate with more number of vacuoles in the fish collected from polluted site compared to that from the less polluted site. The impact of pollution was also assessed in different tissues by immunohistochemistry using primary antibody (mouse monoclonal HSP70 antibody 1:2000) and secondary antibody (HRP conjugated antibody) for expression of stress protein. Immunostaining analysis showed expression of HSP70 with high intensity in the tissues of fish collected from polluted site compared to less polluted sites. Further, HSP70 positive cells were analyzed from six locations per fish tissue section. One-way analysis of variance (ANOVA) followed by least significant difference (LSD) was used to check if the expression was significant. Results indicate that the values are statistically significant at the two different sampling sites (P<0.05).     

Heavy metal accumulation in Littoraria scabra along polluted and pristine mangrove areas of Tanzania

The periwinkle Littoraria scabra was collected at polluted and pristine mangrove sites along the Tanzanian coastline, including Msimbazi, Mbweni (i.e. Dar es Salaam) and Kisakasaka, Nyamanzi and Maruhubi (i.e. Zanzibar). Periwinkles were morphologically characterized, sexed and their heavy metal content was determined using ICP-MS. Analysis revealed that L. scabra from polluted areas contained higher soft tissue heavy metal levels, were significantly smaller and weighed less compared to their conspecifics from the unpolluted mangroves. The current morphological observations may be explained in terms of growth and/or mortality rate differences between the polluted and non-polluted sites. Although a variety of stressors may account for these adverse morphological patterns, our data suggest a close relationship with the soft tissue heavy metal content. Compared to soft tissue heavy metal levels that were measured in L. scabra along the same area in 1998, most metals, except for arsenic, chromium and iron have decreased dramatically.     

Sub-tropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema

This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in São Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe, Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment.     

Accumulation of Cu and Ni in successive stages of Lymantria dispar L. (Lymantriidae, Lepidoptera) near ore smelters at Sudbury, Ontario, Canada

The accumulation of Cu and Ni in successive life stages of the gypsy moth (Lymantria dispar), feces, and foliage near an ore smelter at Sudbury, Ontario and a control site was investigated. Higher concentrations of Cu and Ni were found in all larval stages collected at Sudbury than in those from the control site indicating that elevated levels of these metals in the Sudbury environment is an important factor influencing body burdens. These differences were associated with higher metal concentrations in the foliage of host trees from this site. However, differences between sites became less significant for pupae and adult stages. At the Sudbury site, concentrations of Cu (microg g(-1) dry wt) decreased in successive instars and then increased in tissues of pupae and adults. In contrast, Cu content (microg/individual) increased during larval development and then decreased in pupae and adults. The pattern of Ni concentration and content in larvae from the Sudbury site was similar to that of Cu. Patterns of Cu and Ni concentration and content throughout the life stages are explained by changes in weight resulting in a dilution effect in early stages, and a concentrating effect in later stages. Differences in Cu and Ni concentration and content between Sudbury and control populations became less significant following the pupal stage suggesting metal elimination with the exuviae and meconium. This study also illustrates the importance of sampling all stages in an insect's development when measuring accumulation of metals. Fecal and foliar concentrations of Cu and Ni from Sudbury were not significantly different suggesting that metal assimilation is low. Even though gypsy moths from Sudbury contain elevated levels of Cu and Ni, metal burdens in their tissues do not represent a significant route through the food chain. However, the conversion of foliage with high metal content to feces implies that other ecosystem consequences should be investigated.     

Role of vitamin A in the haematotoxicity of hexachlorocyclohexane (HCH) in the rat.

The haematotoxicity of technical hexachlorocyclohexane (HCH) (1000 ppm) was investigated in male albino rats fed with diet free of vitamin A or containing vitamin A at 2000 or 10(5) I.U./kg. Assessment of HCH-induced haematotoxicity at the end of the 7 weeks feeding period was done on the basis of haemoglobin content, total count of red blood cells and white blood cells and the differential counts of the white blood cells as well as by parameters such as packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin content, prothrombin time and clotting time. In the rats fed with vitamin A-free diet containing HCH, significant reductions were noticed in the total white blood cells count, clotting time and prothrombin time indicating severe haematotoxicity. Differential count of the white blood cells of these rats revealed a non-significant reduction in the lymphocyte count. The only indication of haematotoxicity caused by hexachlorocyclohexane in the vitamin A supplemented rats was a slight but statistically significant reduction of the total count of white blood cells. These results demonstrate that the haematotoxicity of hexachlorocyclohexane in the rats is enhanced by vitamin A-deficiency and its supplementation particularly in excess but not at hypervitaminotic level is protective against the toxicity.