Metallothioneins and lysosomes in metal toxicity and accumulation in marine mussels: the effect of cadmium in the presence and absence of phenanthrene

It has been confirmed that metallothioneins play an important role in the accumulation of cadmium (Cd) in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.). The content of Cd in the tissue of mussels exposed for 9 d to the metal (estimated dosage of 180 g Cd mussel^-1 d^-1) was 66.2 ppm. This value is about the same as the metal content found in the digestive gland of Cd-exposed mussels kept in clean water for a recovery period of 28 d. At the end of the recovery period, however, the Cd bound to thionein had increased by approximately 250%. Our data demonstrate that the stability of lysosomes, a biological parameter adopted as a cellular stress index, is extremely low in mussels exposed to Cd for 9 d, but returns to control values in the digestive gland cells of mussels allowed to recover for 28 d in uncontaminated sea water. At this point most of the Cd present in the cytosol is bound to thionein. These data demonstrate the importance of metallothionein induction in the reduction of the cytotoxic effects exerted by high levels of Cd accumulation. The results of tests designed to clarify the reasons for the long biological half-life of Cd demonstrated that, in the digestive gland of mussels, the lysosomes are not able to eliminate Cd either bound to insoluble thionein polymers or to lipid peroxidation products such as lysosomal lipofuscin, both of which are apparently involved in the elimination of copper. The absence of these two mechanisms of metal sequestration and elimination via excretion of residual bodies (tertiary lysosomes) is in agreement with the persistence of cadmium in the digestive gland of mussels. Finally, the results also demonstrate that simultaneous exposure of mussels to Cd and phenanthrene, an established lysosomal membrane destabilizer, did not significantly alter the accumulation of Cd or the kinetics of the metal in mussels.     

Intracellular localization and binding of technetium-95m in the seastar Marthasterias glacialis

The subcellular distribution of technetium was studied using the seastar Marthasterias glacialis which had previously ingested prey labelled with ^95mTc either as pertechnetate (VII) or in the reduced state (IV). The majority of the Tc [98% (IV) and 96% (VII)] was taken up and retained in the pyloric caeca, the gland responsible for digestive and food storage processes. Differential centrifugation and separation techniques have demonstrated that, at the subcellular level, technetium in the pyloric caeca was largely associated with the lysosomes. ^95mTc (IV) was distributed between the lysosomes and the soluble fraction to roughly the same degree, whereas pertechnetate was more strongly associated with the lysosomal fraction. Chromatography of the cytosol by gel filtration indicated the presence of two distinct protein compounds which were responsible for binding virtually all of the technetium in the soluble fraction. Technetium had a greater relative affinity for the low molecular weight compound, more so for Tc (VII) than for Tc (IV). This anionic protein had an apparent molecular weight of approximately 10 000, and thus may play a role in metal detoxification mechanisms at the cellular level.     

Biochemical correlates of dissolved mercury uptake by the oyster Ostrea edulis

From previous work, the equilibrium concentration factor for dissolved mercury in the digestive gland of Ostrea edulis Linnaeus was found to be three to four times higher than that in the gills. In the present study, an analysis of soluble protein revealed values of 49.3±14.2 mg g wet tissue^-1 for the digestive gland and 0.7 ±0.1 mg g wet tissue^-1 for the gills. Starvation significantly reduces the soluble protein level of the digestive gland to 31.1±6.4 mg g^-1 and that of the gills to below the limit of detection. These results suggest that the difference in concentration factors between the gills and digestive gland may be based on a quantitative difference in macromolecular binding sites. However, the uptake of dissolved mercury over a period of 48 h was considerably greater in the gills, so that although the soluble protein content of the tissue may influence the final concentration factor, it does not appear to affect the rate at which this equilibrium is achieved. A more detailed investigation of the mechanism of dissolved mercury uptake by oyster gills has been carried out using isolated tissues. The process is inhibited by 5mM 2–4 dinitrophenol, by the absence of a readily metabolizable substrate (dextrose) in the uptake medium, and by 30mM K⁺. The effect of K⁺ necessitated further investigation with a specific inhibitor of K⁺ transport. Strophanthin G (ouabain), at a concentration of 0.01 mM, caused a significant increase in mercury uptake.     

Interactions of marine plankton with transuranic elements

In a series of laboratory experiments, the biokinetics of ²⁴¹Am, an important transuranium element, was studied in Meganyctiphanes norvegica, a euphausiid common in the northwestern Mediterranean. The euphausiids accumulated Am from water by passive adsorption onto exoskeletons, achieving wet weight concentration factors on the order of 10² after 1 wk exposure; concentration factors varied inversely with the size of the euphausiids and linearly with their surface area:wet weight ratios. Essentially all (96±10%) of the Am taken up from water was associated with the exoskeleton, so that negligible Am was retained by the euphausiids after molting. The retention half-time of Am in molts was 2.9 d. Euphausiids could also concentrate Am from feeding suspensions by ingesting Am-labelled diatom cells, although there was negligible Am assimilation (3±2% after 4 d feeding); after passage through the gut, virtually all (99%) of the ingested Am was defecated within 1 wk. The retention half-time of Am in fecal pellets was 41 and 51 d at 13° and 5°C, respectively. In oceanic waters, where the preponderance of ²⁴¹Am is in the dissolved phase, uptake of Am from water by euphausiids would be the dominant route of bioaccumulation. The results underscore the importance of sinking biogenic debris from zooplankters in mediating the vertical transport of Am in the sea. Given their retention half-times for ²⁴¹Am and their rapid sinking rates, fecal pellets and discarded molts have the potential to deliver most of their Am to the sediments.     

Biokinetics of plutonium,americium and californium in the marine isopod Cirolana borealis,with observations on its feeding and molting behavior

In a series of laboratory experiments carried out during 1982, the biokinetics of ²³⁷Pu (IV), ²³⁷Pu (V), ²⁴¹Am (III), and ²⁵²Cf (III) were studied in the benthic marine isopod Cirolana borealis Lilljeb. Following a 3 wk exposure to labelled seawater, isopods reached non-equilibrium concentration factors of 52±15, 54±12, 176±14, and 185±53 for Pu (IV), Pu (V), Am and Cf, respectively. Uptake of these radionuclides occurred to a great extent by surface adsorption, with the major fraction of the accumulated radionuclides being associated with exoskeleton and lesser percentages located in gut, digestive gland, muscle and haemolymph. The pattern of radiotracer depuration was in all cases biphasic, with the long-lived retention compartment retaining the largest fraction of whole-body radioactivity. Biological half-lives for radionuclide turnover in the long-lived compartment were 60±3 d, 87±5 d, 261±12 d and 288±60 d for Pu (IV), Pu (V), Am and Cf, respectively. Examination of radionuclide distribution among tissues following longterm depuration showed that the radionuclides were eliminated from internal tissues more rapidly than from the exoskeleton. Uptake and depuration studies indicated that the biokinetics of americium and californium were similar to each other, but that they differed significantly from those of oxidized and reduced plutonium. In general, these experimental results support previous reports that americium and californium are more bioavailable than plutonium in the marine waters. The fraction americium and californium assimilated into tissue from food was very low and did not exceed 5%. Unassimilated labelled food was often retained in the gut for more than 2 mo and released only infrequently in a loosely bound condition. In the event of contamination, this particular feeding-digestion strategy in highly mobile, scavenging isopods could act as a potential biological mechanism for the widespread dispersion of radioactive wastes.     

Influence of americium-241 on the microbial population and biodegradation of organic waste

The present study investigated the influence of ²⁴¹Am on microbial growth and the degradation of organic waste. Leachate samples collected in a lysimeter were periodically analyzed for bacterial growth, under both aerobic and anaerobic conditions. ²⁴¹Am inhibited bacterial growth, and the degradation of organic matter was delayed in comparison with the control. Minimal inhibitory concentration assays and survival curves revealed that it inhibits the growth of Pseudomonas putida F1. The assay also revealed that ²⁴¹Am is more toxic than ²³⁸U, Zn²⁺ and Cd²⁺. This study further led to the finding of four new radionuclide-tolerant bacterial strains: Flavobacterium spp., Pseudomonas gladioli, Chryseobacterium indologenes and Ochrobactrum anthropi. The survival curves of P. gladioli, C. indologenes revealed that these bacteria are resistant to metal as consortia.     

Temperature effects on accumulation and retention of radionuclides in the sea star,Asterias forbesi: implications for contaminated northern waters

Radioactive waste disposal and nuclear testing concentrated in high latitudes in the northern hemisphere have resulted in the accumulation of radionuclides in Arctic marine ecosystems, but little is known of the consequences for marine biota in these waters. Under controlled laboratory conditions in May through September 1994, we examined the bioaccumulation in sea stars, Asterias forbesi (Desor), or the radionuclides ²⁴¹Am, ⁵⁷Co and ¹³⁷Cs, all of which are important components of disposed radioactive wastes. Experiments at 2 and 12°C determined the relative importance of food (the bivalve, Macoma balthica) and water as sources of radionuclides and assessed the influence of temperature on radionuclide influx and efflux rates. The lower temperature greatly increased the retention of radionuclides ingested with food; for instance, the biological half-life (tb _1/2) of ²⁴¹Am in the sea stars was 31 d at 12°C, but was virtually infinite at 2°C. Retention of ingested ⁵⁷Co was also increased at 2°C (tb _1/2=41 d). ¹³⁷Cs was not accumulated from food. Low temperature significantly reduced net influx rates of ¹³⁷Cs from water, but did not affect net uptake of ²⁴¹Am or ⁵⁷Co. Temperature had little effect on the retention of all three isotopes obtained from the dissolved phase. These experiments suggest that extrapolation of results of previous radioecological studies, conducted at warmer temperatures, to polar or temperate winter environments may be problematic, and that nuclear waste isotopes obtained through trophic transfer may be retained far more efficiently in high latitude marine biota than by fauna from warmer ecosystems.     

镉和铅对菲律宾蛤仔脂质过氧化及抗氧化酶活性的影响

为了研究亚致死浓度的重金属对海洋贝类的毒性效应,探讨其可能的作用机理,在实验生态条件下以菲律宾蛤仔(Ru-ditapes philip pinarum)为目标生物,采用半静态毒性实验方法,研究了不同浓度Cd2+(0.0948、0.237和0.474mg·L-1)和Pb2+(0.276、0.690和1.380mg·L-1)对菲律宾蛤仔鳃和消化腺组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性以及丙二醛(MDA)含量的影响。结果显示:(1)鳃和消化腺中的抗氧化酶及MDA的变化呈现出类似的趋势,在胁迫初期,各浓度处理组菲律宾蛤仔鳃和消化腺组织SOD和CAT与对照组相比活性都显著升高(P<0.01),呈现出明显的诱导效应,而MDA含量与对照组相比差异不显著(P>0.05);随着胁迫时间的延长,高浓度Cd2+(0.474mg·L-1)和Pb2+(1.380mg·L-1)处理组中SOD、CAT活性快速下降,与对照组相比差异显著;低浓度处理组中的抗氧化活性虽然也较对照组有所下降,但总体下降幅度不如高浓度组明显,并且所历暴露时间较长。各浓度处理组中MDA含量变化一致,均呈现出先升高后降低的趋势,且含量均高于对照组。(2)通过相关性分析,菲律宾蛤仔消化腺组织中的SOD活性与Cd2+浓度的相关性大于鳃组织,与Pb2+浓度的相关性则小于鳃组织;消化腺组织中的CAT活性与Cd2+、Pb2+浓度呈抛物线型相关,与鳃组织CAT活性相关性不十分显著。这说明消化腺组织中SOD活性对Cd2+的敏感性大于鳃组织,消化腺组织中CAT活性对Cd2+、Pb2+的敏感性大于鳃组织。因此,菲律宾蛤仔消化腺中SOD、CAT对水环境中的重金属反应敏感,且存在一定的剂量-效应关系,消化腺组织中SOD和CAT活性与其他敏感性指标一起可以作为指示早期海洋重金属污染的生物标志物。     

Assimilation,inter-organ transfer and excretion of americium in two teleost fish

Radiotracer experiments were performed (February–April, 1982) to study the assimilation and metabolism of the transuranium nuclide americium-241 in the marine teleosts Serranus scriba (Linnaeus, 1758) and Scorpaena notata Rafinesque, 1810, caught off the Monaco coast. Fish fed with ²⁴¹Am-labelled food showed that assimilation of this radionuclide takes place through the gastrointestinal walls and that the small fraction accumulated is incorporated mainly in the skin, muscle and skeleton. Gut-transfer coefficients were similar in both species and averaged 0.7% (range 0.1 to 1.7%) of the ingested activity. The calculated biological half-lives for loss of the absorbed fraction ranged between 49 and 61 d for Serranus scriba and 12 and 117 d for Scorpaena notata. Results from an intramuscular injection experiment indicated that ²⁴¹Am was retained mainly in the liver, skin and skeleton; the fraction accumulated by muscle was very low. Liver displayed a relatively short biological half-time for ²⁴¹Am loss of roughly 24 d. Routes of ²⁴¹Am excretion from the teleosts appear to be through the kidneys, gills and feces with bile serving as a possible excretion route from the liver. From the limited amount of published information available for comparison, experimental evidence is presented which suggests that ²⁴¹Am taken up via the food chain is more biologically available to marine fish than is plutonium.     

Tissue and sub-cellular distribution of Fe,Cu, Zn and 210Po in the abalone Haliotis rubra

The tissue and sub-cellular distribution of Fe, Cu, Zn and the naturally occurring radionuclide polonium-210 was determined in the gastropod mollusc Haliotis rubra collected from Western Port Bay, Australia, between March and July 1988. The highest concentrations of the metals, with the exception of Cu, were found in the digestive gland. Copper was more uniformly distributed, with tissues that are more vasculated having higher concentrations. Ultrastructural examination of the digestive gland, gill and kidney showed dense membrane-bound granules within the cytoplasm. Elemental analysis of the granules by electron probe x-ray microanalysis indicated that the granules in the digestive gland and gill contained high concentrations of iron, with small amounts of copper and zinc. In contrast, the metal-containing granules in the kidney were predominantly composed of iron, copper and phosphorus, with variable contributions of sodium potassium, and calcium. Homogenisation and fractionation of the digestive gland by differential centrifugation confirmed that approximately 80, 10, 90 and 50% of the total homogenate Fe, Cu, Zn and ²¹⁰Po, respectively, sedimented at 1200xg. In the haemolymph, all the elements studied were associated with the soluble high molecular weight component of the serum, not with the amoebocytes. ²¹⁰Po was present in the mucus-secreting hypobranchial glands at about half the concentration found in gill tissue.     

Artificial radionuclides in sediment of the Yenisei River

Releases from the nuclear facility Mining-and-Chemical Combine (MCC) located at Zheleznogorsk have contributed to the radionuclide contamination of the Yenisei River since operations commenced in 1958. The aim of this study was to assess the activity concentrations of artificial radionuclides and the strength of their binding in Yenisei River sediments. Investigation of Yenisei River sediment samples revealed the presence of artificial radionuclides typical of the MCC radioactive discharge: namely, isotopes of europium, caesium, ⁶⁰Co and transuranium elements. The concentrations of artificial radionuclides in the sediment layers remain relatively high as far as 200 km downstream of the MCC. In sediment cores collected upstream of the MCC, γ-spectrometric measurements registered only one artificial radionuclide, ¹³⁷Cs, with a maximal activity of 8 Bq·kg^?1. Sequential extraction performed on samples of the upper layers of the sediment core showed different degrees of potential environmental availability for artificial radionuclides: the highest was recorded for ²⁴¹Am and ¹⁵²Eu (up to 85% of initial activity), followed by ⁶⁰Co (up to 32%), and finally, ¹³⁷Cs (up to 15%). In a few samples, ²⁴¹Am was present in the unextractable form, which may be accounted for by the presence of reactor fuel microparticles.     

Factors affecting the flux of arsenic through the mussel Mytilus galloprovincialis

Radiotracer experiments were designed to study the effects of certain environmental and biological factors on arsenic accumulation and elimination processes in the Mediterranean mussel Mytilus galloprovincialis. Arsenic (as arsenate) uptake increased with increasing arsenic concentration in the water; however, the response was not proportional, indicating that accumulation was partially suppressed at higher external arsenic concentrations. In general, approximately 80% of the ⁷⁴As taken up was associated with the soft parts, with small mussels concentrating ⁷⁴As to a greater degree than larger individuals. The highest ⁷⁴As concentrations were recorded in the byssus and the digestive gland. Increased temperature enhanced both arsenic uptake and loss. Mussels in sea water at 19 S accumulated approximately three times more ⁷⁴As than those held at 38 S. Arsenic loss was much less affected by salinity, with only a tendency for greater arsenic retention noted at lower salinities. Studies carried out in the laboratory and in situ revealed that arsenic turnover was significantly more rapid in actively growing individuals living under natural conditions. Arsenic-74 loss from the in situ group was essentially biphasic, with biological half-times of approximately 3 and 32 days for the fast and slow compartments, respectively. The active secretion of arsenic in the byssal threads contributed to the total elimination of the element from the mussels.     

Effects of copper on the latency of lysosomal hexosaminidase in the digestive cells of Mytilus edulis

Mytilus edulis collected from Tomales Bay, California, USA, during mid-winter 1979 were exposed to increased concentrations of dissolved copper under controlled laboratory conditions. A dose-dependent reduction in the latency of lysosomal hexosaminidase activity in digestive cells was induced after a 30 d exposure to copper. The half-time of the hexosaminidase staining reaction in sections of digestive gland from control mussels was 15.5 min; for mussels exposed to 25, 50, and 75 g Cu l^-1 it was 11.8, 8.5 and 5.5 min, respectively. In addition, the dyecoupled reaction product was seen earlier in sections from individuals exposed to 50 and 75 g Cu l^-1 (after 30 s) and 25 g Cu l^-1 (1 min) than in sections from control individuals (2.5 min). Copper accumulations were demonstrated histochemically to have the same distribution as the hexosaminidase reaction product, indicating that copper is sequestered in lysosomes. Copper concentrations in digestive gland tissue, were related to the concentrations of copper in the water to which the mussels were exposed.     

Effects of lead exposure on redox status,DNA and histological structures in Venus verrucosa gills and digestive gland

ABSTRACT

Lead (Pb) is one of the most toxic heavy metals that affect the physiological status of aquatic organisms. The present investigation evaluated the possible toxic effect of lead chloride (PbCl₂) on biomarkers responses, DNA damage and histological alterations in Venus verrucosa gills and digestive gland. Three concentrations of PbCl₂ (D1:1µgL^?1, D2: 10µgL^?1 and D3: 100µgL^?1) were chosen for V. verrucosa exposure during six days. At the end of the trial, it was found that Pb tended to accumulate in both gills and digestive gland in a dose-dependent manner. However, gill tissues exhibited the highest metal burden. Our results showed an increase of malondialdehyde, protein carbonyls and advanced oxidation protein product levels in both organs following PbCl₂exposure. The induction of both non-enzymatic and enzymatic antioxidant systems; as well as the decrease of the acetylcholinesterase activity and degradation of DNA structure was recorded in the gills and digestive gland. The histopathological alterations observed in gills (disruption of lamellas and cilia filaments?…) and digestive gland (lumens occlusion, necrosis and fibrosis) confirmed the aforementioned results. Our data highlighted the short-term toxicity effects of PbCl₂ on V. verrucosa and pointed out a high sensitivity of gills towards this metal.     

Petroleum hydrocarbons in the marine bivalve Venus verrucosa: accumulation and cellular responses

Cellular and subcellular responses of the marine burrowing bivalve Venus verrucosa collected from the north-eastern coastline of Malta from January to June 1985, after exposure to petroleum hydrocarbons (PHC) were investigated. After long-term exposure to 100 gl^-1 of water-accomodated fractions (WAF) of crude oil, PHC were found to accumulate most rapidly in the digestive gland and then in the gills, with saturation levels being reached within 100 d of exposure in both cases. PHC accumulation, both in the mantle and muscle tissues, was more gradual and consistent throughout the whole exposure period. After 150 d of exposure, the digestive cells of the digestive gland were significantly reduced in height (atrophy) and exhibited reduced lysosomal membrane stability. After 144 h of exposure to higher concentration of PHC (820 and 420 gl^-1), several cytological effects were recorded, including an increase in cell volume and activity of gill mucocytes as well as in the number of haemocytes in gill blood sinuses. There was also evidence of damage to the epithelial lining of the foot, stomach and style sac and marked atrophy of the digestive cells of the digestive gland. The significance of such responses is discussed.     

Acid and alkaline phosphatase activities in the clam Ruditapes philippinarum

Acid and alkaline phosphatase activities have been partially characterized in Ruditapes philippinarum (Adams and Reeve, 1850). Two activity peaks at pH=4.5 and pH 10.5 were detected in the gill, digestive gland, mantle, siphon and foot. Acid phosphatase activity was higher than that of alkaline phosphatase. The highest activity for both enzymes was observed in the digestive gland and, in decreasing order, the gill, foot, siphon and mantle. Alkaline phosphatase activity was similar in the mantle, siphon and foot. K _m values were determined for both enzymes in the gill and digestive gland. Hill coefficients were near 1, indicating no allosteric behaviour for either enzyme in the two organs. The optimum temperature was the same for acid phosphatase in both gill and digestive gland (50 °C), while for alkaline phosphatase it differed for these two organs (gill, 40 °C; digestive gland, 35 °C). The apparent activation energy was obtained from Arrhenius plots, and ranged from 8.61 kcal/mol for alkaline phosphatase in the gill, to 10.84 kcal/mol for acid phosphatase in the digestive gland. The effects of metals (1 mM conc) on both enzyme activities were assayed in vitro. Hg strongly inhibited the enzyme activities in the gill and digestive gland, probably because of its affinity for the sulphydryl group. Histochemically, acid phosphatase in the gill was located in a granular form throughout the gill cells, but was undetectable in the ciliate epithelium of the gill filaments. Alkaline phosphatase was located in the gill skeleton. Clam size and phosphatase activities were inversely related, probably reflecting a decrease in shell deposition with inereasing size. As a function of season, both enzymes were present in lowest amounts in winter, when undifferentiated sex cells were predominant in the germinative epithelium, and highest in summer, when ripe individuals of both sexes were more frequent.     

Ontogenetic changes in digestive enzyme activity of the spiny lobster,<Emphasis Type="Italic"> Jasus edwardsii</Emphasis> (Decapoda; Palinuridae)

Digestive enzyme profiles of puerulus, post-puerulus, juvenile and adult stages of the spiny lobster Jasus edwardsii Hutton were determined in order to identify ontogenetic changes in digestive capabilities and assess capacity to use dietary components and to exploit diets to meet nutritional requirements. Juvenile and adult lobsters exhibited a diverse range of enzymes, suggesting they can exploit varied diets. Proteolytic activity of trypsin, chymotrypsin and carboxypeptidase A (1.86–3.70 U mg^–1 digestive gland protein) was significantly higher than carbohydrase activity of amylase, -glucosidase, cellulase and chitinase (0.0014–0.38 U mg^–1 digestive gland protein), thus showing that dietary protein was more important than carbohydrate and that the lobster is carnivorous. These conclusions are consistent with other studies that found spiny lobsters to feed predominantly on crabs, bivalves, ophiuroids and sponges. Lipase activity (0.371 U mg^–1 digestive gland protein) was also relatively high, thus showing the importance of dietary lipid. Total activities (units per digestive gland) of every enzyme assayed increased significantly with lobster carapace length. There were few significant ontogenetic trends in specific enzyme activity (U mg^–1 digestive gland protein). Amylase and laminarinase specific activity was significantly higher in small lobsters than large lobsters (regression analyses, respectively, F_(1,23)=9.84, P=0.005; F_(1,11)=19.65, P=0.001), suggesting that carbohydrates including laminarin are more important in the diet of small juveniles. Trypsin, amylase and lipase activities were detected in all non-feeding puerulus stages, suggesting that feeding is not a cue for digestive enzyme production in J. edwardsii. Significant variations in total and specific activities of amylase (F_(1,3)=14.2, P=0.00; F_(1,3)=14.2, P=0.00) and trypsin (F_(1,3)=8.8, P=0.00; F_(1,3)=21.41, P=0.00) and a declining trend in lipase total activity between non-feeding puerulus stages suggests that they may be hydrolysing endogenous energy reserves to sustain their onshore swimming activity prior to settlement. Profiles suggest carbohydrate and lipid are utilised first, followed by protein. Consistently high levels of lipase in all puerulus stages (0.24–0.7 U digestive gland^–1; P>0.05) confirm the importance of lipid as a major energy substrate.Communicated by G.F. Humphrey, Sydney     

Quantitative determination of metallothionein-like proteins in mussels. Methodological approach and field evaluation

Electrochemical quantitation of metallothionin-like proteins (MLP) in mussels was based on the determination of their constituent cysteinyl residues according to Brdika's catalytic reaction. Calibration was performed by an internal MLP standard isolated from the digestive gland of Mytilus galloprovincialis for which protein concentration had been estimated by Bradford's spectrophotometric method. For that purpose three metal-binding proteins [MLP-I, MLP-II and Cu-BP (binding protein)] were separated by DEAE-Sephadex A-25 chromatography from the digestive gland of mussels previously exposed to Cd. The most negatively changed MLP-II fraction was characterized by the fact that it contained the largest amount of both total metal and sulphydryl (-SH) content per mass of protein, although this was approximately two times lower than the-SH level of commercially available MT from rabbit liver. Exposure of mussels to a relatively low level of cadmium (0.2 g Cd l^-1) added into the seawater either by itself or as a mixture with other metals (2 g Cu l^-1 and 1.6 g Pb l^-1) resulted in a measurable level of MLP induction within 14 d in comparison to the control specimens. The effect of the metal mixture on MLP synthesis appears to be less than additive, suggesting a competitive interaction between metal ions for uptake and binding sites as well as differing potentials for MLP induction. Variations in the MLP content observed in the digestive gland of mussels seasonally collected from the same location are in the range 2.1±0.4 mg g^-1 on a wet weight basis. The methodological and conceptual aspects of the application of MLP induction in the Mytilus sp. as a biomarker in seawater trace metal monitoring are critically evaluated.     

Uptake and loss of radioactive cadmium by the sea-skater Halobates robustus (Heteroptera: Gerridae)

Sea-skaters, Halobates robustus Barber, caught around the Galápagos Islands, were exposed over a period of 3 days to radioactive cadmium dissolved in seawater. Although ^115mCd (metastable ¹¹⁵Cd) was accumulated by the insects, they did not concentrate it above the seawater level. The experiments clearly showed that cadmium is taken up into the body, not merely adsorbed onto surfaces. The uptake of Cd followed a biphasic time course. When sea-skaters labelled in this way were transferred back into unlabelled seawater, the rate of loss of Cd was likewise biphasic. During uptake and loss, the biological half-life times for the first compartments were almost identical (0.64 and 0.79 h), indicating that the same compartment might be involved. On the other hand, the biological half-life times for the second compartments were different (1.7 days during uptake, 19.5 days during loss), indicating that the site or chemical form of bound Cd in the sea-skater had changed. For an adult female, weighing about 8.5 mg wet weight, the drinking rate of seawater was estimated at 2.5 l day^-1, equivalent to 29% body weight.     

Sequestration of As by iron plaque on the roots of three rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars in a low-P soil with or without P fertilizer

A pot experiment was carried out in a greenhouse to investigate the sequestration of As in iron plaques on root surface of three rice (Oryza sativa L.) cultivars. Phosphate (P) fertilization increased both plant biomass and tissue P concentrations significantly, indicating that the soils used in this study was highly P-deficient. Results from this study confirmed that low P supply improved the formation of iron plaque on rice roots. As a consequence, arsenic (As) concentrations in DCB-extracts with no P addition were significantly higher than those with P fertilization. Arsenic was highly sequestrated in iron plaque; arsenic concentration in iron was up to nearly 120 mg kg⁻¹, while arsenic concentrations in roots were just several mg kg⁻¹. Both arsenic and phosphate concentrations in iron plaque were highly positively correlated with the amounts of iron plaque (DCB-extractable Fe). Contrary to normal understanding that increasing P supply could reduced As accumulation in plants, results from the present study showed that P fertilization did not inhibit the As uptake by plants (As accumulation in aboveground), which was probably due to the fact that iron plaque formation was improved under low P conditions, thus leading to more As sequestration in the iron plaque. Thus results obtained in this study indicated that the iron plaque may inhibit the transfer of As from roots to shoots, and thus alter the P–As interaction in plant As uptake processes.