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Archives of Environmental Contamination and Toxicology (v.32, #2)

Metabolism of Metolachlor by the Fungus Cunninghamella elegans by J. V. Pothuluri; F. E. Evans; D. R. Doerge; M. I. Churchwell; C. E. Cerniglia (pp. 117-125).

The metabolism of metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] by the fungus Cunninghamella elegans ATCC 36112 was determined. The six metabolites identified comprised 81% of the total [¹⁴C]-metolachlor metabolized by C. elegans. These metabolites were separated by reversed-phase high-performance liquid chromatography and identified by ¹H nuclear magnetic resonance, UV, and atmospheric pressure chemical ionization (APCI) mass spectral techniques. Metabolites I and II were identified as stereoismers of 2-chloro-N-[2-ethyl-6-hydroxymethylphenyl)]-N-(2-hydroxy-1-methylethyl)acetamide. Metabolites III and IV have been tentatively identified as stereoismers of 2-chloro-N-[2-(1-hydroxyethyl)-6-methylphenyl]-N-(2-methoxy-1-methylethyl)acetamide. Metabolites V and VI were identified as stereoismers of 2-chloro-N-(2-ethyl-6-hydroxy-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide and 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-hydroxy- 1-methylethyl)acetamide, respectively. The fungus Cunninghamella elegans was able to biotransform metolachlor. Multiple site oxidation of metolachlor by C. elegans occurred predominantly by O-demethylation of the N-alkyl side chain and benzylic hydroxylation of the arylalkyl side chain.

Sensitivity of Common Tern (Sterna hirundo) Embryo Hepatocyte Cultures to CYP1A Induction and Porphyrin Accumulation by Halogenated Aromatic Hydrocarbons and Common Tern Egg Extracts by A. Lorenzen; J. L. Shutt; S. W. Kennedy (pp. 126-134).

Experiments were conducted to compare the sensitivity of primary cultures of common tern (Sterna hirundo) and chicken (Gallus domesticus) embryo hepatocytes to cytochrome P4501A (CYP1A) induction and porphyrin accumulation after exposure to halogenated aromatic hydrocarbons (HAHs) or tern egg extracts. The HAHs tested were 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF) 3,3′,4,4′-tetrachlorobiphenyl (PCB 77; IUPAC nomenclature), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169) and Aroclor® 1254 (a commercial mixture of PCBs). Extracts were prepared from common tern eggs collected from three sites in the Great Lakes basin and one reference site on the east coast of Canada. CYP1A induction was assayed as ethoxyresorufin-O-deethylase (EROD) activity and/or immunodetectable CYP1A protein, and total intracellular porphyrin accumulation was measured fluorometrically. Unlike chicken embryo hepatocyte (CEH) cultures, no intracellular porphyrin accumulation was observed after treatment of tern embryo hepatocyte (TEH) cultures with HAHs or egg extracts. TEH cultures were ∼50 to >1600 times less sensitive than CEH cultures to HAH-mediated CYP1A induction. In contrast, TEH cultures were either approximately equally sensitive or only 3.5–15 times less sensitive than CEH cultures to CYP1A induction mediated by tern egg extracts. These data suggest that common tern embryos may be more susceptible to the CYP1A inducing effects mediated by complex mixtures of environmental contaminants than indicated by their response to individual HAHs.

Evaluation of Bioassays to Monitor Surface Microlayer Toxicity in Tropical Marine Waters by D. G. Rumbold; S. C. Snedaker (pp. 135-140).

Bioassays were developed, using embryos of: coral, Montastraea faveolata; graysby, Epinephelus cruentatus; grouper, Epinephelus adscensionis x gruttatus (hybrid); queen conch, Strombus gigas; rock-boring urchin, Echinodermata lucunter; spotted seatrout, Cynoscion nebulosus; variegated urchin, Lytechinus variegatus; winged pearl oyster, Pteria colymbus; and yellowtail snapper, Ocyurus chrysurus. Relative sensitivities and precison of various species-endpoint combinations were evaluated using three reference toxicants: copper, sodium dodecyl sulfate, and Dibrom®. The 24-h P. colymbus embryo test had the best overall sensitivity and exhibited a high degree of precision. However, oyster embryos were difficult to obtain and did not aggregate at the air–water interface. Therefore, the P. colymbus embryo test was deemed unsuitable for use as a bioassay for monitoring sea-surface microlayer (SSML) toxicity. Tests based on normal development of L. variegatus to the early pluteus 3 stage and percent normal-live C. nebulosus larvae at 48 h were relatively sensitive and exhibited good replicability and repeatability. The L. variegatus urchin embryo test was also found to be highly reproducible. The results of this comparative study indicated that L. variegatus and C. nebulosus were suitable surrogates for coral-reef species in toxicity assessments of the SSML.

Metabolites of Arsenic Induced Tetraploids and Mitotic Arrest in Cultured Cells by N. Eguchi; K. Kuroda; G. Endo (pp. 141-145).

The toxic effects of arsenic compounds on cell division were studied, using Chinese hamster V79 cells. Seven arsenic compounds were tested. Inorganic arsenic compounds (arsenite and arsenate), which have been found in drinking water, inhibited cell growth at very low concentrations. Monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and trimethylarsine oxide (TMAO), which are methylated metabolites of inorganic arsenics, were less cytotoxic than the inorganic arsenics themselves. The cytotoxicity of the three methylated metabolites decreased as the number of methyl groups increased. Arsenobetaine (AsBe) and arsenocholine (AsC), which have been found in some marine products, did not show any cytotoxicity. Three methylated metabolites; MMA, DMA and TMAO induced mitotic arrest. Tetraploidy production was observed in cells exposed to DMA or TMAO. Arsenite, arsenate, AsBe and AsC did not induce mitotic arrest or tetraploids. These results suggest that MMA, DMA and TMAO exert some effect on cell division in metaphase and may thereby give some clue as to the carcinogenic mechanism of arsenic.

Use of Stress-Inducible Transgenic Nematodes as Biomarkers of Heavy Metal Pollution in Water Samples from an English River System by M. H. A. Z. Mutwakil; J. P. Reader; D. M. Holdich; P. R. Smithurst; E. P. M. Candido; D. Jones; E. G. Stringham; D. I. de Pomerai (pp. 146-153).

Transgenic strains of the nematode Caenorhabditis elegans, which carry stress-inducible lacZ reporter genes, are measurably stressed by exposure to heavy metals in aqueous solution. This stress response can be quantified, using enzymatic assays for the reporter gene-product (Escherichia coliβ-galactosidase), or estimated approximately by in situ staining for β-galactosidase in exposed worms. Stress responses to heavy metals have been demonstrated both in laboratory tests using Cd²⁺ or Hg²⁺, and also in water samples taken from a metal-polluted river system in southwest England. The River Carnon flows through an area with an ancient mining history, principally for Sn, but also for Cu and other metals; As, Cd, Al, Mn, and Zn, as well as large amounts of Fe, are all present in these ore bodies. Four sites in the Carnon river basin were compared with respect to their macroinvertebrate diversity, physical and chemical characteristics (including the concentrations of As, Cd, Al, Cu, Mn, Zn, and Fe). Transgenic worms were exposed to water samples from these four sites, and also to a 0.33% (v/v) dilution of metal-laden minewater from the principal local mine (Wheal Jane). Transgene expression was induced in all five cases, though markedly less so for the least polluted of the sites (which also supported a richer macroinvertebrate fauna). Two different transgenic strains were tested in this study; strain PC72 (using a homologous hsp16 promoter) is slightly more sensitive to most metal-containing water samples than strain CB4027 (using a heterologous Drosophila hsp70 promoter). Both transgenic strains and two different assay methods gave essentially similar results. These findings demonstrate that transgenic nematodes could provide a rapid and simple assessment of aquatic pollution, in that the transgene response is inducible by mixtures of dissolved metals at concentrations actually encountered in metal-polluted watercourses.

Physiological and Ultrastructural Effects of Cadmium on Wheat (Triticum aestivum L.) Leaves by G. Ouzounidou; M. Moustakas; E. P. Eleftheriou (pp. 154-160).

The effects of a 7-day exposure of 3-day-old wheat plants to increasing Cd concentrations are described, with special attention being given to chloroplast ultrastructural changes, chlorophyll fluorescence responses, chlorophyll and nutrient concentration changes as well as growth changes of the whole plant. The plants treated with 1 mM Cd showed symptoms of heavy metal toxicity. The root, shoot-leaf length and the root, shoot-leaf biomass progressively decreased with increasing Cd in nutrient solution and in 1 mM of Cd an almost complete inhibition of growth was found. Shoot-leaf Cd accumulation increased under Cd-treatments, while a Fe, Mg, Ca, and K decline in the above ground parts was observed. The growth reduction and the inhibition of chlorophyll content and photosynthesis observed in the upper plant parts seemed principally due to indirect Cd effects on the content of essential nutrients. Cadmium treatment was shown to damage the structure of chloroplasts, as manifested by the disturbed shape and the dilation of the thylakoid membranes. These ultrastructural changes suggest that Cd probably induced premature senescence.

The Accumulation of Cadmium by the Yellow Pond Lily, Nuphar variegatum, in Ontario Peatlands by E. S. Thompson; F. R. Pick; L. I. Bendell-Young (pp. 161-165).

Cadmium concentrations of a common macrophyte, the yellow pond lily (Nuphar variegatum) were investigated from peatlands with a range in pH (4.4–6.3), alkalinity (0–181 μeq/L Ca), DOC (5.1–16.8 mg/L), and sediment organic content (20–88%). Cd concentrations in Nuphar ranged from 0.3 to 1.51 μg/g in the leaves and from 0.46 to 1.51 μg/g in the petioles, and was significantly higher in the petioles than in the leaves (p = 0.014; t-Test). Significant and negative correlations between Nuphar leaf Cd and pH (r²= 0.76; p < 0.001), alkalinity (r²= 0.41; p = 0.034), and DOC (r²= 0.46; p = 0.022) were obtained. In addition, a significant and negative correlation was found between Nuphar petiole Cd and pH (r²= 0.46; p = 0.023). These results indicate that the leaves of Nuphar found in peatlands of low pH, low alkalinity, and low DOC, and the petioles of Nuphar found in peatlands of low pH are more susceptible to accumulating potentially toxic levels of Cd. The organic content of the peatland sediments was not significantly correlated with either the leaf or petiole Cd concentration. Nuphar is an important food source for many wetland animals; any Cd that is present in these plants may be passed onto other trophic levels because diet is considered to be the major source of Cd to animals.

Application of Plant and Earthworm Bioassays to Evaluate Remediation of a Lead-Contaminated Soil by L. W. Chang; J. R. Meier; M. K. Smith (pp. 166-171).

Earthworm acute toxicity, plant seed germination/root elongation (SG/RE) and plant genotoxicity bioassays were employed to evaluate the remediation of a lead-contaminated soil. The remediation involved removal of heavy metals by a soil washing/soil leaching treatment process. A portion of the soil after remediation was rinsed with water in order to simulate exposure to rainfall. The bioassay results showed that the soils before treatment (BT) and after treatment plus water rinsing (RT) were not toxic to earthworms in a 14-day exposure, while after treatment (AT) showed significant toxicity. The LC50 values for Eisenia fetida and Lumbricus terrestris were 44.04 and 28.83 (as % AT soil supplemented in artificial soil), respectively. The phytotoxicity data indicated that all three test soils significantly inhibited lettuce SG/RE in a dose-related manner, with AT being the most phytotoxic. In oats, RT had no effect on SG/RE and AT was more toxic than BT. For the two local site grass seeds tested (blue grama and sideoat grama), the AT soil was the most phytotoxic followed by BT and RT. In Allium cepa (common onion), BT and AT induced similar levels of genetic damage to root tip cells, whereas RT was not genotoxic. High salt levels generated during the remediation process appeared to be responsible for the increased toxicity of AT soil for both plants and earthworms. The rinsing of the AT soil with water effectively removed both acutely toxic and genotoxic components of the soil.

Tissue-Specific Accumulation Patterns of Pb, Cd, Cu, Zn, Fe, and Mn in Workers of Three Ant Species (Formicidae, Hymenoptera) from a Metal-Polluted Site by W. B. Rabitsch (pp. 172-177).

The present study compares the metal levels (Pb, Cd, Cu, Zn, Fe, Mn) in different tissues of workers of the ant species Formica pratensis Retzius, Formica polyctena (Förster), and Camponotus ligniperda (Latreille) collected from a metal-polluted site. In all species, highest metal levels were found in the midgut, followed by the malpighian tubules and the hindgut. Additional target sites were the

Mortality, Preference, Avoidance, and Activity of a Predatory Leech Exposed to Cadmium by D. Wicklum; D. E. C. Smith; R. W. Davies (pp. 178-183).

The effects of cadmium on the hatching success of the cocoons of the freshwater predatory leech Nephelopsis obscura were examined together with the survivorship of hatchlings, changes in 96-h LC₅₀ with biomass, preference-avoidance responses and changes in activity. The 96-h EC₅₀ for cocoons was 832.6 μg Cd/L with a decreasing bounded monotonic function best describing hatchling success as a function of Cd concentration. Exposure of cocoons to Cd had a highly significant effect on post-hatchling survivorship with survivorship of hatchlings from the 0–500-μg Cd/L concentrations not significantly different from each other but higher than survivorship of hatchlings from cocoons exposed to 1,000–4,000 μg Cd/L. Resistance to acute Cd toxicity, measured as LC₅₀, increased with leech biomass. In preference-avoidance tests large leeches (>450 mg) spent more time in 100- and 200-μg Cd/L than in control water or in 50-μg Cd/L, while small leeches (<250 mg) spent more time in 200-μg Cd/L compared to control water or 50–100-μg Cd/L. Leeches exposed to 100- and 200-μg Cd/L exhibited a significant decrease in activity compared to the leeches in the control and 50-μg Cd/L treatments.

Distribution and Association of Trace Metals in Soft Tissue and Byssus of Mytilus edulis from the East Coast of Kyushu Island, Japan by P. Szefer; K. Ikuta; S. Kushiyama; K. Szefer; K. Frelek; J. Geldon (pp. 184-190).

Concentrations of Cd, Pb, Zn, Cu, Ag, Cr, Co Ni, Mn, and Fe in soft tissues and byssi of blue mussel (Mytilus edulis) from three sites along the east coast of Kyushu Island, Japan, were determined by AAS method. Large inter-regional differences in metal concentrations in both soft tissues and byssi (Cu, Cd, and Pb and Cu, Pb, Co, Ni, Mn, and Fe, respectively) were recorded. Highly significant correlations (p < 0.01) were observed between tissue and byssal concentrations of Pb, Cu, Zn, and Mn. The tissue concentrations of Cu, Cd, and Pb were two orders of magnitude greater in Mytilus edulis from expected to be the most contaminated locations compared to those from a nonindustrialized area. Intercomparison of the present study data with those published previously indicates that the soft tissue and especially byssus are useful in detecting some areas of some metallic contaminants. The high concentrations of Cd, and especially Pb and Cu, in Saganoseki mussels and moderately elevated concentrations of these metals in Akamizu mussels may be attributed to the anthropogenic emissions from a metallic refinery and an artificial fiber factory, respectively. It is evident that, compared to the soft tissue, the increase of Cu levels relative to Zn levels in the byssi of M. edulis are eight times higher, with a slope b (Cu/Zn) of 7.5 for byssus and 0.93 for soft tissue. This suggests that byssus, as compared to soft tissue, is a more sensitive bioindicator for Cu. From the data obtained, the soft tissue and especially byssi of M. edulis appear to be good bioindicators for identification of coastal areas exposed to metallic contaminants.

Uptake of Nickel and Zinc by the Zebra Mussel Dreissena polymorpha by P. L. Klerks; P. C. Fraleigh (pp. 191-197).

Short-term experiments with nickel and zinc radioisotopes showed that the zebra mussel Dreissena polymorpha takes up both “dissolved” (<0.45 μm) and particulate fractions of these metals in water. Uptake of particulate nickel was significant (despite a relatively low affinity of nickel for particulate matter), though less important than uptake of dissolved nickel. The relative importance of dissolved and particulate zinc varied from an almost exclusive uptake of dissolved zinc to uptake of particulate zinc only. This variability may reflect a dependence on the composition of the suspended particulate material, in line with the observation that zinc uptake and bioaccumulation were higher in high-turbidity water than in low-turbidity water. Metal excretion differed between the two metals; more than half of the accumulated zinc was excreted in twenty four hours, while no nickel excretion was evident. The mussels removed a larger proportion of total watercolumn zinc than of total watercolumn nickel. Of the metal removed from the watercolumn, a majority of the zinc was biodeposited (as feces/pseudofeces) while most of the nickel was bioaccumulated. These results indicate that the introduction of the zebra mussel will result in element-specific decreases of watercolumn metal levels, increases in metal bioaccumulation and increases in metal biodeposition. Results also indicate that D. polymorpha tissue metal levels obtained in biomonitoring programs will generally reflect both dissolved and particulate metal levels.

Survey of Organotin Compounds in the Western Mediterranean Using Molluscs and Fish as Sentinel Organisms by Y. Morcillo; V. Borghi; C. Porte (pp. 198-203).

Tributyltin (TBT) and its degradation products, mono- (MBT) and dibutyltin (DBT) as well as triphenyltin (TPT) were determined in molluscs and fish collected along the Catalan coast (Western Mediterranean). Marine molluscs (mussels, clams and snails) were sampled from three harbors with different characteristics (small vs. large boats). Two fish species were studied (a) the grey mullet Liza aurata sampled in Barcelona harbor and (b) the red mullet Mullus barbatus sampled along the coast; different tissues (muscle, liver, gills and digestive tube) were analysed separately. The composition of butyltin compounds was different according to the organism and sampling point, but in general elevated concentrations of TBT were noticed in molluscs. The highest organotin residue levels (5.4 μg/g d.w. as Sn) were detected in mussels from Masnou, a recreational marina, followed by those collected in Barcelona harbor (1.2 μg/g d.w. as Sn). In contrast, no organotin compounds were detected in fish muscle and very low levels in the other organs, being TPT the major organotin in red mullet liver.

Toxicity of Binary Chemical Mixtures: Effects on Reproduction of Zebrafish (Brachydanio rerio) by U. Ensenbach; R. Nagel (pp. 204-210).

A complete life-cycle test with zebrafish was carried out with different concentrations of the binary mixture 3,4-dichloroaniline and lindane under flow-through conditions. Length and weight of fish of the F₁-generation were reduced, even in the lowest test concentration of 2 μg/L 3,4-dichloroaniline and 40 μg/L lindane. The same effects were found in the early life stage test for the F₂-generation. In the mixture of 100 μg/L 3,4-dichloroaniline and 40 μg/L lindane, fish which were exposed for their whole life time stopped spawning, irreversibly; the fish population will become extinct. In an additional experiment, fish were exposed to the same xenobiotic concentrations after reaching maturity. In this case, egg production was reduced. Cessation of egg production occurs in a concentration of 200 μg/L 3,4-dichloroaniline and 40 μg/L lindane. Nevertheless, effects on spawning are influenced by duration of exposure and the life stages of exposure.

Mercury in Feathers of Little Egret Egretta garzetta and Night Heron Nycticorax nycticorax Chicks and in Their Prey in the Axios Delta, Greece by V. Goutner; R. W. Furness (pp. 211-216).

Mercury concentrations were measured in feathers of little egret and night heron chicks and in their prey in the Axios Delta, Greece. Significantly higher concentrations occurred in night heron than in little egret in 1993. In the night heron the mercury content of feathers was negatively correlated to the size of chicks, possibly due to inhibition of growth. Mercury concentrations were higher than reported for heron feathers in seriously polluted sites in North America and Japan, but the toxic hazard is unclear. Diets differed considerably between the two species due to use of different foraging habitats and this seems responsible for different mercury contents of feathers. Mercury concentrations in the pumpkinseed sunfish Lepomis gibbosus, goldfish Carrassius auratus, and in dragonfly Odonata larvae were the highest among the prey categories. Frogs and water beetles Dytiscidae had moderate concentrations whereas saltwater fish and terrestrial prey had very low mercury concentrations. The implication is that the deltaic marshes are the habitat most polluted with mercury. Night heron chick feathers, freshwater fish and dragonfly larvae could be used to monitor mercury contamination in this region, but use of bird feathers alone could give misleading results if changes in diet occurred.

Heavy Metal and Selenium Concentrations in Feathers of Egrets from Bali and Sulawesi, Indonesia by J. Burger; M. Gochfeld (pp. 217-221).

Herons and egrets are ideal organisms to use as indicators of heavy metal exposure in an ecosystem because different species feed at different levels of the food chain and live in both coastal and inland habitats. This paper reports on the concentration of heavy metals and selenium in the feathers of cattle egrets Bubulcus ibis that were examined from nesting and roosting sites in Bali and Sulawesi, Indonesia, and in feathers of little egrets Egretta garzetta and intermediate egrets E. intermedia from the same colony in Bali. Mercury and manganese concentrations were significantly higher in cattle egrets from Bali compared to Sulawesi, but otherwise there were no significant differences. There were significant differences in lead, cadmium and mercury among the three egret species nesting on Bali: 1) the cadmium and mercury concentrations related to size and trophic level (insectivorous cattle egrets had the lowest concentrations, fish-eating intermediate egrets had the highest concentrations), and 2) cattle egrets had significantly lower concentrations of lead than the other two species. For cattle egrets, secondary flight feathers had significantly higher levels of cadmium and mercury, and lower levels of manganese, than mixed breast and tertiary feathers, reflecting temporal differences in exposure.

Fluoride Content and Mineralization of Red Deer (Cervus elaphus) Antlers and Pedicles from Fluoride Polluted and Uncontaminated Regions by U. Kierdorf; A. Richards; F. Sedlacek; H. Kierdorf (pp. 222-227).

Fluoride, calcium, and phosphorus content as well as ash percentage and ash density of primary antlers and pedicle bones were studied in nine yearling red deer stags from a fluoride polluted region in North Bohemia (Czech Republic) and in nine control animals from two uncontaminated areas in West Germany. Fluoride levels in antlers (845 ± 257 mg F⁻/kg ash, mean ± SD) and pedicles (1,448 ± 461 mg F⁻/kg ash) of the N-Bohemian specimens exceeded that of the controls (antlers: 206 ± 124 mg F⁻/kg ash, pedicles: 322 ± 157 mg F⁻/kg ash) by factors of 4.1 and 4.5, respectively. Antler and pedicle fluoride concentrations of the deer (n = 18) were closely correlated (r = 0.975, p < 0.001). Analyses of ash percentage and ash density revealed that the antlers of the N-Bohemian deer contained significantly less mineral and were significantly less dense than both their pedicles and the control antlers. In the pooled antler samples (n = 18), bone fluoride concentration was negatively correlated with ash density (r = −0.826, p < 0.001) and ash percentage (r = −0.759, p < 0.001), whereas non significant, positive correlations existed for the pooled pedicle samples. Ash percentage and ash density of the antlers and their corresponding pedicles were uncorrelated. It is concluded that increased fluoride exposure of deer leads to reduced mineral content and mineral density of antler bone and that it is the rapidity of their growth and mineralization that makes antlers especially susceptible to fluoride action. Due to their ability to accumulate high amounts of fluoride during a defined, limited timespan and the apparently dose-dependent negative effect of fluoride on their density and mineral content, (primary) antlers can be recommended as monitoring tools for studying environmental pollution by fluorides.

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Archives of Environmental Contamination and Toxicology (v.32, #2)