Responses of the mussel Mytilus edulis to copper and phenanthrene: Interactive effects

Most investigations of the responses of marine organisms to xenobiotics have concentrated on single contaminants and little is known of possible interactive effects of different classes of xenobiotics. As these latter seldom occur in environmental isolation, it is important to understand any interactions (synergistic or antagonistic) which may occur. This problem has been approached in the mussel Mytilus edulis by exposing estuarine mussels to copper (20 μg litre⁻¹) and phenanthrene (100 μg litre⁻¹) both individually and in combination, and measuring cytochemical subcellular and physiological responses after 3 days exposure and 3 days and 12 days recovery period. Results showed that mussels accumulated both xenobiotics during 3 days exposure. Depuration of copper was complete in 3 days recovery period, while loss of phenanthrene ranged from 30% to 70% of the concentration reached after 3 days exposure. There were no interactive effects on depuration.Both copper and phenanthrene reduced lysosomal hydrolase latency in digestive cells, and copper appeared to have a synergistic effect in preventing recovery of latency of lysosomal N-acetyl-β-hexosaminidase during the recovery period. There was evidence, in the digestive cells, of an antagonistic effect of copper on stimulation of activity of the microsomal respiratory chain (measured as NADPH-neotetrazolium reductase) by phenanthrene. Stimulation of this system by phenanthrene persisted after 12 days recovery period. There was a synergistic interaction of copper and phenanthrene on elevation of oxygen consumption and ammonium excretion. Clearance rates and scope for growth (physiological condition) were depressed by copper but not by phenanthrene after 3 days exposure.These findings are discussed in terms of known effects of copper and phenanthrene and the interactions are considered in terms of environmental effects measurements.     

Metal pollution assessment of the marine environment by determination of metal-binding proteins in Mytilus sp.

Adult mussels (Mytilus galloprovincialis) (shell length 5–7 cm) exposed to elevated concentrations of Cd (0.1–1.3 mg l⁻¹), in a flowing seawater aquarium under laboratory conditions, responded by elevated synthesis of metal-binding proteins in a digestive gland. Response depended on the Cd concentration level as well as on duration of the exposure time. Results obtained show that inducible proteins (isolated by gel-filtration column chromatography and characterized by electrochemical and spectrophotometric methods) should be considered metallothionein-like proteins.The induced metal-binding proteins of mussels in specific organs or tissues are considered to be a potential biological indicator of metal pollution, taking into account specific qualities of the mussel response to different metals. The advantages of the analytical methods applied (gel-chromatography and polarography) and the relevance of the laboratory data when transferred to environmental conditions are discussed. Previously reported results dealing with metal-binding proteins of Mytilus species are also summarized.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

Effect of in vivo cadmium exposure on the respiratory burst of Marine Fish (Limanda limanda L.) phagocytes

Following the in vivo exposure of dab (Limanda limanda L.) to cadmium chloride, kidney phagocytes were collected and their respiratory burst measured in vitro using chemiluminescence. Fish were exposed to mean measured concentrations of 1.3, 2.7 and 5.5 mg Cd litre⁻¹ (as total cadmium ion) for a total of nine weeks, followed by a three week depuration period in clean sea water. Compared with control fish, the respiratory burst of kidney phagocytes from dab sampled after six weeks was significantly reduced in the 2.7 and 5.5 mg Cd litre⁻¹ treatments (Steel's test, p < 0.05). Significant reductions were observed in the respiratory burst of phagocytes from all cadmium exposed fish compared with control fish after nine weeks (Steel's test, p < 0.05). After a further three week depuration period in clean sea water, the respiratory burst of phagocytes from fish previously exposed to 1.3 and 2.7 mg Cd litre⁻¹ were still significantly less than in the control group (Steel's test, p < 0.05). Muscle tissue cadmium concentrations were also analysed, although there was no clear relationship between the muscle total cadmium levels and kidney phagocyte chemiluminescence. The results are discussed with respect to the possible mechanism(s) of cadmium immunotoxicity in dab and recommendations made for future work.     

Significance of metallothioneins and lysosomes in cadmium toxicity and homeostasis in the digestive gland cells of mussels exposed to the metal in presence or absence of phenanthrene

Metallothioneins and lysosomes are known to be involved in cellular detoxication and sequestration of certain metals^1–3 and both have been identified in this role in elimination of copper from marine mussels (Mytilus edulis/galloprovincialis).³ Cadmium (Cd), however, has been shown to persist in the cells of the digestive gland for long periods with only minimal elimination. An experiment was designed to test the effects of Cd on the fragility of lysosomal membranes in the digestive cells as a measure of cellular injury,^4,5 metallothionein content of the digestive gland and cadmium concentration in this organ. Phenanthrene was used also to destabilise lysosomal membranes⁶ in order to test if increased lysosomal fragility interfered with cadmium metabolism and detoxication. The results demonstrated that Cd induced metallothionein synthesis and that elimination of Cd was minimal after 28 days in clear seawater. Lysosomal fragility was initially increased but this effect was soon reversed, even with continued exposure to Cd. The lysosomal destabiliser, phenanthrene, did not appear to affect accumulation of Cd or levels of metallothionein.     

Adaptation of the polychaete Neanthes Arenaceodentata to copper

Neanthes arenaceodentata were exposed to 292, 146, 92 and 56 μg litre⁻¹ Cu (measured) and control seawater after a 27-day pre-exposure to a sublethal concentration of Cu (10, 16 and 28 μg litre⁻¹ and control) to determine if the worms increased their tolerance to Cu after the pre-treatment. The worms pre-exposed to 28 μg litre⁻¹ Cu were significantly more resistant to Cu toxicity than control and 10 and 16 μg litre⁻¹ Cu pre-exposed worms. For example, the time to 50 % mortality at 92 μg litre⁻¹ Cu was 18 and 11 days for worms pre-exposed to 28 μg litre⁻¹ Cu and control conditions, respectively. The net rate of Cu uptake during the toxicity test was lower for worms pre-exposed to 28 μg litre⁻¹ Cu than for the control and 10 and 16 μg litre⁻¹ Cu pre-exposed worms. For example, the net rate of Cu uptake at 292 μg litre⁻¹ Cu by worms pre-exposed to 28 μg litre⁻¹ Cu and control conditions was 42 and 102 μg g⁻¹ day ⁻¹, respectively.     

Effect of oil and dispersants on the growth of Mussels

Mussels (Mytilus edulis L.) were exposed to North Sea crude oil, microencapsulated oil and dispersants, singly and in combination, and growth rates measured at 24–48 h intervals.Exposure to microencapsulated pure oil (2·0–2·1 mg litre⁻¹) and to microencapsulated mixtures of oil (2·2−2·5 mg litre^-1+5 % of the different dispersants (FINASOL OSR 5, COREXIT 9527, DISPOLENE 36 S) gave approximately the same reduction in growth rate (80–90%) within 170h.Oil chemically dispersed with DISPOLENE 36 S and a pure oil mechanically dispersed in water were significantly less toxic. In high concentrations (2 mg litre⁻¹) all disperants are toxic, DISPOLENE 36 S ssignificantly more than the others.Mussels exposed for 170 h to microencapsulated oil and to microencapsulated oil dispersant mixtures recovered to control growth within 300 h in clean seawater, while in those given pure oil-in-water suspension, the recovery was slower.It is concluded that the toxicity of oil is mainly related to size and concentration of oil particles, while the effect of 5% dispersants added is negligible.     

Role of metallothioneins in Cu and Cd accumulation and elimination in the g gill and digestive gland cells of mytilus galloprovincialis lam.

The data presented in this paper demonstrate that exposure of mussels to Cu (40 μg/liter) or Cd (200 μg/liter) for a period of 2 days is sufficient to induce Cu or Cd thioneins in the gills of the metal-exposed animals. The concentrations of Cu and Cd thioneins increase during the period of metal exposure (3–4 weeks), confirming the fact that metallothioneins play a fundamental role in the accumulation of these metals in the tissues examined (gills and digestive gland). Moreover, it has been demonstrated that when metal-loaded mussels are returned to the field for the recovery period, Cu is rapidly eliminated from the gill and digestive gland cells, showing a biological half-life of 9–10 days, whereas Cd is released much more slowly from the tissues, only about 50% of the total metal being lost after 4 months of detoxification. Interestingly enough, during the detoxification period the concentration of the metal bound to thioneins also follows the same pattern as the total metal present in the tissues, in fact the concentration of Cu thioneins decreases near the control level in about 24 days but, in contrast, the Cd-thionein concentration in the cytisol of the gill and digestive gland cells remaains high, decreasing only by about 40% at the end of the 4 month recovery period.The data presented also demonstrate that when Cd-loaded, detoxified mussels are exposed to Cu (100 μg/liter) for 6 h, the metal taken up by the gill cells is able to displace the Zn which is always present in the Cd-thionein fraction, but it does not, however, displace the Cd present. In this case also, period (20 days), whereas the Cd-thionein content decreases slightly. Such data seem to indicate that the metabolic characteristics of thioneins are closely related to the metal bound to them, particularly where the elimination rate of the metals from the cells is concerned.     

Age-related differences in the recovery of lysosomes from stress-induced pathological reactions in marine mussels

This study was conducted to investigate the adaptability of marine mussels Mytilus edulis of increasing age to induced stress and subsequent recovery. Lysosomes, present in large numbers in the molluscan digestive gland, play a major role in intracellular digestion, and the stability of their membranes provides a sensitive biomarker for generalised cell injury which is correlated with the stress response of the whole animal. Lysosomal stability was measured in mussels of three age groups (2–4, 6–8 and ≥ 10 years) during exposure to hypoxia/hyperthermia and, in a separate experiment, to copper (50 ppb: where billion = 10⁹). The lysosomal reactions of all three age groups to both experimental stressors were similar. However, recovery from the induced pathological reactions was most pronounced in the youngest animals and least apparent in the oldest group. These findings indicate that the stress reaction is independent of age but that the potential for recovery of lysosomal integrity is age-related.     

Relationships between macroalgal biomass and nutrient concentrations in a hypertrophic area of the Venice Lagoon

Macroalgae biomass and concentrations of nitrogen, phosphorus and chlorophyll a were determined weekly or biweekly in water and sediments, during the spring-summer of 1985 in a hypertrophic area of the lagoon of Venice. Remarkable biomass production (up to 286 g m⁻² day⁻¹, wet weight), was interrupted during three periods of anoxia, when macroalgal decomposition (rate: up to 1000 g m⁻² day⁻¹) released extraordinary amounts of nutrients. Depending on the macroalgae distribution in the water column, the nutrients released in water varied from 3·3 to 19·1 μg-at litre⁻¹ for total inorganic nitrogen and from 1·8 to 2·7 μg-at litre⁻¹ for reactive phosphorus. Most nutrients, however, accumulated in the surficial sediment (up to 0·640 and to 3·06 mg g⁻¹ for P and N respectively) redoubling the amounts already stored under aerobic conditions, Phytoplankton, systematically below 5 mg m⁻³ as Chl. a, sharply increased up to 100 mg m⁻³ only after the release of nutrients in water by anaerobic macroalgal decomposition. During the algal growth periods, the N:P atomic ratio in water decreased to 0·7, suggesting that nitrogen is a growth-limiting factor. This ratio for surficial sediment was between 6·6 and 13·1, similar to that of macroalgae (8·6–12·0).     

Assessing site-specific effects of TBT contamination with mussel growth rates

During nine field transplant tests in San Diego Bay (1987–1990), juvenile mussels were exposed to mean concentrations of tributyltin (TBT) in ambient seawater ranging from 2 to 530 ng liter⁻¹ for 12 weeks under natural conditions. A total of 79 cages with 18 mussels each were monitored at 18 different sites. Growth and seawater TBT concentrations were measured weekly or on alternate weeks (biweekly). Mean growth rates ranged from 17 to 505 mg week⁻¹ (0·2 to 2·5 mm week⁻¹). Accumulation of TBT in mussel tissues was measured at the end of each 12-week test exposure and ranged from 0·1 to 3·2 μg g⁻¹ TBT wet weight. The frequency of the measurements and the integration of chemical and biological measurements improved the accuracy of the assessment over more traditional approaches. Growth was significantly related to seawater and tissue TBT. The statistical relationships with growth effects were used to estimate chemical effect zones for TBT in San Diego Bay. Site-specific differences were distinguished by additional statistical analyses and consideration of environmental significance.     

Cholinesterase activities in the Antarctic scallop Adamussium colbecki: Tissue expression and effect of ZnCl2 exposure

Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl₂ was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25–29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10⁻⁴–10⁻¹⁰ M (IC₅₀ 10⁻⁶) while in vivo exposure to ZnCl₂ did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.     

Total oxyradical scavenging capacity and cell membrane stability of haemocytes of the Arctic scallop, Chlamys islandicus, following benzo(a)pyrene exposure

Industrial activities, notably oil and gas industries, are expanding in the Arctic. Most of the biomarkers were developed using temperate organisms living at temperatures above 10 °C. Little is known about the biomarker responses of organisms living between −1.88 and 5 °C. Therefore, assessment of the toxicity of chemicals to cold-water adapted species is required. In this study, the Arctic scallop, Chlamys islandicus, was selected as a key species for bio-monitoring because of wide distribution in Arctic waters and its commercial value. Test animals, stored in seawater at 2 °C, were injected with benzo(a)pyrene (diluted in cod liver oil 5 mg ml⁻¹) in the adductor muscle every 24 h for four days giving a final dose of 0, 74 and 90.6 mg kg⁻¹ wet weight for control, low and high dose, respectively. The biomarkers used were total oxyradical scavenging capacity (TOSC) in the digestive gland and cell membrane stability of haemocytes. TOSC values were significantly reduced (ca. 30%) in exposed groups (P<0.05), indicating a depletion in oxyradical molecular scavengers. The antioxidant defences appeared to be overwhelmed by the reactive oxygen species as the plasma membranes of haemocytes were destabilised (P<0.05) probably due to lipid peroxidation. These data indicate that reactive oxygen species (ROS) were produced by Arctic scallops via the metabolisation of benzo(a)pyrene at 2 °C.     

Cadmium in Mytilus spp.: Worldwide survey and relationship between seawater and mussel content

A worldwide literature survey of data on cadmium concentration in the soft tissue of the mussel, Mytilus spp., from 591 stations is presented. These stations are from 13 regions. Geometric means for the regions vary from 0·6 to 3·3 μg g⁻¹ (dry weight) for the Barents Sea and the Northeastern Pacific coast, respectively.The averages of seven of these regions, for which reliable cadmium concentrations in seawater were available, were used to calculate a relationship between cadmium concentrations in seawater and mussel soft tissue. The relationship was highly significant: (Cd) mussel (μg g⁻¹, dry weight) = 0·074 (Cd) water (ng litre⁻¹) + 0·39 (P ≤ 0·0005).This model has been successfully applied in the context of the contamination of the Gironde estuary (France). It can also be used to define a water quality criterion for mussel maturing parks consistent with the quality criterion defined for shellfish for human consumption.     

Some effects of copper on the veliger larvae of the mussel Mytilus edulis and the Scallop Pecten maximus (Mollusca, Bivalvia)

During 1983 and 1985 several batches of laboratory reared veliger larvae of Mytilus edulis and Pecten maximum here subjected to a rank of concentrations of added copper (CuCl₂) over a 15-day period. M. edulis larvae were less sensitive, measured both as mortality (15-day LC₅₀) of 400 μglitre⁻¹) and reduced growth, than P. maximus larvae (15-day LC₅₀ of 85μg litre⁻¹). Both species appeared less sensitive to Cu than other bivallve larvae previously studied. Veliger larvae of M. edulis are from 7 to 10 times more tolerant of Cu than juveniles or adults and this unexpected finding is discussed in relation to the recent literatures on Cu toxicity and accumulation in mussels.     

Effects of mercury on the prosobranch mollusc Crepidula fornicata: Acute lethal toxicity and effects on growth and reproduction of chronic exposure

Exposure to a mercury-equilibrated algal suspension containing 0·25, 0·42 and 1 μg Hg litre⁻¹ as mercuric chloride in solution reduced the growth and condition of pairs of adults of the slipper limpet Crepidula fornicata in a 16-week period. Reproduction rates and larval survival to settlement were also reduced over the first three spawnings when the exposed pairs reached sexual maturity. The adult and larval 96-h LC₅₀s were 330 and 60 μg Hg litre⁻¹, respectively, as mercuric chloride in solution, indicating that a ‘safety factor’ of 10⁻¹ needs to be applied to adult data to protect the most sensitive stage in the life cycle. However, the chronic exposure of the maturing adults showed that levels of inorganic mercury below the ‘safe’ concentration derived from the adult 96 h LC₅₀ affected growth and reproductive success.     

Influence of chronic exposure to silver and mercury in the field on the bioaccumulation potential of the bivalve Macoma balthica

In order to assess the adaptation to metals previously observed in the bioindicator organism, Macoma balthica, subjected to chronic contamination by silver and mercury in the French Loire estuary, the bioaccumulation potential of individual organisms originating from the contaminated Loire estuary and a relatively uncontaminated control estuary (Somme) was evaluated using both radiotracers and stable isotopes of Ag (80 μg Ag litre⁻¹) and Hg (100 μg Hg litre⁻¹). Clams from the contaminated estuary were more sensitive to Ag (LT₅₀ = 9d) than those originating from the Somme estuary (LT₅₀ > 15d), even though the former bioaccumulated Ag to a significantly lower degree. This is attributed to a consequence of the chronic stress induced by Ag while clams were living in their natural environment. Therefore, past history of trace metal contamination should be considered when evaluating the susceptibility of M. balthica to heavy metal exposure. Lower uptake rates obtained for Hg (during the initial uptake phase only) and for Ag in clams from the polluted estuary suggest the presence of an adaptive trait for survival in contaminated areas. However, the lower degree of bioconcentration observed for Ag was not sufficiently low to reduce the sensitivity of the organisms to Ag and allow them to resist the toxic stress. Clams that survived Ag or Hg exposure at LT₅₀ did not protect themselves against metal toxicity by accumulating a significantly lesser amount of these metals than clams which did not survive metal stress. The results suggest that the bioaccumulation potential of each individual was not a factor which can explain the survival ability of M. balthica exposed to chronic Ag and Hg contamination in estuaries. In this case, cellular, biochemical and genetic levels of adaptation are presumed to be of greater importance.     

Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Butyltins in marine waters of The Netherlands in 1988 and 1989; Concentrations and effects

Specific effects of tributyltin (TBT) on Crassostrea gigas—valve thickening, and Nucella lapillus—imposex, were measured on local populations, relatively clean unaffected species from England were transferred to the Netherlands and exposed during six weeks to ambient TBT concentrations. Near marinas 50% of the exposed species were sterile after six weeks. In general, no dissolved butyltins were detected in the Rhine and Scheldt estuaries. In 1988 TBT concentrations in marinas ranged from 120 to 4000 ng litre⁻¹. In sediments (fraction <60 μm) and suspended particulate matter TBT concentrations reached up to 1200 ng g⁻¹. TBT concentrations in mussel tissue ranged from <1 to 2300 ng g⁻¹ based on a dry weight. In 1989 concentrations of dissolved TBT ranged from <0·1 to 7200 ng litre⁻¹. In 1989 a seasonal study in the marina of Colijnsplaat showed that dissolved butyltins increased from April to the end of May due to the launching of freshly painted boats, and decreased afterwards.