Effects of linear alkylbenzene sulphonate (LAS) and cadmium in the digestive gland of mussel, Mytilus sp.

A laboratory study was carried out exposing mussels (Mytilus sp.) to linear alkylbenzene sulphonate (LAS) (6 mg litre⁻¹), Cd (0.05 mg litre⁻¹) and LAS plus Cd at the same concentrations. The aim was to assess the use of several histopathological and biochemical indices as potential biomarkers of the impact of these xenobiotics in the digestive gland of molluscs. Treated mussels actively accumulated Cd in the digestive gland compared with controls (p 0.01), the highest levels occurring after 30 days of exposure in the group treated with Cd plus LAS. Among several histological alterations screened in digestive gland tissues the thickness of digestive tubules in Cd treated animals decreased more markedly (p 0.01) than in LAS exposed mussels. As for biochemical parameters, the investigated antioxidant enzyme activities, superoxide dismutase (SOD), catalase, DT-diaphorase and glutathione peroxidase (GPX) did not show any significant induction due to these xenobiotics. However, a slight decrease of the antioxidant defences of the animals was detected after 30 days of exposure to contaminants.     

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.     

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.     

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.     

Accumulation and effects of organotin compounds in oysters and mussels: Correlation with serum biochemical and cytological factors and tissue burdens

Oysters and mussels exposed to a concentration of 0·7 ppb (μg/liter) tributyltin from painted panels in flowing seawater accumulated tin in the digestive glands to comparable levels. The mussels experienced approximately 50% mortality during the 60-day test period, but the oysters suffered virtually no deaths. There was no evidence from either bivalve of elevated numbers of hemocytes during the test period and no evidence for cellular disruption as detected by increased levels of serum lysosomal hydrolases. Serum protein of exposed mussels relative to controls increased with time of exposure to the toxicant, while oyster serum protein, normally 10 x higher than in mussels, did not. No evidence was found for elevated stress proteins (heat shock proteins) or metallothioneins in the serum hemocytes of either bivalve. Responses by these animals to fatal or near fatal doses of TBT were thus very different from responses to copper that we have reported elsewhere.^1,2     

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.     

The combined effect of copper and hydrocarbons on the length growth of Mytilus edulis

The combined effect of copper and North Sea crude oil is tested on juvenile Mytilus edulis. Dissolved CuSO₄ x 5H₂O is added to the seawater in concentrations of 3 and 6 μg Cu litre^-1. Crude oil, microencapsulated in a gelatine/acacia structure, is added in concentrations of 1·5 and 3·0 mg oil litre^-1. The shell length growth is measured every 24 h with laser diffraction. Linear effects were observed for single components within 24 h of exposure and significant antagonistic interaction appeared within 48 h. Reduced growth rate coincided with reduced filtering and defaecation, and reduced byssus production.     

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.     

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.     

Lysosomal reaction to xenobiotics in mussel hemocytes using BODIPY-FL-verapamil

Many cellular and sub-cellular biomarkers associated with mussel (Mytilus edulis) digestive gland and kidney have been characterised. The lysosomal compartment of these tissues have been recognised as being particularly sensitive, exhibiting pollutant induced responses which could be potentially used as a ‘biomarker’. However, relatively few studies have investigated the lysosomal response within molluscan hemocytes. This study was conducted to test whether lysosomal reactions, in live hemocytes isolated from mussels, can be used as a biomarker of pollutant exposure and deleterious effect. Lysosomal responses to a number of hydrocarbons, including anthracene and phenanthrene, and to the amphiphilic heterocylic chemical, chlorpromazine, were examined. The supravital dye neutral red (NR) was used to examine lysosomal membrane fragility, following xenobiotic exposure. NR was also used to verify the lysosomal compartment as the reported accumulation site of a new molecular probe, BODIPY-FL-verapamil (BFLV). The use of BFLV, with confocal laser microscopy and image analysis enabled visualisation and quantification of lysosomal distribution and perturbation. BFLV showed that exposure of molluscan hemocytes to xenobiotics (20 ppb–10 ppm) induced the formation of pathologically enlarged lysosomes. The internal trafficking of lysosomes was shown to be severely compromised after exposure to chlorpromazine. Exposed molluscan hemocytes exhibited significantly reduced lysosomal retention times, for neutral red. Preliminary data is presented demonstrating the opportunity for these non-destructive biomarker techniques to detect pollution gradients in situ.     

Recovery of Mytilus edulis L. from chronic oil exposure

Previous laboratory studies¹ have shown that physiological and cellular processes of Mytilus edulis are affected by exposure to low and environmentally realistic concentrations of oil. However, there is little information concerning the rate of recovery from oil exposure and the extent to which physiological recovery may be related to the depuration of hydrocarbons from the tissues. The present study has shown a marked reduction in the feeding rate and scope for growth of mussels exposed to two concentrations of diesel oil (30 and 130 μg/litre) for 8 months. During recovery from oil exposure the depuration of hydrocarbons from the tissues was concomitant with the recovery of physiological performance. Mussels exposed to high oil concentrations (‘high-oil’ mussels) were found to recover more rapidly than those exposed to low oil concentrations (‘low-oil’ mussels), both in terms of depuration and scope for growth, and there was evidence of ‘catch-up’ growth. Recovery of both low- and high-oil mussels was complete after approximately 55 days.     

Ammonium input to the sea via large sewage outfalls—part 1: Tracing sewage in Southern California waters

Ammonium concentrations were found to be elevated near Southern California sewage outfalls; concentrations exceeding 3 μg-atom litre⁻¹ were measured 5 km from the discharge area. In stratified water, high values were found below 15 m, but in well-mixed water, high levels were detected at the surface. Subsurface high concentrations were associated with turbid layers, coliform bacteria and reduced oxygen levels. The distribution of ammonium correlated well with measured subsurface currents. The maximum concentration at the Whites Point outfall was 155 μg-atom litre⁻¹ at 27 m, about 2 km from the diffuser. Measurements of ammonium in sewage, compared with that in seawater at Whites Point, suggested that sewage was diluted up to 400-fold. Ammonium may be a useful tracer of the discharge of sewage in seawater.     

Ammonium input to the sea via large sewage outfalls-Part 2: Effects of ammonium on growth and photosynthesis of southern California phytoplankton cultures

Cultures of six marine phytoplankton were grown at ammonium concentrations ranging up to 200 μg-atom NH₄---N litre⁻¹. Only the growth of dinoflagellates, Gymnodinium splendens and Gonyaulax polyedra was inhibited at the two highest concentrations used. In 3-h photosynthetic ¹⁴CO₂ uptake experiments, only Gymnodinium was inhibited at concentrations of NH₄---N greater than 100 μg-atom litre⁻¹. We conclude that the increased ammonium concentrations found near Southern California sewage outfalls would not be inhibiting to phytoplankton in the vicinity of such outfalls.     

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.     

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.     

The effect of low, chronic levels of no. 2 fuel oil on natural phytoplankton assemblages in microcosms: 1. Species composition and seasonal succession

No. 2 fuel oil-sea water dispersions were added to three large scale (13 m³) microcosms twice weekly for a period in 1977 and a 4 month period in 1978. Water column concentration of total hydrocarbons averaged 190 μg litre⁻¹ in 1977 and 93 μg litre⁻¹ in 1978. Several responses of t the phytoplankton community in the oiled microcosms, relative to control microcosms, were similar during both periods of oil addition: total phytoplankton abundance and chlorophyll concentration were elevated, species diversity was higher and diatoms represented a greater proportion of the total p phytoplankton abundance than flagellates during 4 months of oil addition in 1977 and the entire period of oil addition in 1978. Phytoplankton species composition in the oiled microcosms was similar to the source water, Narragansett Bay, but the microcosm assemblages were numerically dominated by populations of the diatom Chaetoceros spp. and nanoflagellates in 1977 and several Chaetoceros spp. in 1978. Reduced predation pressure and altered herbivore feeding behaviour combined with the low level, non-lethal (to the majority of phytoplankton populations), concentrations of No. 2 fuel oil in the water column are postulated as mechanisms that can account for the elevated phytoplankton abundance in the oiled microcosms relative to the controls.     

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).     

Ethoxyresorufin-O-deethylase activity and fixed wavelength fluorescence detection of PAHs metabolites in bile in turbot (Scophthalmus maximus L.) Exposed to a dispersed topped crude oil in a Continuous Flow System

Long term effects of sublethal concentrations of oil on the marine environment have become of general concern. Cytochrome P4501A activity (EROD) in liver and fixed wavelength fluorescence detection of PAHs metabolites (FF) have in this study been used as biomarkers for dispersed oil exposure on a long term period of juvenile turbot (Scophthalmus maximus L.). A Continuous Flow System was used to carry out the study. The fish were continuously exposed to 0.125, 0.5 or 2.0 mg litre⁻¹ dispersed topped crude oil for 6, 15, 24 h, 4 and 21 days followed by a 9 days recovery period in clean seawater. No induction of the cytochrome P4501A was measured. A maximum level in bile metabolites (4- to 5-fold) was recorded after 24 h of exposure revealing thereby a detoxification process, but a decline occurred from day 4 to day 21. This study demonstrated that FF detection of PAHs metabolites in bile could be a more sensitive biomarker than EROD activity in a long term exposure to sublethal concentration of oil.     

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.     

Combined effects of salinity and cadmium chloride upon embryos and larvae of the Japanese oyster, Crassostrea gigas

Following the observation of high concentrations of cadmium in Gironde estuarine waters, the response of embryos and larvae of the Japanese oyster, Crassostrea gigas to this heavy metal at various salinity regimes was studied for 10 days in the laboratory. The experimental design was a 4 x 4 factorial experiment using concentrations of cadmium of 0, 10, 20 and 50 μg litre⁻¹ and salinities of 20, 25, 30 and 35‰. Statistical analysis indicated that the salinity changes have important effects on the embryonic development, survival and growth of C. gigas larvae. No deleterious effect of the toxin at all cadmium concentrations and no interaction between cadmium and salinity were found.