An alkaline unwinding assay for the detection of DNA damage in aquatic organisms

This paper reports a rapid and facile procedure for obtaining of DNA from small, whole aquatic organisms (fathead minnow, Pimephales promelas) as well as from the liver of another species (bluegill sunfish, Lepomis macrochirus) and the subsequent estimation of its double strandedness. DNA was isolated from various tissues by homogenization in a basic pH solution containing detergent followed by differential extraction using organic solvents. Additional purification was accomplished by molecular sieve chromatography. Under defined alkaline unwinding conditions, the amount of double-stranded and single-stranded DNA was determined using the bisbenzimidazole dye Hoechst 33258. The technique was used to detect the occurrence of strand breaks in DNA of fathead minnows chemically exposed to benzo[a]pyrene.     

Effects of toxicants on developing oocytes and embryos of the blue crab, Callinectes sapidus

Oocytes (90 μm in diameter) were isolated from ovaries of 14-day post-molt adult female crabs and maintained in culture media. The addition of cadmium (20 μg/ liter) or tributyltin (2 μg/liter) to developing oocytes resulted in decreases in the growth of oocytes as measured by protein and lipovitellin accumulation relative to controls. Crab embryos isolated from yellow ‘sponge’ of female blue crabs were maintained in seawater until hatching (6–8 days). Toxicants tested included cadmium, copper, tributyltin and endosulfan. Some of the processes or events which were followed included water uptake, lipovitellin utilization rate, formation of appendages, formation of a heart, formation of eyespots and hatching to zoea stage. The primary effects from addition of toxicants were deformed eyespots and reduced hatching success. The hatching EC₅₀ (concentration at which 50% of embryos failed to hatch) for copper, cadmium, tributyltin and endosulfan were 3.1, 0.25, 0.047 and 450 μg/liter, respectively. Crab embryos appear to be suitable for testing of the effects of a variety of toxicants with advantages including low cost, reproducibility, low variability and sensitivity.     

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.     

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.     

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.     

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.     

Response of the mixed-function oxidase system to toxicant dose, food and acclimation temperature in the bluegill sunfish

The degree of induction of hepatic mixed function oxidase (MFO) enzymes in fish is modulated by environmental conditions. This study was designed to investigate the influence of water temperature, presence or absence of food, and exposure to benzo(a]pyrene (BaP) on the inductive response of bluegill sunfish (Lepomis macrochirus). The results show an increase in 7-ethoxyresorufin O-deethylase (EROD) activity with an increase in acclimation temperature and dose. This activity appears to be associated with a very small fraction of the total cytochrome P-450 induced. Major changes were observed in the 53- and 57-kilodaltons (kDa) electrophoretic bands.     

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.     

Interactions of cadmium with sulfhydryl-containing compounds in striped mullet (mugil cephalus L.)

Interactions between cadmium and sulfhydryl-containing compounds in striped mullet, Mugil cephalus, were examined. Exposure to 10 mg Cd/liter for up to 6 weeks caused an increase in the acid-soluble thiol (AST) content of the liver. Although the AST concentration was elevated at 1·4 weeks, increases in the glutathione and cysteine contents were not observed until after 4 weeks of exposure. Homogenates of livers were fractionated on a Sephadex G-75 column. A homogenate of livers of mullet injected with ¹⁰⁹Cd 2 days prior to sampling had an elution profile for thiols similar to controls. After 3 weeks exposure to 10 mg Cd/liter the 9400 MW thiol peak increased 3·6-fold and the peak with the same retention volume as glutathione increased 4·3-fold. Greater than 70% of the thiols in these two peaks were acid-soluble. In both experiments, almost all the cadmium was associated with the 9400 MW peak. The ratio in the 9400 MW peak declined from 14·1 (controls) to 3·2 after 3 weeks exposure. No cadmium was associated with glutathione in the bile.     

Measurement of alkaline and earthy ions in fish otolith and sea water using a high performance ion chromatography

Fish growth and the relation between growth and environmental conditions offer a good opportunity for measuring alkaline and earthy ions in fish otoliths.The analytical method must involve high sensitivity when attempting to discriminate between fish growth and environmental conditions.The aim of this paper is to propose a chromatographic method, with low detection limits, as a new approach in determining some important micronutrients present in sea water and fish otoliths.The work samples are: coastal, off-shore and sediment waters and fish otoliths (Engraulis encrasicholus, Mullus barbatus, Umbrina cirrhosa, Sciaena umbra, Pagellus erythrinus) in the Adriatic Sea and the Canal of Sicily.The analytical method includes an IONPAC CS12A chromatographic column and a 18 mM methanesulfonic acid eluent.The detection limit readings obtained with this method, for one E. encrasicholus fish otolith, weighing 2.6 mg are equal or inferior to 0.1 μg/L for lithium (Li), 59 μg/L for sodium (Na), 46 μg/L for ammonium (NH₄), 23 μg/L for potassium (K), 13 μg/L for magnesium (Mg), 88 μg/L for manganese (Mn), 2.567 μg/L for calcium (Ca) and 13 μg/L for strontium (Sr).The HPIC method minimizes overlaps such as Na on Li, and NH₄ in seawater and Ca on Mg and Sr in fish otolith. These elements are an essential constituent present in otoliths when describing the relation between growth and environmental conditions.Good separation among analytes is achieved within 16 min.     

Zinc and cadmium metabolism in euglena gracilis: Metal distribution in normal and zinc-deficient cells

The effect of cadmium (5 μ/ml) on the growth of Euglena gracilis is moderated by the addition of supplemental zinc levels above the normal 1 μ/ml level found in the growth medium. Gel chromatography reveals that th majority (ca. 85%) of cytosolic cadmium is localized in a cadmium-binding protein (CdBP). The CdBP appears to elute in a position slightly lower in molecular weight than mammalian metallothionein, contains thiol, and upon ion-exchange chromatography elutes in multiple peaks at ionic strengths greater than mammalian metallothioneins. The remaining cadmium is associated with high molecular weight components. Cytosolic zinc is found primarily (> 80%) in a low molecular weight peak distinct from cadmium, with the remainder as high molecular weight species. As the cells become zinc-deficient and cease proliferation, zinc is lost from the low molecular weight peak while, based on cytosolic protein, the zinc content of the high molecular weight peak remains essentially constant.     

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.     

Influence of cadmium exposure on plasma calcium, vitellogenin and calcitonin in vitellogenic rainbow trout

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre⁻¹ for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.     

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.     

Acute and sublethal toxicity of naphthalene and three methylated derivatives to the estuarine copepod, Eurytemora affinis

The bi-cyclic aromatic hydrocarbon naphthalene (N) and its alkylated derivatives 2-methylnaphthalene (MN), 2,6-dimethylnaphthalene (DMN) and 2,3,5-trimethylnaphthalene (TMN) were used to investigate possible relationships between increased methylation and toxicity during acute high level and chronic low level exposure experiments using the calanoid copepod Eurytemora affinis Poppe. The 24-h LC₅₀ levels determined indicated an approximate twofold increase in toxicity per methyl group addition: N-3798 μg/litre; MN-1499 μg/litre; DMN-852 μg/litre and TMN-316 μg/litre. Low level chronic exposure of E. affinis to the various naphthalenes in sea water concentrations of about 10 μg/litre for the duration of their adult life (maximum twenty-nine days) resulted in significant reductions in length of life, total numbers of nauplii produced and mean brood size. Exposure to all the naphthalenes at this level (10 μg/litre) resulted in reduced rates of egg production which were, on average, about 50% of those of control animals. Results are discussed in relation to previous work on the fate and effect of petroleum hydrocarbons in marine copepods.     

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.     

三丁基锡对文蛤外套膜代谢酶活性的影响

用实验生态学方法，观察浓度为0．1、1、10ng／dm^3的三丁基锡暴露2、8、20d以及恢复7、20d后对文蛤外套膜酸性磷酸酶、碱性磷酸酶以及Na^＋、K^＋-ATP酶活性的影响．结果表明各浓度组的三丁基锡在暴露早期（2d），外套膜中酸性、碱性磷酸酶和Na^＋、K^＋-ATP酶活性与对照组比较没有显著差异，随着暴露时间的延长，高浓度的TBT暴露对AKP表现出激活作用，对ACP和Na^＋、K^＋-ATP酶活性则具有抑制作用．结果显示Na^＋、K^＋-ATP酶可作为有机锡暴露的生物标志物．     

A simplified resorcinol method for direct spectrophotometric determination of nitrate in seawater

A direct, spectrophotometric method has been adapted for quantitative determination of nitrate concentrations in seawater. The method is based on nitration of resorcinol in acidified seawater, resulting in a color product. The absorption spectrum obtained for the reaction product shows a maximum absorption at 505 nm, with a molar absorptivity of 1.7 × 10⁴ L mol^− 1 cm^− 1. This method has a detection limit of 0.5 μM and is linear up to 400 μM for nitrate. The advantage of this method is that all reagents are in aqueous solutions without involving cadmium granules as a heterogeneous reactant, as in conventional methods, and therefore is simple to implement. Application of the resorcinol to seawater analysis demonstrated that the results obtained are in good agreement with the conventional approach involving the reduction of nitrate by cadmium followed by diazotization.     

Reproductive success, xenobiotic contaminants and hepatic mixed-function oxidase (MFO) activity in Platichthys stellatus populations from San Francisco Bay

To identify some specific effects of organic contaminants on fisheries in an urbanized estuary we compared the reproductive success of starry flounder from San Francisco Bay with concentrations of tissue contaminants and hepatic mixed-function oxidase (MFO) activity. We found significantly lower (P < 0·05) sediment concentrations of total identified polynuclear aromatic hydrocarbons (PAHs) in the less urbanized San Pablo Bay (SP) area (Fig. 1) than in the more urbanized central bay (CB) stations (Table 1). For flounder in early gametogenesis (August and September) the SP fish (n = 20) had significantly lower (P < 0·01) liver concentrations of Aroclor 1260 (0·34 ± 0·14 μg/g) than those at the CB stations: Berkeley (BK, n = 20, 1·6 ± 1·6 μg/g); Oakland (OK, n = 16, 2·3 ± 2·8 μg/g); and Alameda (AL, n = 4, 2·2 ± 1 μg/g). A similar pattern existed for DDT concentrations: SP = 0·2 ± 0·16 μg/g; BK = 0·1 ± 0·34 μg/g; OK = 0·4 ± 0·53 μg/g; and AL = 0·4 ± 0·33 μg/g. Total PAHs in livers were as follows: SP = 0·14 μg/g; BK = 2·6 μg/g; OK = 1·4 μg/g; and AL = 14 μg/g. Although gonad index, liver index, and presence of fin rot are inversely related to aryl hydrocarbon hydroxylase (AHH) activity, healthy fish in a similar reproductive state have lower AHH activities in the SP area. For example, in August and September, 1984, mean AHH activities were as follows: SP = 203 ± 89, and CB = 355 ± 200 pmol 3-OH-B[a]P mg microsomal protein min. We found a log-linear relationship for AHH activity and its percent inhibition by 7,8-benzoflavone (10⁻⁴m) and only a few fish from SP showed enhanced AHH activity after addition of 7,8-benzoflavone. This suggests that most of the starry flounder in San Francisco Bay are induced.     

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