Evaluation of the lethal and sub-lethal toxicity and potential endocrine disrupting effect of nonylphenol on the zebra mussel (Dreissena polymorpha)

Nonylphenol (NP) is commonly found in surface waters nearby municipal wastewater treatment plants and was shown to have endocrine disrupting effects in aquatic organisms. The purpose of this study was to investigate the toxicity and potential endocrine disrupting effects of NP on the freshwater zebra mussel (Dreissena polymorpha). Toxicity assessment yielded LC(50) values of 3.68, 2.19 and 1.62 mg L(-1) after 15, 35 and 50 days of exposure, respectively. LC(10) values of 1.6, 1.11 and 0.68 mg L(-1) were respectively obtained for similar exposure periods. At concentrations >5 mg L(-1), mortality effects were significant, as were those relating to attachment and siphon extension (indicating filtration), both general indicators of health. Endocrine disruption effects were investigated after a prolonged exposure (112 d) to 5 and 500 microg L(-1) NP by measuring Vitellin (Vn)-like protein levels using the alkali-labile phosphate (ALP) assay and gel electrophoresis (GE). An increase in ALP levels was observed in both male and female mussels, although only marginal owing to a significant decrease in the mussels' health indicated by its condition, during the experiment. These levels, however, increased proportionally with NP concentration. Using solid phase thin-layer chromatography, we confirmed increased levels of the steroid cholesterol and evidence of NP uptake. Cholesterol levels in gonad tissue proved to be a more responsive biomarker of exposure to NP than levels of ALP. Further implications relating to the occurrence of endocrine disruption in the zebra mussel are discussed.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Regeneration of lost siphon tissues in the tellinacean bivalve Nuttallia olivacea

The inhalant siphon of the tellinacean bivalve Nuttallia olivacea is an important prey item for juvenile stone flounder Platichthys bicoloratus in estuaries in Japan. We examined quantitative siphon regeneration of N. olivacea in rearing experiments of siphon-removed bivalves (> 30 mm shell length) both in the laboratory and in their natural habitat. Under laboratory conditions, siphon-removed bivalves regenerated lost tissues quantitatively at 15 and 25 °C 1 mo after siphon removal, although regeneration was incomplete. A 3-mo caging experiment in the field showed that great regeneration occurred in siphon-removed bivalves. However, the siphon weight of removed bivalves was significantly smaller than that of non-amputated bivalves, suggesting the incomplete regeneration. In a 1-mo caging experiment, bivalves that had approximately 15% of their siphons amputated were selected at some intervals to illustrate the quantitative regeneration process. Estimated daily siphon production was remarkably high only a few days after amputation. It decreased greatly thereafter, but regeneration was not completed within 30 d. These results indicate that bivalves regenerate siphons rapidly just after losing siphon tissues and then regeneration is slowed down before it is completed.     

Bioaccumulation and toxicity of aluminium in the pond snail at neutral pH

The low solubility of aluminium (Al) at neutral pH means that it largely exists as colloidal particulates in aquatic systems. However, the pond snail Lymnaea stagnalis accumulates significant amounts of Al following exposure to water containing added Al (up to 500 microg l(-1)) at pH 7. This is accompanied by depression of behavioural activity (locomotion, feeding) which subsequently recovers, suggesting tolerance to the metal. The presence of silica ameliorates behavioural toxicity of Al, but does not prevent uptake of the metal. In vitro studies using the isolated central nervous system demonstrate toxicity at the cellular level. Extracellular application of Al (100 microM) led to membrane depolarisation, bursts of action potentials and action potential broadening. The chemical form in which Al is applied influences the extent of bioaccumulation and toxicity. Detailed knowledge of its solution chemistry is therefore essential.     

Evaluation of PAH bioaccumulation and DNA damage in mussels (Mytilus galloprovincialis) exposed to spilled Prestige crude oil

We analyzed the hydrocarbon composition of the Prestige oil as it reached the shores, its solubility in sea water, its bioaccumulation, and the genotoxic damage associated to oil exposure, using Mytilus galloprovincialis as sentinel organism. Mussels were exposed to two oil volumetric ratios (1:500 and 2:500) for 12 days. Great concentrations of total polycyclic aromatic hydrocarbons (TPAH) have been obtained, being in general higher in the samples from the dose of 1:500, both in sea water (55.14 vs. 41.96 microg/l) and mussel tissue (16,993.80 vs. 17,033.00 microg/kg), probably due to the great tendency of these compounds to link to particles in water. Comet assay results reflected an increase in the DNA damage associated to oil exposure, higher in the mussels exposed to the higher aqueous TPAH content. In the view of our results, the importance of the evaluation of biodisponibility, bioaccumulation and DNA damage in the assessment of the effects of xenobiotic pollutants to marine environments could be highlighted.     

Interactive effects of sublethal predation and body size on siphon production of the bivalve Nuttallia olivacea

This paper is intended to reveal effects of siphon cropping on siphon production of a tellinacean bivalve Nuttallia olivacea, an important prey for juvenile stone flounder. We carried out a two-way field experiment in which bivalves of three treatments (3-times siphon removal, 1-time removal, no removal) and four size classes (9-50 mm shell length) were caged and placed in the field for 3 mo. Growth of repeatedly-removed bivalves was inhibited, indicating reduced siphon growth (natural increase of siphon size according to somatic growth). However, siphon production of removed bivalves was larger than non-removed bivalves, possibly because of siphon regeneration. Juveniles (N. olivacea < 20 mm shell length) showed high growth performance. Their siphon growth was greater than their siphon regeneration. In all bivalves except juveniles, siphon regeneration was greater than siphon growth and engendered high siphon production. Siphon growth was dependent on bivalve size and was only slightly reduced by siphon loss, but siphon regeneration seemed to be dependent mostly on the extent of siphon loss. Greater siphon removal enhanced larger siphon production. These results indicate that intensive siphon cropping by juvenile stone flounder induces high siphon production without serious impact on N. olivacea.     

Comparative evaluation of the toxicity induced by mussels (Mytilus galloprovincialis) from two sites of the Moroccan Atlantic coast in mice

Atlantic coast in mice. Preliminary studies showed that seawater contains heavy metals from domestic, agricultural and industrial wastes. Marine bivalves concentrate these pollutants by filtration and serve as vectors in human exposure. The objective of this study was to determine the concentration of heavy metals; cadmium (Cd); chromium (Cr), and lead (Pb) in mussels (Mytilus galloprovincialis) collected in two coastal sites; Jorf Lasfar (JL) (neighbouring a phosphate processing platform) and Oualidia (OL) (a vegetable growing area) located at 120 and 190 km south of Casablanca, respectively. Another objective was to test and compare the toxicity of these mussels on mice. The results indicated the presence of heavy metals (Cd, Cr, and Pb) in mussels at different concentrations, depending on the collection period. Higher concentrations were obtained at JL than at OL: for example, Cd concentrations were 80 +/- 15 to 199 +/- 28 versus 23 +/- 5 microg/g mussel dry weight, respectively. Cramming with mussel powder did not increase Cd, Cr, or Pb concentration in either liver or kidneys of treated mice. The relative kidney weights were reduced. Increased glucose urea was observed in animals' urine. Treatment with mussels from OL induced significant reduction (20%) in mice body weight, together with an increase in creatinuria. These results indicate that mussels collected from OL are more harmful than those obtained from JL are. All these mussels should not be recommended for human consumption.     

The effects of copper on the microbial community of a coral reef sponge

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators for sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha-proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 microg l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 microg l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 microg l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction in the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 microg l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morpho-type actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 microg l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 microg l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 microg l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 microg l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.     

Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals

Contaminant related changes in behavioral, phase I and II metabolizing enzymes and pro-oxidant/antioxidant processes in the freshwater mussels Dreissena polymorpha exposed to metals and PCBs were assessed. Behavioral and biochemical responses including filtering rates, key phase I, II and antioxidant enzymes and levels of metallothioneins, glutathione, lipid peroxidation and DNA strand breaks were determined in digestive glands of mussels after being exposed to sublethal levels of mercury chloride, methyl mercury, cadmium and Aroclor 1260 during 5 days. In 7 out of 12 responses analyzed, mussels showed significant differences across treatments. Unusual properties of measured ethoxyresorufin-O-deethylase (EROD) activities indicated that mussels lack an inducible CYP1A enzymatic activity. Despite of using similar exposure levels, inorganic and organic mercury showed different biomarker patterns of response with methyl mercury being more bio-available and unable to induce metallothionein proteins. Mussels exposed to Cd presented higher levels of metallothioneins and an enhanced metabolism of glutathione, whereas those exposed to Aroclor showed their antioxidant glutathione peroxidase related enzyme activities inhibited. Although there was evidence for increased lipid peroxidation under exposure to inorganic and organic mercury, only mussels exposed to Aroclor had significant greater levels than those in controls.     

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus.

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 microg l(-1)) and ferric iron (20 and 40 mg l(-1)), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe3+ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe3+ treatment. No significant dose-response relationship was found with the two compounds and no synergistic effect was observed between Fe3+ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose-effect relationships and synergistic effects were not observed.     

The effects of zinc oxide nanoparticles on the metallome in freshwater mussels

The use of zinc oxide nanoparticles (nanoZnO) as sunscreens has raised concerns about their safety and release in the aquatic environment through swimming activities and within municipally treated wastewaters. This study's purpose was to examine the effects of nanoZnO on the elemental composition (metallome) in exposed freshwater mussels, Elliptio complanata. Mussels were exposed for 21 days to an environmentally realistic (low) concentration (2 μg/L) of nanoZnO and zinc chloride. The mussels were also exposed to a physically and chemically treated municipal effluent (ME), both alone and in the presence of both forms of Zn. The metallome profile was characterized by the following 15 elements in gills, digestive gland and gonad tissues: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, V and Zn. The levels of metallothioneins (MT) and lipid peroxidation (LPO) in the digestive gland were also measured as biomarkers of toxic effects. The data revealed that exposure to nanoZnO increased the total levels of Zn, MT and LPO in the digestive gland. Discriminate function analysis revealed that the digestive gland responded the most to exposure to either nanoZnO or Zn^2 +. For nanoZnO, the observed changes in Al, As and Mo in the digestive gland offered the best discrimination from dissolved Zn^2 +. Co-exposure of nanoZnO with the ME changed the metallome profile closer to dissolved Zn^2 +, suggesting a common interaction site within the ME. This was observed in changes in Ni, Cu, Se and Zn in the digestive gland of exposed mussels. Canonical analysis of essential and non-essential elements revealed that exposure to nanoZnO increased the relationships between LPO and the sum of essential elements in the digestive gland. Conversely, exposure to dissolved Zn^2 + and the ME decreased the relationship between the sum of non-essential elements and LPO and MT. In conclusion, the use of a “metallomic” approach was used to discriminate changes following exposure to nanoZnO and dissolved Zn in freshwater mussels and provided insights into the interaction of forms of Zn in ME towards mussels.     

Metal accumulation,filtration and O(2) uptake rates in the mussel Perna perna (Mollusca: Bivalvia) exposed to Hg(2+), Cu(2+) and Zn(2+)

Tissue metal concentrations, filtration and oxygen uptake rates were investigated for Perna perna (Bivalvia: Mollusca) during exposure to Hg(2+), Cu(2+) and Zn(2+) (50 microg/l for 24 days, and 24 days recovery with no metal). Hg and Cu tissue levels increased with exposure time, reaching maximum levels after 24 days (87.5 microg Hg/g dry mass and 45 microg Cu/g dry mass, respectively). Zn levels peaked after 4 days exposure (to 233 microg Zn/g dry mass) and stabilized thereafter. Accumulated metal was rapidly lost from tissues when mussels were returned to uncontaminated seawater, suggesting that tissue concentration data may be of limited use in biomonitoring situations where environmental metals fluctuate to low levels. Filtration rates fell below control rates during Hg(2+) exposure, and became elevated again during the recovery period. Cu(2+) and Zn(2+) exposure had little effect on filtration, but suppressed oxygen uptake. During recovery, oxygen uptake of Cu(2+) and Zn(2+) exposed mussels was elevated above the controls. Filtration and oxygen uptake rates were not correlated, but rather responded in different ways to metal pollution. While these physiological responses of P. perna may be of limited use in biomonitoring, they could indicate how populations may respond to marine pollution.     

Detection of micronuclei in haemocytes of zebra mussel and great ramshorn snail exposed to pentachlorophenol

The frequency of micronuclei (MN) induced by pentachlorophenol (PCP) in haemocytes of zebra mussel, Dreissena polymorpha Pall. and great ramshorn snail, Planorbarius corneus L. was determined over a 14 days of exposure (sampling after 4, 7 and 14 days) under laboratory conditions. PCP doses for zebra mussel ranged from 10 to 150 microg/l, and for ramshorn snail from 10 to 450 microg/l. Micronuclei were detected after bisbenzimide fluorescent staining. Positive responses were observed in both species. The mean MN frequencies in treated mussels ranged between 0.69 and 7.50 per thousand, and between 2.07 and 13.80 per thousand in treated snails. The spontaneous MN levels in mussels averaged from 0.5 to 2.75 per thousand, and in snails from 1.56 to 2.00 per thousand. Our results suggest that haemolymph of both species represent an appropriate test tissue in environmental genotoxicity assessment.     

Tracing Consumer-Derived Nitrogen in Riverine Food Webs

The flux of consumer-derived nutrients is recognized as an important ecosystem process, yet few studies have quantified the impact of these fluxes on freshwater ecosystems. The high abundance of bivalves in both marine and freshwater suggests that bivalves can exert large effects on aquatic food webs. The objective of our study was to determine the importance of unionid mussel-derived nitrogen (MDN) to the food web. We used a stable isotope tracer approach in conjunction with nutrient uptake and excretion experiments. We fed mussels (Lampsilis siliquiodea, n = 249) a ¹⁵N-enriched algal diet and placed them into a N-limited stream for 63 days. Mussel hemolymph was non-lethally sampled over the course of the experiment to measure tissue turnover of δ¹⁵N and excretion experiments were done to model the amount of N mussels provided in comparison to stream N uptake demand. Multiple food web pools were sampled twice prior and five times following the mussel addition to trace the ¹⁵N through the food web. Our mussel excretion rates in comparison to areal uptake demand suggested that mussel excretion can account for 40% of the total N demand in this stream. Our enrichment showed that MDN was entering the food web and supplied up to 19% of the N in specific compartments of the food web near the mussel bed. When scaled to a natural mussel aggregation, our results suggest up to 74% of N in the food web may be mussel-derived. Our results show that N supplied by mussels can be an important nutrient subsidy that provides food web support.     

Nylon microfibers develop a distinct plastisphere but have no apparent effects on the gut microbiome or gut tissue status in the blue mussel,Mytilus edulis

Ingestion of microplastics (MP) by suspension-feeding bivalves has been well-documented. However, it is unclear whether exposure to MP could damage the stomach and digestive gland (gut) of these animals, causing ramifications for organism and ecosystem health. Here, we show no apparent effects of nylon microfiber (MF) ingestion on the gut microbiome or digestive tissues of the blue mussel, Mytilus edulis. We exposed mussels to two low concentrations (50 and 100 particles/L) of either nylon MF or Spartina spp. particles (dried, ground marsh grass), ca. 250–500 μm in length, or a no particle control laboratory treatment for 21 days. Results showed that nylon MF, when aged in coarsely filtered seawater, developed a different microbial community than Spartina spp. particles and seawater, however, even after exposure to this different community, mussel gut microbial communities resisted disturbance from nylon MF. The microbial communities of experimental mussels clustered together in ordination and were similar in taxonomic composition and measures of alpha diversity. Additionally, there was no evidence of damage to gut tissues after ingestion of nylon MF or Spartina spp. Post-ingestive particle processing likely mediated a short gut retention time of these relatively large particles, contributing to the negligible treatment effects.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

DNA damage measured by the single-cell gel electrophoresis (Comet) assay in mammals fed with mussels contaminated by the 'Erika' oil-spill

This research aimed to estimate potential genotoxicity for consumers resulting from the ingestion of seafood contaminated with polycyclic aromatic hydrocarbons (PAHs) released into the marine environment after the 'Erika' shipwreck along the coasts of south Brittany, in France. Mussels (Mytilus sp.) collected from sites on the Atlantic coast that were affected by the oil slick in various degrees, were used to feed rats daily for 2 and 4 weeks. DNA damage was measured by use of the Comet assay in the liver, bone marrow and blood of rats receiving food contaminated with 312 microg of 16PAHs/kg dry weight (d.w.) equivalent to 33.8 microg TEQs (toxic equivalent quantities to benzo(a)pyrene (BaP))/kg d.w. mussels, 569 microg/kg d.w. (83.6 microg TEQs/kg) and 870 microg/kg d.w. (180.7 microg TEQs/kg). A dose-effect-time relationship was observed between the amount of DNA damage in the liver and bone marrow of the rats and the PAH contamination level of the mussels. Genotoxicity increased during the period between 15 and 30 days in rats that received food at the highest two PAH levels. On the other hand, no significant change in liver and bone marrow of rats fed with mussels containing 33.8 microg TEQs/kg d.w. was recorded at 30 days compared with 15 days, indicating efficient DNA repair capacity at low levels of exposure. No signs of genotoxicity were found in peripheral blood. Globally, the observed effects were rather moderate. These results show that oil-contaminated food caused DNA damage in predators, and underline the bioavailability to consumers of pollutants in mussels contaminated with fuel oil. The usefulness of the Comet assay as a sensitive tool in biomonitoring studies analyzing responses of PAH transfer through food webs was also confirmed.     

Exposure to Thiodan results in lipofuscin accumulation in hepatocytes of the freshwater catfish Tandanus tandanus

We investigated the effect of time after pulse exposure to 1.0 microg l(-1) endosulfan (applied as Thiodan) on endosulfan residues in the liver and ultrastructural changes in the hepatocytes of the freshwater catfish Tandanus tandanus. Time after exposure did not affect the mean residue level in the liver. After exposure to endosulfan, residues in the liver were 227.47 microg kg(-1) after 1 d and 282.83 microg kg(-1) after 28 d; residues in the bile were 313.97 microg kg(-1) after 1 d and 334.53 microg kg(-1) after 28 d. At the end of 28 d exposure, lipofuscin was present in up to 69% of hepatocytes of fish containing residues of endosulfan, but absent from control fish. There was a statistically significant increase in the percentage of pyknotic nuclei and altered rough endoplasmic reticulum 28 d after exposure. The mean percentage of cells with altered endoplasmic reticulum ranged from 12.93% (Day 1) to 7.50% (Day 28) for control fish, while for exposed fish it increased from 14.30% (Day 1) to 35.00% (Day 28). The mean percentage of cells with pyknotic nuclei increased from 1.1 to 2.1% in control fish and from 3.8 to 9.6% in exposed fish. Other ultrastructural changes included increased ultrastructural heterogeneity, progressive vacuolation and fractionation of rough endoplasmic reticulum, accumulation of lysosomes and residual bodies, intranuclear inclusions and pseudoinclusions, membrane whorls and myelinated bodies. Protracted senescence was one of the main features of endosulfan toxicity to T. tandanus hepatocytes.     

Influence of the glass packing on the contamination of pharmaceutical products by aluminium. Part I: salts, glucose, heparin and albumin.

The presence of aluminium (Al) in pharmaceutical products used parenterally as sodium and potassium chlorides, glucose, heparin and albumin were investigated with respect to their storage in glass containers. As glasses can have aluminium in their composition, the aluminium may be released from the glass into the solution. The action of the substances above mentioned were investigated storing their solutions in glass and plastic containers, and measuring the aluminium in solution at determined time intervals. The aluminium present in the commercial pharmaceutical products, stored in both plastic and glass containers were also measured. All glass containers were analysed to determine their aluminium content. The aluminium determinations were done by atomic absorption spectrometry. The resuLts showed that aluminium is present in all analysed glasses in a percentage of 0.6 to 3%. Although all substances already have a residual aluminium contamination, the major contribution comes from the glass containers in which their solutions were stored. The contamination arising from glass depends too much on the nature of the substance. While the salts extracted about 400 microg Al/l in 60 days, glucose extracted 150 microg Al/l, and albumin and heparin about 500 microg Al/l in the same time interval. Commercial solutions of glucose contain about 10 microg Al/l when stored in polyethylene and from 350 to 1,000 microg Al/l when in glass ampules. Considering all commercial products, solutions stored in plastic containers contained no more than 20 microg Al/l whereas in glass the aluminium contamination reached 1,000 microg/l, and in all of them the aluminium increases with the age of the product.     

The bitterling–mussel symbiosis: a model for host‐parasite adaptation

Bitterlings (Acheilognatinae) are a monophyletic group of cyprinid fishes that lay their eggs in the gill chamber of freshwater mussels. They have evolved many behavioural, morphological and physiological adaptations to the symbiosis. Female bitterling develop a long ovipositor that insert into the exhalant siphon of a mussel and males fertilize the eggs by releasing sperm over the inhalant siphon of the mussel. Embryos hatch within 2 days but develop inside the mussel for further 3 to 6 weeks. Embryos are adapted to the low oxygen environment in the mussel's gill chamber. Both males and females discriminate among mussels in relation to their quality as host for developing embryos. On the other hand, mussels used for oviposition have larvae that obligate ectoparasites on fish. Here I review current knowledge on the status of the symbiosis, developmental and behavioural adaptations by bitterling and mussel and summarize costs and benefits to both symbionts. Further, I use a recent well‐resolved bitterling phylogeny to emphasize the potential of this model system to study the evolution of this symbiosis, which is a part of the ongoing study.