Effect of reproductive toxicants on lipovitellin in female blue crabs, Callinectes sapidus

Changes in the concentration of lipovitellin, a lipoprotein that is the major protein in mature oocytes of crustaceans, were determined during ovarian development in female blue crabs, Callinectes sapidus. To test the effects of reproductive toxins on vitellogenesis, crabs were fed food containing cadmium or injected with the juvenile hormone antagonist, precocene II. Monoclonal antibodies prepared against one of the lipovitellin peptides (peptide A—107 kDa) were used in an enzyme linked immunosorbent assay to determine lipovitellin concentrations. Recently molted female crabs fed food containing cadmium (0.02 mg/g) for 18 days had oocytes with the same lipovitellin concentration as controls, but after 24 days the oocytes in cadmium treated crabs had significantly lower lipoprotein contents than controls (30 ± 4 ng/oocyte—cadmium treated; 45 ± 4 ng/oocyte—control). Precocene II-treated crabs had significantiy lower lipovitellin concentrations after 18 and 24 days. Diameters of oocytes were the same in both treated and control crabs. The results suggest that some pollutants may affect reproduction by acting during secondary vitellogenesis i.e., period when there is a large increase in lipovitellin synthesis. Since lipovitellins serve many of the nutritional needs of recently emerged crab embryos, poor larval survival may occur as a result of lower lipovitellin content.     

Effects of long-term exposure to silver or copper on growth, bioaccumulation and histopathology in the blue mussel Mytilus edulis

The purpose of this study was to determine the long-term accumulation of either silver or copper from low concentrations in seawater by blue mussels, Mytilus edulis. Mussels raised from eggs in the laboratory to the age of 2·5 months (approximately 4·5 mm in length) were continuously exposed to 0, 1, 5 and 10μg/liter of either silver (nitrate) or copper (chloride) and sampled at 12, 18 and 21 months for growth studies, measurements of metal accumulation and histopathological examination.Whole-body soft tissues were analyzed for the presence of both silver and copper, as background levels of copper in the incoming seawater averaged 2–4 μg/liter. Mussels exposed to silver had accumulated significant amounts of silver only at the highest test concentration (10 μg/liter Ag) after 12 months, but at 18 and 21 months significant levels were accumulated at all three test concentrations. Mussels exposed to copper accumulated significant amounts of copper at 5 and 10 μg/liter Cu after all three sampling periods, but not at 1μg/liter. Silver-exposed animals also accumulated significantly greater amounts of copper than control animals.In a comparative study, field-collected juvenile mussels (approximately 16·1 mm in shell length) and adult mussels (approximately 53·4 mm in shell length) were exposed for 12 months to 0, 5, 25 and 50 μg/liter silver only and subsequently sampled for metal-accumulation analyses and growth measurements. Juvenile mussels accumulated significant amounts of silver at all test concentrations, with the exception of mussels exposed to 5 μg/liter Ag for 6 months. Copper accumulation in the silver-exposed juveniles was significant only at 50 μg/liter Ag after 6 months, but at all test concentrations after 12 months. Adult mussels exposed to silver accumulated significant levels of both silver and copper, but at somewhat lower levels than juveniles.In the growth study, silver had no effect on laboratory reared mussels at the highest concentration of 10 μg/liter tested, whereas copper at 10 μg/liter did appear to affect growth as early as 4 months after the start of experimental exposure. Field-collected juvenile mussels did show inhibition in growth after 6 months' exposure to 25 and 50 μg/liter Ag, with some growth occurring after 12 months. Adults also showed inhibition in growth after 6 months but not at 12 months.Histopathological examination of mussels exposed to either 5 or 10 μg/liter of copper for 18 months showed changes in the digestive diverticula, gastrointestinal tract, reproductive tract and muscle tissues. These changes were more noticeable in mussels exposed to 5 μg/liter Cu than in those exposed to 10 μg/liter. Mussels exposed to silver for 21 months showed yellowish to black particulate deposition in the basement membrane and connective tissue of the various organs and tissues. Silver deposition increased with increasing test concentration.     

Response of hematological and biochemical parameters to heavy metal exposure: Implications in environmental monitoring

The use of selected hematological and biochemical parameters as indicators of metal exposure in aquatic organisms was evaluated. The hematological and biochemical parameters examined include glucose, hematocrit and aminotransferase levels in golden shiners exposed to cadmium. Cadmium exposure produced significant alterations in the levels of glucose, aspartate aminotransferase and alanine aminotransferase; however, hematocrit was not altered by exposure to cadmium. In addition, the comparative activity of Na/K adenosine triphosphatase (ATPase) was evaluated in the fathead minnow, golden shiner and bluegill sunfish. Basal Na/K ATPase activity was lowest in golden shiner (1·01 μmol Pi/mg protein/h) and highest in bluegill sunfish (1·45 μmol Pi/mg protein/h). While a stimulation of Na/K ATPase activity was observed at an exposure concentration of 1 μg Cd/liter in the fathead minnow and bluegill sunfish, inhibition of enzymatic activity was observed at higher exposure concentrations (10 and 100 μg Cd/liter). Gill Na/K ATPase activity in golden shiner was not significantly influenced by cadmium exposure. The observed insensitivity of ATPase in shiner may, in part, be related to higher background and accumulated concentrations of cadmium in gill tissue.     

Metabolism of tributyltin oxide by crabs, oysters and fish

Bis(tributyltin) oxide (TBTO) is widely used as an antifouling agent in various antifouling paints. Thus, some marinas have TBTO concentrations as high as 2 μg/liter.¹ These concentrations can be toxic to zooplankton.² The objectives of the present study were to determine the ability of a number of marine animals, including crabs, oysters and fish, to metabolize TBTO. Earlier work showed that extracts of rat liver were able to metabolize TBTO to a variety of metabolites, e.g. β-hydroxybutyldibutylin.³ The role of the cytochrome P-450 dependent mixed-function oxygenase system in oxidizing TBTO in marine animals was also of interest. Both in vivo (uptake of ¹⁴C-TBTO from food or water) and in vitro studies demonstrated that all the animals were able to metabolize TBTO. The oysters metabolized TBTO at a much slower rate than the other animals. The mixed-function oxygenase system from hepatic tissues of the various animals was able to metabolize TBTO by forming a number of hydroxylated metabolites.     

Long-term effects of tributyltin compounds on the baltic amphipod, Gammarus oceanicus

Amphipod larvae, Gammarus oceanicus, were exposed for 8 weeks to bis(tributyltin) oxide (TBTO) or tributyltin fluoride (TBTF), or for 5 weeks to leachates from tributyltin-containing antibiofouling paints. No larvae survived 8 weeks' exposure to 3 μg/litre TBTO or TBTF. Differential larval survival occurred during exposure to 0·3 gmg/litre of these same compounds. Paint leachates were shown to cause similar toxicity responses at comparable aqueous tributyltin concentrations. An examination of sublethal responses showed slight decreases in growth for larvae exposed to TBTO and to paint leachates, although responses at tributyltin levels below 1 μg/litre were not marked. Whole-animal oxygen consumption rates were not affected by low tributyltin exposure levels. While these studies demonstrate low level toxicity of tributyltins, effects on oxygen consumption and growth do not indicate a mode of action for these compounds.     

Cadmium-binding proteins in the blue crab, callinectes sapidus: laboratory-field comparison

The blue crab, Callinectes sapidus, is distributed along the east coast of the United States from Cape Cod, Mass., through the Gulf of Mexico, including both relatively unpolluted coastal areas and estuaries contaminated with trace metals. Cadmium is of particular concern because it is concentrated in the digestive glands of blue crabs and can be passed on to consumer organisms. Tissue concentrations and partitioning of trace metals from crabs exposed in the laboratory to 10 ppb dissolved cadmium for 40 days were compared with blue crabs collected from two locations on the Hudson River, NY, Foundry Cove and Haverstraw Bay, Foundry Cove and Haverstraw Bay, both of which have elevated trace metal levels relative to estuarine areas near Beaufort, NC. Crab digestive glands, gills and muscle were removed and analyzed for total cadmium, copper, zinc and nickel concentrations using acid digestion and atomic absorption spectrophotometry, and metal-binding (metallothionein-like) proteins were determined by gel-filtration chromatography. In crabs exposed to cadmium in the laboratory, the cytosolic partitioning was similar to previous investigations at our laboratory where higher levels of cadmium (100 ppb) and shorter exposure times (14 days) were used. The similarity in cadmium partitioning from these two separate experiments indicates dose independence. In crabs from polluted environments the digestive glands contained the highest concentrations of trace metals. Chromatograms of the cytosol from the digestive glands and gills from both field and laboratory exposed crabs showed similar distributions of cadmium, copper and zinc. The gills of both field and laboratory-exposed crabs had metal-binding proteins that contained mostly cadmium, and the digestive glands had metallothionein-like proteins that contained cadmium, copper and zinc. Estimated molecular weights for these proteins were similar to the metallothioneins found in other crustaceans and mammals.     

Effects of tributyltin (TBT) exposure on the reproduction and embryonic development of the bivalve Scrobicularia plana

Field and laboratory work was carried out during the summers of 1990 and 1991 on bivalves Scrobicularia plana from sites moderately-affected and relatively-unaffected by tributyltin (TBT) contamination (i.e. a mean concentration in clam tissues of 0.4 and 0.02 μg Sn g⁻¹ dry wt, respectively). Standard cultures after artificial fertilizations with broodstock of either site did not result in dissimilar survival of embryos nor in larvae of different quality at the time of hatching. Static 48 h toxicity tests on S. plana embryos showed that an EC₅₀ of less than the nominal 250 ng Sn litre⁻¹ (a range of 178–198 ng Sn litre⁻¹, as analysed) can be set for TBT independently of the origin of broodstock. The results are discussed in relation to the reported disappearance of S. plana throughout northern Europe and the simultaneous presence of toxic levels of butyltin. It is concluded that TBT has probably reduced the recruitment into some UK clam populations by preventing the successful development of a significant proportion of their embryos.     

Acute and chronic toxicity of the rice herbicides thiobencarb and molinate to opossum shrimp (Neomysis mercedis)

Neomysis mercedis were exposed to the rice herbicides molinate and thiobencarb, which have been detected in the Sacramento-San Joaquin Delta. Continuous-flow acute and chronic toxicity studies were performed. For thiobencarb, the 4-, 7- and 14-day LC_sos were 304, 214 and 91 μg/liter, respectively. For molinate, these values were 9910, 2530, and 820 μg/liter, respectively. Mortality stabilized after 18 and 28 days exposure, respectively, for thiobencarb and molinate. The incipient lethal concentrations were 53 and 230 μg/liter, respectively. Joint toxicity studies indicated additive toxicity for the two chemicals. Chronic no observable effects concentrations (NOECs) were estimated at 3·2 and 25·6 μg/liter for thiobencarb and molinate, with measurable effects occurring at 6·2 and 45·2 μg/liter, respectively. Environmental monitoring data suggests that concentrations of these chemicals in the Delta prior to 1985 may have exceeded the threshold for chronic toxicity, under conditions of joint exposure. However, due to continued improvements in pesticide management practices, environmental concentrations decreased to approximately 30 percent of the chronic threshold for joint toxicity by 1988. Baseline data on growth and reproduction relevant to toxicity tests are also presented for this species.     

Cadmium, copper, lead and zinc in three species of planktonic crustaceans from the east coast of Corsica

Results of trace metal analyses performed on two species of Euphausiacea, Meganyctiphanes norvegica and Stylocheiron longicorne, and one species of Decapoda, Sergestes arcticus, collected off the east coast of Corsica, are reported. Analyses were carried out by atomic absorption spectrophotometry and by differential pulse anodic stripping voltammetry.S. arcticus contained lower concentrations of phosphorus (which was also analysed as a biological indicator), cadmium (0.33 μg g⁻¹), copper (17.7 μg g⁻¹), lead (2.13 μg g⁻¹) and zinc (51 μg g⁻¹) than the two Euphausiacea (0.50 μg Cd g⁻¹, 25.4 μg Cu g⁻¹, 4.03 μg Pb g⁻¹ and 59 μg Zn g⁻¹). Moreover, manganese concentrations were low in all the samples.When the results presented here are compared with previous results on phytoplankton and mesozooplankton, there appears to be no trend of trace metal enrichment from phytoplankton to the Decapoda.     

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     

Toxicity of sea-surface microlayer: Effects of hexane extract on baltic herring (Clupea harengus) and atlantic cod (Gadus morhua) embryos

This study was designed to determine whether contaminated sea-surface microlayer was toxic to marine fish embryos in its ntaive form and as a hexane extract. Developing embryos of Atlantic cod (Gadus morhua) and Baltic herring (Clupea harengus) were exposed to hexane extracts of sea-surface microlayer collected from five locations in the North Sea and Baltic Sea. Extracts from two of these locations produced significant embryos mortality as well as severe deformities in live hatched larvae. A control sample of bulk water collected from 20 cm under the surface and extracted in the same way produced no significant mortality or deformities. Significaant changes in timing of hatching were also observed in those samples which produced embryo toxic effects. A comparison of these data with those obtained from code embryos exposed to unextracted microlayer showed a similar biological effect with both unextracted samples and hexane extracts.Chemical analyses revealed the greatest biological effect in samples with petroleum hydrocarbon concentrations between 180 and > 200 μg liter⁻¹. The bulk water control had 1 μg liter⁻¹ while the three samples that showed no biological activity had 3 to 8 μg liter⁻¹ petroleum hydrocarbons. Phthalic acid esters were detected in four samples and chlorinated hydrocarbons in one, but could not be positively correlated with any of the toxic responses. No other chemical contaminants were detected in the five samples.The data presented here show that some sites contain sea-surface microlayer which can be toxic to marine fish embryos: that Baltic herring and Atlantic cod embryos respond similarly to the toxic effects of contaminated microlayer: and that unextracted microlayer and hexane extract of microlayer produce essentially the same toxic effect(s) if only organic contaminants are considered.     

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.     

The relationship between the copper complexing capacity of sea water and copper toxicity in shrimp zoeae

Mortality of larval coon-stripe shrimp, Pandalus danae, was related to labile copper and the copper complexing capacity of sea water, as measured by differential pulse Anodic Stripping Voltammetry (ASV). In nominal treatments of control, 5 and 10μg/litre Cu which contained less than 1·0μg/litre ASV labile Cu, zoeal development was usually complete. In treatments of 20 and 50μg/litre Cu which contained more than 7·0μg/litre ASV labile Cu, most larvae died while in the first zoeal stage. ASV labile Cu was related to the copper complexing capacity of sea water. Copper toxicity at less than 1·0 μg/litre ASV labile Cu is indicated by moulting delay and apparent Cu accumulation in zoea of the 5 and 10μg/litre Cu nominal treatments.     

Acclimation to cadmium by Fundulus heteroclitus as demonstrated by fin regeneration and cadmium accumulation

There is a body of literature indicating that pre-exposure to low levels of metals may increase tolerance during subsequent exposure.¹ We have previously demonstrated that exposure to cadmium at 100 to 500 μg/liter will retard the rate of fin regeneration in the killifish Fundulus heteroclitus.² These experiments were designed to investigate the effects of pre-exposure to cadmium, using fin regeneration as the parameter of effect. The results indicate that pretreatment allows this species to regenerate faster during cadmium treatment as compared to non-pretreated fish, and that the relationship between the pretreatment and treatment concentrations is relevant to pretreatment effects.     

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.     

Gonadal development in Japanese medaka (Oryzias latipes) exposed to 17 β-estradiol

The purpose of this research was to evaluate the Japanese medaka (Oryzias latipes) as an ecotoxicological model for the rapid evaluation of environmental estrogens. A novel short-term (48-h) exposure to 17 β-estradiol is proposed in development of a positive control for disruption of gonadal development. Recently hatched medaka fry (30 fry per dose group) with undifferentiated gonads were exposed to 4.0, 29.4, and 115.6 μg/liter of 17 β-estradiol (acetone carrier) for 48 h in a water bath at 25 °C. The fry were then grown-out in spring water for 2 weeks, killed, and processed for histological evaluation. High lethality was encountered during the grow-out period in the 115.6 μg/liter dose group. Fry in the spring water and acetone (carrier) control groups developed into females or males. Fry exposed to 17 β-estradiol developed primarily into females or had testis-ova.     

Movement patterns and trajectories of ovigerous blue crabs Callinectes sapidus during the spawning migration

Female blue crabs (Callinectes sapidus Rathbun) migrate from low salinity estuarine regions to high salinity regions near the ocean to release larvae. During this migration, ovigerous females use ebb-tide transport, a vertical migratory behavior in which they ascend into the water column during ebb tides, to move seaward to larval release areas. In order to determine the relationship of ebb-tide vertical migrations to local currents and the influence of these vertical migrations on the horizontal transport of blue crabs in the estuary, ovigerous females with mature embryos (1–3 days from hatching) were tracked near Beaufort Inlet, North Carolina (USA), in July and August 2001 and 2002. Crabs were tagged and tracked using ultrasonic telemetry, and currents near the crabs were measured simultaneously with a shipboard acoustic Doppler current profiler.During the two seasons, eight crabs were successfully tracked for periods ranging from 3.9–37.0 h and for distances ranging from 1.9–10.6 km. All crabs migrated seaward during the tracking periods. Crabs moved episodically during all tidal phases with periods of movement on the order of minutes to an hour. They moved with local currents in terms of both speed and direction during ebb tides, consistent with ebb-tide transport, and moved down-estuary (seaward) in opposition to local currents during flood tides. The percentage of time that crabs were active was higher during night ebb tides than during day ebb tides or flood tides and increased with increasing ebb-tide current speed. Mean migratory speeds were 0.11, 0.04, 0.08 and 0.02 m s⁻¹ during night ebb, night flood, day ebb and day flood tides, respectively, and net migratory speeds were on the order of 5 km day⁻¹. Due to the episodic nature of the crabs' movements, the total distances that crabs traveled during ebb tides ranged from 10–40% of the distances that passive particles could have traveled under the same conditions.     

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.     

Antagonistic effects of xenobiotics on steroid-induced final maturation of Atlantic croaker oocytes in vitro

The antagonistic effects of Kepone (chlordecone) and o,p'-DDD on maturation-inducing steroid (17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S) induced final maturation of Atlantic croaker (Micropogonias undulatus). Oocytes were investigated in an in vitro germinal vesicle breakdown (GVBD) bioassay. Exposure to Kepone or o,p'-DDD (100 nM-100 μM) prevented the majority of the oocytes from completing GVBD in response to 20β-S; many of them were arrested at the lipid coalescence or germinal vesicle migration stages when exposed to high xenobiotic concentrations (100 μM). In addition, clearing of the ooplasm, oil droplet formation and hydration were incomplete in xenobiotic-exposed oocytes that underwent GVBD. The xenobiotics inhibited GVBD in a concentration-dependent manner. One minute exposure to either of the xenobiotics was sufficient to block GVBD. Washing the follicle-enclosed oocytes after xenobiotic exposure restored their ability to undergo GVBD in response to 20β-S. These results suggest that o,p'-DDD and Kepone exert antagonistic effects on 20β-S action, possibly by competing for the ovarian 20β-S membrane receptor.     

Behavioral effects in mummichog larvae (Fundulus heteroclitus) following embryonic exposure to methylmercury

Embryos of the mummichog, Fundulus heteroclitus, were exposed to concentrations of methylmercury (meHg) below those that can cause morphological abnormalities. After hatching, larvae were kept in clean water and tested periodically for ability to capture prey (Artemia salina) or for their swimming performance and predator avoidance. Embryonic exposure to 10 μg/liter meHg resulted in reduced prey capture ability by early larvae, measured as total Artemia caught in 1 and 5 min. However, older larvae recovered from the behavioral deficits, perhaps owing to a compensation for early CNS insult. Fish from a chronically polluted environment (Piles Creek (PC), a tributary of the Arthur Kill), previously shown to have high tolerance to the teratogenic effects of higher concentrations of meHg, proved to be less susceptible to these behavioral effects than fish from a relatively uncontaminated site (EH). Also, the PC control larvae captured prey far more effectively than the EH larvae, although they made more miscues. When tested for swimming performance after embryonic exposure to 10 μg/liter, EH larvae had increased performance when compared to controls. This effect was also transitory. The improved swimming performance did not correspond to improved ability to avoid predation by grass shrimp (Palaemonetes pugio). The increased swimming performance may correspond to greater total activity, making them more likely to attract the predator's attention and resulting in increased capture. Thus, negative, though temporary, effects were observed in both prey capture and predator avoidance after embryonic exposure to meHg.