Behavioural,developmental and morphological responses of Chironomus gr. thummi larvae (Diptera,Nematocera) to aquatic pollution

Populations of Chironomus gr. thummi larvae from two differently polluted lowland streams (Dommei, high cadmium and zinc; Ijse, medium copper and organic xenobiotics) were screened for behavioural and morphological responses to pollution. Behaviours such as locomotion (swimming and looping), respiration movements (ventilation) and inactivity were quantified with impedance conversion technique. Chironomids from the Dommel were more active than larvae from Ijse. In Ijse, deformed larvae showed less emergence, less locomotion and more ventilation than non-deformed larvae. In Dommel, deformed and normal larvae were equally fit (behaviour, emergence).     

Cadmium,zinc, lead and copper inChironomus riparius (Meigen) larvae (Diptera,Chironomidae): uptake and effects

Cadmium, Zn, Pb and Cu uptake and effects in larvae ofChironomus riparius (Meigen) were studied in an integrated laboratory investigation, in which metal analyses in different instar stages, uptake kinetics and effects on development and growth were considered in three separate experiments.In short-term experiments with fourth instar larvae, it was demonstrated that only a minor portion of metals was adsorbed on the larval exoskeletons. No conclusive evidence on the uptake mechanism was found, but active uptake of trace metals seemed highly unlikely.In partial life cycle experiments, all four metals studied were readily accumulated in chironomid larvae. Uptake could be described satisfactorily utilizing a first-order one-compartment uptake model which incorporated growth. In all cases steady state conditions were approached and high uptake and elimination rate constants were estimated. Distinct differences between essential (Zn and Cu) and non-essential (Cd and Pb) metals were noted. Larval growth was significantly impaired upon exposure.Finally, long-term exposure experiments with low Cd concentrations (0.010 and 0.025 mg 1^{–1) resulted initially in growth impairment and high mortality in first instar stages, but surviving larvae restored growth and adults emerged even before control adults. In similar experiments with Zn (0.1 and 1.0 mg 1–1}), development of larvae was significantly retarded. High Zn concentrations in larvae were noted and almost no adult midges emerged.     

Maternal and neonatal scalp hair concentrations of zinc, copper, cadmium, and lead

Postpartum scalp hair samples from 82 term-pregnancy mother/neonate pairs were analyzed for their concentration of zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb), using inductively coupled plasma-mass spectrometry. Maternal and neonatal Zn concentrations had geometric means (and 99% confidence intervals) of 122.5 μg/g (117.9–131.5 μg/g) and 146.9 μg (141.5–156.7 μg/g) respectively. Corresponding Cu values were 18.4 μg/g (17.6–23.8 μg/g) and 6.7 μg/g (6.3–7.6 μg/g). Those of Cd were 0.49 μg/g (0.47–0.69 μg/g) in the mothers and 0.57 μg/g (0.55–0.86 μg/g) in the neonates. For Pb, they were 7.95 μg/g (7.60–9.32 μg/g) and 4.56 μg/g (4.39–5.56 μg/g). Cigaret smoking, despite its relatively low prevalence (19.5%), was associated with lower Zn and higher Cd and Pb concentrations and in lower Zn/Cd and Zn/Pb molar concentration ratios. Smoking also altered interelemental relationships, particularly those of Zn with Cd and Pb and those between Cd and Pb. Smoking frequency appeared to show negative dose-response effects on maternal and neonatal Zn concentrations, Zn/Pb molar concentration ratios, and birth weight. Mothers with a history of oral contraceptive (OC) usage had significantly higher Cu concentrations and lower Zn/Cu molar concentration ratios than nonusers, with the highest Cu concentrations and lowest Zn/Cu values being associated with third-generation OCs. No similar effects were elicited in the respective neonatal Cu concentrations. Neither alcohol consumption nor prenatal supplementation with iron and/or folic acid had discernible effects on the maternal or neonatal elemental concentrations. The data from this study suggest that in a given population of term-pregnancy mothers and neonates, significant interindividual variations in hair trace element concentrations can occur, irrespective of commonality of general environment, and that lifestyle factors, including cigaret smoking and OC usage history, can be significant contributory factors to such variations. The data are discussed in relation to the effects of smoking-associated exposure to Cd and Pb exposure on Zn availability for placental transfer, as well as on the quantitative maternal Zn supply levels to the fetus resulting from the known tendency of smokers to have lower dietary intakes of Zn. The higher Cu concentrations in OC users are discussed in relation to altered Cu metabolism, characterized by increased synthesis of the Cu-binding protein, ceruloplasmin, as an acute-phase antioxidant response to altered lipid profile and increased lipid oxidation.     

Uptake of lead,cadmium and zinc by the fairy shrimp,Branchinecta longiantenna (Crustacea: Anostraca)

Individuals of the fairy shrimp, Branchinecta longiantenna, were subjected to 5 concentrations (0.1 to 15 mg l^–1) of Pb in diluted habitat water at 13 °C. Lead concentrations (mg kg^–1 wet weight) in the animals were determined at 2-day intervals by digestion in nitric acid followed by atomic absorption analysis. The shrimp were also subjected to 0.1 mg l^–1 media of Cd and Zn, separately.Uptake rates by the fairy shrimp for the three metal ions at 0.1 mg l^–1 were: 0.111, 0.0885, and 0.0460 mg kg^–1 day^–1 for Zn, Pb, and Cd, respectively. After 2 days in 1.0 mg l^–1 Cd or Zn, the animals expired; but they surviced for 8 days in a 10 mg l^–1 Pb medium and for 2 days in 25 mg l^–1 Pb. Lead uptake demonstrated a linear dependence on the Pb concentration in the media.Shrimp survived at much higher tissue accumulations of Pb compared to Zn and Cd. Estimated lethal doses were 20, 1.2–2.4, and 0.4–1.4 mg kg^–1 wet weight for Pb, Zn, and Cd, respectively. Pb was found to be at much lower concentration than Cd or Zn in the natural pond water but between Cd and Zn levels in the sediment. Thus Cd and Zn probably present a greater threat to B. longiantenna than Pb, although Pb may be in higher concentration in the environment.Contribution 47, Laboratory of Ecology, The Claremont Colleges, Claremont, CA 91711, USA. Send reprint requests to Clyde Eriksen.     

Perturbation of monovalent cation composition in Ulva lactuca by cadmium, copper and zinc

Discs of thallus cut from the macroalga Ulva lactuca were incubated in filtered seawater containing cadmium, zinc, copper or cobalt (30 m). The metal uptake rates differed for each metal in the order Cu > Zn > Cd > Co. Exposure of the macroalga to metals resulted in a disruption of intracellular monovalent cation composition. Intracellular potassium was irreversibly lost and sodium was accumulated by cadmium- or copper-treated U. lactuca, which was assumed to indicate irreversible disruption of the plasmalemma. Exposure to zinc caused an increase in sodium concentrations, whereas potassium concentrations were not significantly different from the controls, suggesting that the integrity of the plasmalemma had been maintained at the zinc concentration used. Intracellular magnesium was also lost from copper-treated algae, which again indicated a loss of integrity of the cell membrane.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

Effect of chronic exposure to cadmium and copper on Asellus aquaticus (L.) (Crustacea,Isopoda)

The effects of chronic exposure to 5 g·1^–1 cadmium or copper on the crustacean Isopod Asellus aquaticus (L.) were studied by analyzing survival and body growth in the first stages of the life-cycle and by determining fecundity and survival of embryo-bearing females. Juveniles survival is differently affected by the two metals in that embryonic development is more sensitive to cadmium while juvenile development is more sensitive to copper. Juvenile body growth is stimulated by cadmium and depressed by copper. Embryo-bearing female survival and fecundity are significantly reduced by cadmium but are not affected by copper. The consequence of environmental contamination by a sublethal cadmium or copper concentration is discussed.     

On the structure and function of the ventral tubuli of some Chironomus larvae (Diptera,Nematocera)

The ultrastructure of the walls of the tubuli of larvae of two Chironomus species living in lakes and four species from small ponds were studied, in order to decide whether the function of the tubuli is primarily a respiratory one or not.Data suggest a reinforcement of the concept of a respiratory function and a hypothesis is discussed which might explain the differences found between the tubuli of lake- and pond-dwelling species.     

Macromolecular interactions with cadmium and the effects of zinc,copper, lead,and mercury ions

Interactions of cadmium (Cd) ions with bovine serum albumin (BSA), bovine hepatic metallothionein (MT), calf thymus histone and deoxyribonucleic acid (DNA), and bovine hepatic chromatins were studied in the presence and absence of divalent zinc (Zn), copper (Cu), mercury (Hg), or lead (Pb) ions, using equilibrium dialysis at pH 7 and at 37°C. The BSA had 3.5 Cd-binding sites with an apparent affinity constant of 1×10⁵. The other metal ions inhibited the binding by reducing the affinity constant and the number of Cd-binding sites in BSA. There were 6 high affinity and 13 low affinity Cd-binding sites in the MT. Zinc ions had poor efficacy in reducing the binding of Cd to the MT. However, the Cu²⁺ and Hg²⁺ ions inhibited the Cd binding to a considerable extent, the former ions being more potent in this respect. Histone did not bind Cd. There were two kinds of Cd-binding sites in DNA: One mole of Cd per four moles DNA-phosphorus at low affinity sites, and one mole of Cd per 6.7 moles DNA-phosphorus at high affinity sites. Their apparent association constants were 8.3×10⁵ and 4.4×10⁶ M, respectively. The other metal ions had inhibitory effects on the binding of Cd to DNA. Histone reduced the Cd-DNA interactions to only a minor extent. The other metal ions reduced the binding of Cd to DNA-histone complex to a small extent. Cadmium binds to the euchromatin (Euch), heterochromatin (Het), and Euch-Het mixture almost equally. The other metal ions reduced the binding maximally in Euch-Het followed next in order by Het and Euch. Cupric ions were the most potent inhibitors of the interactions of Cd with the nuclear materials.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

Accumulation,regulation and toxicity of copper,zinc, lead and mercury in Hyalella azteca

Zinc, lead and mercury accumulation in the amphipod Hyalella azteca increases with increasing exposure to metals. During 10 week chronic toxicity tests, metal accumulated at the highest non-toxic/lowest toxic concentration was 126/136 µg Zn g^–1, 7.1/16 µg Pb g^–1 and 56/90 µg Hg g^–1 dry weight. Concentrations of lead and mercyry in control animals were substantially lower (1.3 µg Pb g^–1 and 0.4 µg Hg g^–1), but concentrations of zinc in controls (74 µg g^–1) were about one half those of the lowest toxic concentration. Copper was completely regulated. Accumulated copper concentrations after 10 weeks exposure to all waterborne copper concentrations resulting in less than 100% mortality were not significantly different from controls (79 µg g^–1). Lead and mercury concentrations in wild H. azteca should be useful indicators of potential toxicity. Zinc accumulation may also be a useful indicator of zinc toxicity, but careful comparison with control or reference animals is necessary because of the small differences between toxic and control concentrations. Copper is not accumulated by H. azteca under chronic exposure conditions and body burdens of field animals cannot be used as an indicator of exposure or potential toxic effects. Short term exposures to copper, however, result in elevated copper concentrations in H. azteca, even at concentrations below those causing chronic toxicity. Short term bioaccumulation studies might, therefore, provide a useful indication of potential chronic copper toxicity.     

The influence of cadmium,copper, lead,and zinc on the distribution and evolution of metallophytes in the British Isles

Mine spoils and other soils contaminated with cadmium, copper, lead and zinc show natural colonization by species which have strategies of avoidance or tolerance of metal toxicities. The distribution of plants on such substrata in the British Isles is examined in the light of present knowledge of such strategies. Evolutionary processes mediating the selection of tolerant individuals and ecotypic differentiation of adapted populations on metalliferous soils are considered. Other factors determining which species can and which cannot evolve tolerance include constitutional differences in species sensitivity to toxic metals, and phenotypic (environmentally-induced) tolerances. The importance of constitutional properties and phenotypic responses in providing explanations for plant distribution on metalliferous soils is assessed.     

The effect of calcium concentration on the toxicity of copper, lead and zinc to yolk-sac fry of brown trout, Salmo trutta L., in soft, acid water

Yolk-sac fry of brown trout were exposed to three levels of single trace metals (Cu, 20,40,80 nmol 1^-1; Pb, 12·5,25,50 nmol 1^-1; Zn, 75,150,300 nmol 1^-1) typical of concentrations reported for acid soft waters, in flowing, artificial, soft water media maintained at pH 4·5 and [Ca] of 20 or 200 μmol 1^-1for 30 days.
Mortalities were high in fry subjected to all levels of the three trace metals at [Ca] 20 μmol 1^-1, with 80% of the total deaths occurring between days 11 and 15 of the experiment. 25% mortality occurred at low [Ca] and pH 4·5 in the absence of trace metals, with only one death recorded at [Ca] 200 μmol1^-1'(Cu, 80 nmol 1^-1). At high [Ca] all three levels of Cu and Pb impaired net Na and K uptake; Cu was the only metal to reduce the uptake of Ca. The Zn treatments had no significant effect on mineral uptake. Calcification of centra was reduced by all three Cu treatments at [Ca] 200 μmol 1^-1. The lowest Zn concentration (75 nmol 1^-1) was the only other treatment to impair skeletal development. In the absence of trace metals, low [Ca] significantly reduced Ca, Na and K uptake, skeletal calcification and dry mass at pH 4·5.
The deleterious effects of low Cu, Pb and Zn concentrations at low pH and low [Ca], and the ameliorative effect of higher ambient [Ca], are discussed in relation to fishery status in soft, acid waters.     

Silver,copper, lead and zinc accumulation byPseudomonas stutzeri AG259 andStreptomyces albus : electron microscopy and energy dispersive X-ray studies

Metal accumulation by a silver-resistant Pseudomonas stutzeri AG259 strain and a Streptomyces albus strain was investigated in a mixed metal solution of silver, copper, lead and zinc. The location of silver, lead and copper on cells was determined by transmission electron microscopy coupled with an X-ray analysis system. In P. stutzeri cells silver was detected as dense deposits on the cells. Copper and lead were distributed over the cells. S. albus accumulated these metals only on part of cells with a higher concentration per cell than in P. stutzeri.     

Adaptive and cross resistance to cadmium (II) and zinc (II) by Pseudomonas aeruginosa BC15

Cadmium and zinc appear in the combined forms and they are co-pollutants. Cd is the most hazardous metal ion for human beings and causes renal dysfunction, liver and lungs damage, bone degeneration and blood damage. Though Zn is an essential nutrient, excess of Zn is toxic. Biological process was more important because conventional methods fail to remediate these pollutants due to high costs and less affordability. The screening and understanding of the functioning of microorganism plays an important role in removal and recovery of metals from heavy-metal-polluted water and soil. In our study, the strain Pseudomonas aeruginosa BC15 was isolated from oil-mill-treated waste water and it showed to be highly resistant to 6 mM Cd and 20 mM Zn in the solid and liquid media. The growth studies of BC15 strain in the medium without induction exhibited high tolerable capacity when compared to other microbes. Pretreatment of P. aeruginosa BC15 with sub-lethal concentrations of Cd induced adaptive resistance to lethal doses of Cd. Cadmium-induced cells also showed cross resistance to lethal concentration of zinc. The organism had high resistance against Cd and Zn. This has been clearly proven through biosorption studies: Cd was absorbed up to 62% and Zn about 60% in single solution, whereas in binary solution Cd was biosorbed up to 82% and Zn 85%. In conclusion, this study reveals the significance of using the strain P. aeruginosa BC15 in the bioremediation of Cd and Zn from industrial waste water and contaminated soil.     

Accumulation, depuration and distribution of cadmium and zinc in the green-lipped mussel Perna viridis (Linnaeus) under laboratory conditions

Ecotoxicological tests were conducted in the green-lipped mussel Perna viridis under laboratory conditions. Different rates of accumulation and depuration in soft tissues are found and this might be due to different mechanisms of metal binding and regulation. At the end of depuration, Cd levels in soft tissues of P. viridis were 10–30 times higher than before exposure, while Zn levels in soft tissues were almost similar to levels before exposure. These results indicate that P. viridis is a good biomonitoring organism for Cd but Zn levels might be actively regulated. It remains uncertain whether P. viridis is a good biomonitoring organism of environmental Zn contamination. However, the positive patterns, although different rates, of accumulation and depuration for Cd and Zn support the use of P. viridis as a biomonitoring agent for such metals.     

Nonrandom chromosomal distribution of spontaneous breakpoints and satellite DNA clusters in two geographically distant populations of Chironomus riparius (Diptera: Chironomidae)

Two geographically distant populations of Chironomus riparius (syn. C. thummi) from two environmentally polluted sites (Santena, Italy and Varna, Bulgaria) show numerous somatic and inherited chromosomal aberrations (inversions, deletions and deficiencies). Fifty-five percent of the observed breakpoints occurred in at least two larvae from both populations. Breakpoints occurring twice or more were considered as common structural chromosomal breakpoints. We tested whether such common breakpoints in larvae of the two polluted populations had a random chromosomal distribution or occurred preferentially in specific heterochromatic regions. Distribution of common breakpoints was not random, and proximal regions of first and third chromosome had significantly more common breakpoints than distal ones. By FISH we identified and mapped 56 chromosomal sections containing clusters of two tandem-repetitive satellite DNA families called Hinf and Alu elements. Like the common breakpoints, these repetitive DNA clusters appeared to be significantly more abundant in regions of constitutive heterochromatin such as the pericentromeric regions, while in distal sections of chromosomal arms they were rare or absent. Twenty-four out of 45 common breakpoints (i.e., 53.3%) occurred in cytogenetic sections where Alu and Hinf satellite DNA probes hybridized. The frequency of co-localization between common breakpoints and repetitive DNA hybridization signals was significantly higher than expected by chance. We hypothesize that spontaneous or induced breaks occur more frequently in sections containing blocks of repetitive DNA.     

Effect of medium copper concentration on the growth,uptake and intracellular balance of copper and zinc in Menkes' and normal control cells

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through normal to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08–8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.     

A structural study of the anal papillae of the midge Chironomus riparius Meigen (Diptera: Chironomidae)

Larvae of the midge, Chironomus riparius Mg., have four anal papillae arranged as two pairs, one dorsal to and the other ventral to the anus. Structural study with light and electron microscopes has revealed that their integument consists of a thin cuticle overlaying a thick, syncytial epidermis which is specialised to facilitate ion transport. There is a distinct neck or collar region at the junction of each papilla with the rest of the body wall. Although in many respects these structures resemble those in mosquitoes, there are both morphological and physiological features which suggest that differences exist in the means by which ion exchange with the environment is controlled and effected in the two families.