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Archives of Environmental Contamination and Toxicology (v.51, #4)

Concentration and Emission Sources of Airborne Metals in Particulate Matter in the Industrial District of Médio Paraíba, State of Rio de Janeiro, Brazil by Josiane Loyola; Pierre Batista de Almeida Jr.; Simone Lorena Quiterio; Célia Regina Sousa; Graciela Arbilla; Viviane Escaleira; Maria Isabel de Carvalho; Alzira dos Santos Amaral Gomes da Silva (pp. 485-493).

Total suspended particles and 12 airborne metals were determined in 4 sampling sites in the industrial region of Médio Paraíba, Brazil. The geometrical means for the four sampling locals were (in units of μg/m³): 65.9 in Barra Mansa, 57.3 in Jardim Paraíba (Volta Redonda), 41.7 in Resende, and 48.9 in Volta Grande (Volta Redonda). These values are lower than levels previously determined in urban and industrial locals of the Metropolitan Area of Rio de Janeiro. For metals, the higher concentrations were obtained for Ca, Zn, Al, Fe, and Mg. Ca, Zn, and Al levels are higher than those determined in other industrial areas. These three metals are used in steel manufacturing, the main economical activity of the region. Enrichment factors for Zn, Cu, Cd, and Pb are higher than 10, suggesting an industrial input. Statistical analysis show a high correlation among Ca, Mg, Zn, Cr, Al, Mn, and Fe, all of them used as raw materials in steel manufacturing and/or accumulated as industrial blast furnace slag and steelworks slag.

Atmospheric Deposition of Polycyclic Aromatic Hydrocarbons in an Urban and a Suburban Area of Korea from 2002 to 2004 by H.-B. Moon; K. Kannan; S.-J. Lee; G. Ok (pp. 494-502).

Atmospheric bulk samples (wet and dry) were collected monthly during 2002 to 2004 from an urban and a suburban area in Korea for assessment of depositional flux and seasonal variations in the concentrations of polycyclic aromatic hydrocarbons (PAHs). PAH depositional flux ranged from 64.1 to 610 μg/m²/y for the urban area and from 65 to 460 μg/m²/y for the suburban area. The fluxes of PAHs measured in this study were comparable with those reported for urban and suburban areas in other countries. The fluxes of particulates and PAHs were higher in winter than in summer, consistent with the greater per capita consumption of fossil fuel in winter than in summer. Ambient temperature played a major role in the seasonal variability in PAH fluxes. Photochemical degradation of PAHs appears to occur during the summer months. The relationship of PAH depositional fluxes with major air pollutants, such as ozone, sulfur dioxide, carbon monoxide, nitrogen dioxide, and presence of particulate matter up to 10 μm in size (PM₁₀), was also investigated. Dominant PAH compounds in both the urban and the suburban locations were benzo[g,h,i]perylene, pyrene, and indeno[1,2,3-c,d]pyrene. Based on the PAH diagnostic ratios and a factor analysis, the major sources of PAHs in the urban and the suburban regions were found to be similar. Diesel exhaust, coal combustion, and gasoline emissions contributed predominantly to atmospheric PAH contamination.

Development of Stable Isotope Dilution Quantification Liquid Chromatography–Mass Spectrometry Method for Estimation of Exposure Levels of Bisphenol A, 4-tert-Octylphenol, 4-Nonylphenol, Tetrabromobisphenol A, and Pentachlorophenol in Indoor Air by K. Inoue; S. Yoshida; S. Nakayama; R. Ito; N. Okanouchi; H. Nakazawa (pp. 503-508).

The potential risks to human health and reproduction of phenolic xenoestrogens, such as bisphenol A (BPA), 4-tert-octylphenol (OP), 4-nonylphenol (NP), tetrabromobisphenol A (TBBPA), and pentachlorophenol (PCP), have not been fully elucidated. It is possible that healthy humans are exposed to phenolic xenoestrogens in their daily lives. It is also possible that the source of human exposure to trace amounts of phenolic xenoestrogens is indoor air. Therefore, we have established an accurate, sensitive, and selective method for determining BPA, OP, NP, TBBPA, and PCP in air samples by using stable isotope dilution techniques using ¹³C₁₂-BPA, 4-(1-methyl) octylphenol-d₅ (m-OP-d₅), ¹³C₁₂-TBBPA, ¹³C₆-PCP, and liquid chromatography–mass spectrometry (LC-MS). Air sampling (71/min, 24 hours), using glass filters (GB-100R, 47 mm) and Empore SDB-XD filters (47 mm/0.5 mm, 3M, Tokyo, Japan) and subsequent extraction by washing were carried out. Then the extracted sample solution was subjected to LC-MS. The percentage recovery of these analytes ranged from 87.0% to 101.9% (SD 0.2% to 4.6%, n = 3) with correction using the stable isotope dilution quantification method. The quantification limit (signal-to-noise ratio, S/N>10) in the air samples was 0.1 ng/m³. Our findings suggest that the LC-MS method can detect low levels of phenolic xenoestrogens in air samples and that these trace levels of analytes exist in indoor air.

Pesticide Detoxifying Functions of N-Halamine Fabrics by Xin Fei; Pengfei Gao; Takayuki Shibamoto; Gang Sun (pp. 509-514).

Halamine structures incorporated on polyester/cotton fabrics were able to detoxify oxime carbamate pesticides that contain thio bonds rapidly upon contact. The reaction was endothermic, and the detoxification rate was in first order to concentrations of the pesticides. Aldicarb was degraded in a much faster rate than that of methomyl by the halamine fabrics. The reactivity of halamine structures was different, and imide halamine was more reactive than amine halamine. The detoxification was an oxidative reaction on the sulfur atom existing in both aldicarb and methomyl. The same halamine structures were unable to effectively react with carbaryl and carbofuran, which are aromatic carbamates and do not contain any thio bonds.

Comparative Cytotoxicity of Alachlor on RTG-2 Trout and SH-SY5Y Human Cells by M. Fernández; J. C. Ríos; A. Jos; G. Repetto (pp. 515-520).

The cytotoxic effects of the herbicide alachlor were compared on rainbow trout gonadal RTG-2 and human neuroblastoma SH-SY5Y cell lines. The end points evaluated in both cells after 24, 48, and 72 h of exposure were total protein content (PC), lysosomal function, and mitochondrial’s integrity by mitochondrial succinate dehydrogenase (SDH) activity. After 24 h, cytoplasmic membrane integrity by cytosolic lactate dehydrogenase (LDH) leakage and LDH intracellular activity were also studied. In addition, acetylcholinesterase activity (AChE) was quantified in SH-SY5Y cells. The possible biotransformation of alachlor by RTG-2 cells was investigated by analyzing the exposure culture medium by liquid chromatography–mass spectrometry. In RTG-2, EC₅₀ values on PC, lysosomal function, and SDH activity after 24 h exposure ranged from 80 to 95 μM and decreased to approximately 40 μM for longer exposure time periods. SH-SY5Y cells were slightly more sensitive than RTG-2 cells, with EC₅₀ values on PC and lysosomal function ranging from 87 to 75 μM at 24 h and decreasing to 47 μM and 34 μM at 72 h, respectively. AChE activity was increased, being the most sensitive marker for SH-SY5Y with an EC₅₀ of 20 μM at 24 h. The metabolic enzyme SDH was stimulated in SH-SY5Y and reduced in RTG-2 cells. At the studied conditions, no metabolites of alachlor were detected in RTG-2 cultures. In conclusion, the proposed battery approach is an effective screening tool for the safety assessment of environmental contaminants as a complement to fish and animal toxicity procedures.

Simultaneous Cd²⁺, Zn²⁺, and Pb²⁺ Uptake and Accumulation by Photosynthetic Euglena gracilis by D. G. Mendoza-Cózatl; E. Rangel-González; R. Moreno-Sánchez (pp. 521-528).

The ability of Euglena gracilis to simultaneously remove and accumulate Zn²⁺, Cd²⁺, and Pb²⁺ from culture up- to media was evaluated. E. gracilis was able to remove up to 80% of the Cd²⁺ present in the medium when cultured with 20 or 50 μM CdCl₂. Higher external Cd²⁺ concentrations increased Cd²⁺ accumulation per cell but decreased cell growth, thus decreasing the capacity of the cell culture to remove Cd²⁺. E. gracilis removed 70% to 80% of the Zn²⁺ present in the medium when cultured with 5 to 50 μM ZnSO₄. Zn²⁺ did not affect Cd²⁺ removal capacity. E. gracilis was much less efficient in removing Pb²⁺ (<15%) when cultured with 100 or 200 μM Pb(NO₃)₂. Moreover, Pb²⁺ decreased the efficiency to remove Cd²⁺, but it did not affect Zn²⁺ removal. Cd²⁺ induced a generalized increase in the cellular thiol compounds, including phytochelatins, and Pb²⁺ had an additive effect only at 200 μM. Zn²⁺ did not stimulate phytochelatin synthesis. Cd²⁺ and Pb²⁺ colocated in the same cytosolic high-molecular-weight fraction. Because Pb²⁺ is a weak phytochelatin inducer, competition between Pb²⁺ and Cd²⁺ for transportation across the plasma membrane and binding to phytochelatins and other thiol compounds is proposed to explain the detrimental effects of Pb²⁺ on the Cd²⁺ removal capacity of E. gracilis.

Morphologic and Growth Responses in Chironomus tentans to Arsenic Exposure by E. A. Martinez; L. Wold; B. C. Moore; J. Schaumloffel; N. Dasgupta (pp. 529-536).

Laboratory bioassays consisting of sediments spiked with three concentrations (30, 130, and 260 μg g⁻¹ As dry wt) of arsenic (As⁺³) were used to assess morphologic responses and growth in Chironomus tentans larvae. Chironomid larvae were raised in contaminated sediments from egg stage to emergence, and mouthpart abnormalities, larval length, and larval head width were used as end points to determine differences between metal-spiked and control specimens. C. tentans exhibited significantly higher mouthpart deformity proportions, smaller body sizes, smaller head widths, and slower development than control larvae. Our results demonstrate a dose–response relationship between As and mentum deformities in C. tentans. However, the proportion of deformed larvae did not increase with time in the treatment tanks. Results demonstrate that As induces mouthpart abnormalities at various concentrations. This research provides more support for the use of chironomid abnormalities as a tool for the assessment of heavy-metal pollution in aquatic systems.

Growth Stimulation and Inhibition Effects of 4-Hydroxybenzoic Acid and Some Related Compounds on the Freshwater Green Alga Pseudokirchneriella subcapitata by Y. Kamaya; S. Tsuboi; T. Takada; K. Suzuki (pp. 537-541).

4-Hydroxybenzoic acid (4-HBA) exhibited low algal toxicity with the 72-h median inhibition concentration (IC₅₀) of 9.9 mmol/L in the standard growth inhibition test using the freshwater green alga Pseudokirchneriella subcapitata. In contrast, it stimulated the algal growth at lower concentrations ranging from 0.1 to 1.0 mmol/L. Comparative studies with benzoic acid and 2- and 3-hydroxybenzoic acids (2-HBA and 3-HBA) indicated that 2-HBA was the most toxic, giving a 72-h IC₅₀ of 0.172 mmol/L, and 4-HBA was the least toxic and that only 4-HBA had the pronounced growth stimulation activity. In a semicontinuous exposure to 4-HBA (0.15 and 0.3 mmol/L), algae maintained increased cell growth compared with controls during up to 10 times consecutive batch cultures, without any indication of adaptive responses to the growth enhancing effect of 4-HBA. Return to the clean standard medium of the exposed cells resulted in the quick recovery from the stimulant effect. Furthermore, 4-HBA (0.3 mmol/L) was found to diminish the toxicity of 2-HBA (< 1.0 mmol/L). The presence of such compounds in the complex mixture might interact with other toxicants to alter the whole toxicity when assessed by the algal growth inhibition test. The effects of 4-HBA on P. subcapitata growth observed in the present study are not expected for planktonic algae in the aquatic environments, because known environmental concentrations are far below the effective concentration range.

Uptake and Elimination of Chlorpyrifos and Pentachlorophenol into the Freshwater Amphipod Gammarus pulex by Roman Ashauer; Alistair Boxall; Colin Brown (pp. 542-548).

Uptake and elimination rates were determined for chlorpyrifos (CPF) and pentachlorophenol (PCP) in the freshwater amphipod Gammarus pulex. Internal concentrations of the two pesticides were measured over a three-day exposure phase and a subsequent three-day elimination phase. Rate constants were obtained by fitting measured internal concentrations to a one-compartment single first-order model. The uptake rate constants were 747 ± 61 [L kg⁻¹ day⁻¹] for CPF and 89 ± 7 [L kg⁻¹ day⁻¹] for PCP. The elimination rate constants were 0.45 ± 0.05 [day⁻¹] for CPF and 1.76 ± 0.14 [day⁻¹] for PCP. The resulting bioconcentration factors at steady state were 1660 and 51 for CPF and PCP, respectively. The parameter estimation method and possible variability due to varying lipid content are briefly discussed.

Toxicity of Synthetic Musks to Early Life Stages of the Freshwater Mussel Lampsilis cardium by M. P. Gooding; T. J. Newton; M. R. Bartsch; K. C. Hornbuckle (pp. 549-558).

Polycyclic musk fragrances are common additives to many consumer products. As a result of their widespread use and slow degradation rates, they are widely found in aquatic environments. This study reports on the lethal and sublethal toxicity of the polycyclic musks AHTN (Tonalide^®) and HHCB (Galaxolide^®) to glochidial (larval) and juvenile life stages of the freshwater mussel Lampsilis cardium (Rafinesque, 1820). In glochidia, 24-h median lethal concentrations (LC50s) ranged from 454 to 850 μg AHTN/L and from 1000 to >1750 μg HHCB/L (water solubility). Results for 48-h tests were similar to the 24-h tests. In 96-h tests with juveniles, we did not observe a dose-response relation between mortality and either musk. However, the growth rate was reduced by musk exposure. The median effective concentrations (EC50s, based on growth) were highly variable and ranged from 108 to 1034 μg AHTN/L and 153 to 831 μg HHCB/L. While all adverse effects occurred at concentrations that are much greater than those reported in natural waters (low μg/L to ng/L), these results indicate the potential for adverse effects on these long-lived organisms from exposure to synthetic musk fragrances.

Detection System of Cryptosporidium parvum Oocysts by Brackish Water Benthic Shellfish (Corbicula japonica) as a Biological Indicator in River Water by T. Izumi; K. Yagita; T. Endo; T. Ohyama (pp. 559-566).

The brackish water benthic shellfish, Corbicula japonica, was experimentally exposed to Cryptosporidium parvum oocysts at 1.51 × 10⁴oocysts/clam/day for 7 or 14 days. Oocysts were predominantly eliminated through the feces of Corbicula japonica in both cases by microscopic and PCR methods. The fecal excretion rates of oocysts within 4 days after the last exposure to Corbicula japonica were 87.6% for the 7-day exposure group and 86.0% for the 14-day exposure group. The tissue residue level of oocysts in the gastrointestinal tract 3 days after the last exposure was 2.7% of total exposed oocysts and that of 7 days was 1.1% for the 7-day exposure case and 1.6 and 0.5% for the 14-day exposure case, respectively, maintaining infectivity to cultured cells (HCT-8) in vitro. At the same time, field tests of Corbicula japonica for collecting oocysts showed that this clam could certainly collect Cryptosporidium parvum oocysts in the natural river and, furthermore, the gene type of C. parvum could be also identified proving its effectiveness as a biological indicator. The present study showed that the brackish water benthic shellfish Corbicula japonica may be capable of gathering and preserving Cryptosporidium parvum oocysts to a considerable extent under the natural ecological conditions, and further suggests the effectiveness of Corbicula japonica as a practical and general bioindicator for estimates of river water contamination by oocysts of Cryptosporidium parvum.

Cadmium Concentration and Subcellular Distribution in Organs of the Mussel Crenomytilus Grayanus from Upwelling Regions of Okhotsk Sea and Sea of Japan by Olga V. Podgurskaya; Victor Ya. Kavun (pp. 567-572).

Cadmium (Cd) accumulation and subcellular distribution in the digestive gland and kidney of mussel Crenomytilus grayanus from naturally Cd elevated areas was studied. Mussels were collected from three sites of Okhotsk Sea and Sea of Japan: control area (site 1), seasonal upwelling region (site 2), and stationary upwelling region (site 3). Mussel from site 3 was shown to accumulate a significantly increased Cd concentration in the digestive gland and an extremely high Cd concentration (1780 ± 732 μg g⁻¹ dry weight) in the kidney. Cd was mainly sequestered into the kidney cytosol of the mussels from both upwelling regions and control sites (73% to 96%). However, digestive gland cytosol bound about 55% of Cd for the mussels from sites 1 and 2 and only 22.4% of Cd for the mussel from site 3. In the organs of the mussels from upwelling regions, the most of cytosolic Cd was associated with the protein fraction corresponding to molluscan metallothioneins. MTLP was isolated from kidney cytosol of the mussels from site 3 (powerful upwelling regions) by gel filtration chromatography, whereas in the organs studied of the mussels from other sites, MTLP content was below the detection limit.

Influence of Potentially Confounding Factors on Sea Urchin Porewater Toxicity Tests by R. S. Carr; J. M. Biedenbach; M. Nipper (pp. 573-579).

The influence of potentially confounding factors has been identified as a concern for interpreting sea urchin porewater toxicity test data. The results from >40 sediment-quality assessment surveys using early–life stages of the sea urchin Arbacia punctulata were compiled and examined to determine acceptable ranges of natural variables such as pH, ammonia, and dissolved organic carbon on the fertilization and embryological development endpoints. In addition, laboratory experiments were also conducted with A. punctulata and compared with information from the literature. Pore water with pH as low as 6.9 is an unlikely contributor to toxicity for the fertilization and embryological development tests with A. punctulata. Other species of sea urchin have narrower pH tolerance ranges. Ammonia is rarely a contributing factor in pore water toxicity tests using the fertilization endpoint, but the embryological development endpoint may be influenced by ammonia concentrations commonly found in porewater samples. Therefore, ammonia needs to be considered when interpreting results for the embryological development test. Humic acid does not affect sea urchin fertilization at saturation concentrations, but it could have an effect on the embryological development endpoint at near-saturation concentrations. There was no correlation between sediment total organic carbon concentrations and porewater dissolved organic carbon concentrations. Because of the potential for many varying substances to activate parthenogenesis in sea urchin eggs, it is recommended that a no-sperm control be included with every fertilization test treatment.

Effects of Copper, Cadmium, and Zinc on the Hatching Success of Brine Shrimp (Artemia franciscana) by K. V. Brix; R. M. Gerdes; W. J. Adams; M. Grosell (pp. 580-583).

Previous studies indicate that the hatching success of brine shrimp (Artemia franciscana) cysts is surprisingly sensitive to ambient metal concentrations. These studies estimated median effective concentrations (EC₅₀s) of 7, 5, and 28 μg l⁻¹ for Cd, Cu, and Zn, suggesting that the hatching end point for A. franciscana is the most sensitive tested to date for Cd and Zn in saline environments and comparable in sensitivity with the most sensitive tested to date for Cu. Furthermore, these data suggest that brine shrimp are at significant risk from Cu and Zn in Great Salt Lake (GSL), UT, where ambient concentrations as high as 10 and 14 μg l⁻¹, respectively, have been measured. Given that brine shrimp appear to be successfully reproducing in GSL, we hypothesized that these toxicity values were either biased low as a result of an artifact of the test method used or that site-specific water-quality conditions in the lake had decreased metal bioavailability such that brine shrimp could successfully reproduce. To test these hypotheses, we initiated a step-wise series of experiments. First we investigated the effects of pretreatment of brine shrimp cysts with antibiotics on brine shrimp sensitivity to metals because previous investigators as part of their test methods have used antibiotics. Next we considered the effect of ionic composition of the artificial test media on sensitivity. Finally, we evaluated the effects of the site-specific water quality of the GSL on metal bioavailability and toxicity. Results indicate that pretreatment of cysts with antibiotics had no effect on sensitivity. However, we were unable to repeat the previous values for Cd and Zn, obtaining EC₅₀s of 11,859 and 289 μg l⁻¹ for Cd and Zn, respectively. For Cu, however, we estimated an EC₅₀ of 12 μg l⁻¹, so we conducted further testing on the artificial media, adjusting the media composition to better reflect the Ca²⁺ and HCO ₃ ⁻ concentration of normal seawater. This increased the EC₅₀ to 28 μg l⁻¹. Finally we evaluated the toxicity of Cu in GSL water and obtained an EC₅₀ of 68 μg l⁻¹, suggesting that the increased dissolved organic carbon in GSL has a significant protective effect. Overall, the results of this study suggest that brine shrimp hatching success is not particularly sensitive to Cd and Zn, but it is sensitive to Cu. However, site-specific water-quality conditions ensure that brine shrimp cyst hatching success is not significantly affected by any of these metals at the normal background concentrations that occur in GSL (<15 μg l⁻¹).

Biomarkers of Heavy Metal Contamination in the Red Fingered Marsh Crab, Parasesarma erythodactyla by G. R. MacFarlane; M. Schreider; B. McLennan (pp. 584-593).

Variation in glutathione antioxidant biochemistry in response to metal contamination and accumulation under field conditions was examined in the brachyurid grapsid, Parasesarma erythodactyla. Significant relationships suggesting accumulation were found between sediment metals and metals in crab tissue for Pb, Cu, Cr, Zn, and Se in males and Cd, Pb, Cr Zn, As, and Se in females. Higher pH and lower organic content were associated with greater uptake of selected metals in males and females. Higher salinity was related to increased metal uptake for Cu and Zn in males and lower salinities to increased Se uptake for males and females. When examining metals, which were elevated in crabs, patterns of site discrimination were similar to sediment metal site discrimination for both males and females. In terms of biochemical responses, glutathione levels remained constant while glutathione peroxidase activity was elevated in individuals where metals were elevated. Only females with the highest levels of accumulated metals exhibited increases in lipid peroxidation products. Glutathione peroxidase activity may be a sensitive biomarker of metal exposure and biological effect and lipid peroxides as a secondary marker when accumulated metals are high.

Cd Exposures in Fathead Minnows: Effects on Adult Spawning Success and Reproductive Physiology by Marlo K. Sellin; Alan S. Kolok (pp. 594-599).

The primary objective of this study was to determine whether cadmium (Cd) exposures alter reproduction in fathead minnows (Pimephales promelas). Pairs of sexually mature minnows were exposed to waterborne Cd at 0, 12.5, 25, or 50 μg/L for 21 days. During this exposure period, the reproductive success of pairs was assessed. Following the exposure, gonadosomatic index (GSI), male secondary sexual characteristics, male plasma 11-ketotestosterone (11-KT) concentrations, and female plasma estradiol (E₂) concentrations were determined. Results of this study show that Cd exposures impair reproduction in fathead minnows. Pairs exposed to 50 μg/L experienced a significant decline in spawning frequency and fecundity relative to unexposed minnows. Cd exposures also caused alterations in male secondary sexual characteristics, as males exposed to 25 μg/L had significantly fewer nuptial tubercles than controls. Furthermore, males exposed to 12.5 μg/L Cd experienced a significant increase in plasma 11-KT concentrations relative to controls. No alterations in GSI or female plasma E₂ concentrations were observed.

Modified Japanese Medaka Embryo-Larval Bioassay for Rapid Determination of Developmental Abnormalities by Andrea Farwell; Vincent Nero; Melanie Croft; Prabjot Bal; D. George Dixon (pp. 600-607).

The Japanese medaka (Oryzias latipes) early-life-stage bioassay (18-day test) provides a suitable laboratory model for the evaluation of toxicant impact. The naturally high variability in the time to hatch for medaka embryos is, however, a major limitation in terms of the duration of the test. In this study, the 18-day test was modified to use agitation to synchronize hatch and reduce time to hatch for nonexposed embryos. Then, bioassays were conducted using two different complex mixtures (polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs)) to compare the sensitivity of the measurement endpoints using the 18-day standard protocol (SP) and the modified protocol (MP). Agitation reduced time to hatch by one third or more and improved hatch success (100%). The MP proved to be a more sensitive test method for the sublethal evaluation of a stressor (PAH mixture) that caused reduced hatch length and induced signs of blue sac disease (BSD) including heart deformities, yolk sac–pericardial edema, and cranial–skeletal deformities. In contrast, there was some variability in the sensitivity of the test methods for developmental endpoints for the sublethal evaluation of a stressor (NA mixture) that had no effect on hatch length and was a weak inducer of signs of BSD. Even though the sensitivity of the measurement endpoints were slightly more or less sensitive for the MP vs. SP depending on the mixture tested, the MP using agitation is recommended as a cost-effective and rapid alternative for screening the sublethal impact of toxicants on the early-life stages of fish.

Assessment of Butyltin and Phenyltin Pollution in the Sea Mullet, Mugil cephalus, Along the Moroccan and Spanish Coasts (Mediterranean Sea) by L. Hajjaj Hassani; A. Garrido Frenich; M. H. Benajiba; M. J. González Rodríguez; J. L. Martínez Vidal; A. Debdoubi (pp. 608-614).

Levels of three butyltin (BTs) compounds, monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT), and three phenyltin (PTs) compounds, monophenyltin (MPhT), diphenyltin (DPhT), and triphenyltin (TPhT), were determined in the sea mullet, Mugil cephalus, collected along the northern Mediterranean coast of Morocco, and the south Mediterranean coast of Spain. Also, sediment samples from the Guadalquivir River (Spain) near to its mouth in the Mediterranean Sea were analysed. TBT and TPhT were the predominant compounds in the analyzed samples, and TBT concentrations were higher in liver than in muscle. In general, the total content of BTs in these samples was higher than PT levels. In the Moroccan coast, the highest value of BTs (18.00 mg/Kg wet weight) was found in the west harbour of the Tangier site, while the lowest concentration (0.01 mg/Kg wet weight) was detected in the Oued Laoue site. Concerning PTs compounds, the highest value (4.00 mg/Kg wet weight) was found in the harbour of the M’diq Site and the lowest value (0.01 mg/Kg wet weight) was detected in the Oued Laoue site. On the other hand, organotin (OT) residues were not detected in the water samples of the Spanish coast although TBT was identified in four of the nine sediment samples from the Guadalquivir River at concentrations between 15.45 and 245.07 mg/Kg wet weight.

Dietary Pb Accumulation in Juvenile Freshwater Rainbow Trout (Oncorhynchus mykiss) by L. C. Alves; C. N. Glover; C. M. Wood (pp. 615-625).

Three different diets amended with lead (Pb) nitrate Pb(NO₃)₂ (7, 77, and 520 μg Pb/g dry weight) and a Pb-free control diet (0.06 μg Pb/g dry weight) were fed to juvenile freshwater rainbow trout for 21 days. Accounting for measured food consumption, the calculated doses per fish were 0.02, 3.7, 39.6, and 221.5 μg/day, for the control, low, intermediate, and high Pb treatments, respectively. The patterns of Pb accumulation over time were determined in various tissues (gills, liver, kidney, intestine, carcass), red blood cells (RBC), and plasma, as well as feeding, growth, hematological, and ionoregulatory parameters. Pb accumulation occurred in a dose-dependent manner in all tissues except the plasma, where accumulation was minimal. Overall, when fed the highest Pb diet, the intestine exhibited the greatest Pb burden (17.8 μg Pb/g tissue wet weight), with high concentrations also found in the kidney (2.4 μg Pb/g tissue wet weight) and liver (1.9 μg Pb/g) at the highest dietary Pb treatment by day 21. The RBCs accumulated a substantial amount of Pb (1.5 μg Pb/g) when compared to the plasma (0.012 μg Pb/g) in the high treatment group. The percentage of Pb retained in the fish decreased with increasing dietary Pb concentrations. Growth, survival, plasma protein, and hematocrit were not significantly affected by dietary Pb. Plasma Ca²⁺ levels decreased at the beginning of the experiment, whereas Mg²⁺ levels decreased during the middle of the experiment in both the intermediate and high dietary treatments. Both the Ca²⁺ and Mg²⁺ levels stabilized by day 21. Branchial Ca²⁺ and Na⁺ influx rates were not affected by dietary Pb, except on day 8 where Na⁺ influx rates were significantly elevated. The results of this study show that Pb does accumulate internally from the diet when present at levels within the range reported in contaminated benthic invertebrates in nature. We further identify the intestine as a potential target site of chronic toxicity of Pb via the diet, and RBCs as a reservoir of dietary Pb.

The Effect of Paraquat on Hepatic EROD Activity, Liver, and Gonadal Histology in Males and Females of Nile Tilapia, Oreochromis niloticus, Exposed at Different Temperatures by A. Figueiredo-Fernandes; A. Fontaínhas-Fernandes; E. Rocha; M.A. Reis-Henriques (pp. 626-632).

The activity of fish monooxygenases has been used as a monitoring tool to evaluate contamination by cytochrome P450 inducing agents. In this study ethoxyresorufin O-deethylase (EROD) activity was analyzed in males and females of Nile tilapia exposed to a low concentration of paraquat (PQ) at 17°C and 27°C. PQ-treated fish showed a high hepato-somatic index, except females acclimated at 17°C. No differences were found for the gonado-somatic index (GSI) between males. However, PQ-treated females showed high GSI values (6.46 ± 1.75) when compared with the control group (2.30 ± 0.26) maintained at 27°C. Males and females exposed to PQ showed higher microsomal protein values than the control group (9.46 ± 0.22 vs. 6.20 ± 0.18 at 17°C; 9.51 ± 0.35 vs. 4.70 ± 0.19 mg of protein at 27°C, in PQ-treated and control groups, respectively). The EROD activity was high in females exposed to PQ when compared with the control group at 17° and 27°C. The liver histology showed that PQ also caused some hepatic alterations of parenchyma, like vacuolization, necrosis, and an increase of macrophages aggregates and eosinophilic granular cells. Females exposed to PQ showed a greater increase of late-vitellogenic (22.2 ± 3.2) and mature (12.1 ± 2.0) percentage of oocytes than the control group (9.9 ± 3.0 and 8.0 ± 4.3, respectively), and a lower percentage of primary oocytes (8.0 ± 3.3) at 27°C. In short, this work has advanced new knowledge on the influence of gender in biotransformation activity and the reproductive activity of Nile tilapia exposed to a low concentration of paraquat, and demonstrated that their effects could be observed at different temperatures.

Evaluation of Remediation of Coal Mining Wastewater by Chitosan Microspheres Using Biomarkers by J. C. Benassi; R. Laus; R. Geremias; P. L. Lima; C. T. B. Menezes; M. C. M. Laranjeira; D. Wilhelm-Filho; V. T. Fávere; R. C. Pedrosa (pp. 633-640).

Acidic mine waters have a marked influence on the surrounding environment and pose a serious threat through long-term environmental degradation. Therefore, it is important to improve and monitor water quality with the aim of decreasing the hazard presented by this effluent emission. The aim of this work was to evaluate the remediation of mining wastewater effluents by chitosan microspheres using biomarkers of exposure and effect. DNA damage (Comet assay) and several biomarkers of oxidative stress, such as lipoperoxidation levels (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activities, and contents of reduced glutathione (GSH), were measured in blood and liver of tilapia (Oreochromis niloticus) exposed for 7, 15, and 30 days to dechlorinated tap water, 10% coal mining wastewater (CMW), and coal mining wastewater treated with chitosan microspheres (RCM). The results indicate that hepatic TBARS levels were significantly higher in fish exposed to CMW after 7, 15, and 30 days (100%, 86%, and 63%, respectively), and after remediation there was no significant difference in relation to the control group. Hepatic GSH concentrations were lower than control values for CMW after 7 and 15 days of exposure (34% decrease at both times), and this concentration was normalized by treatment with chitosan. SOD showed increased activity in liver after 15 and 30 days of exposure, 30% and 36%, respectively, and in fish exposed to RCM there was no change in this activity compared with the control group. Increased CAT activity in liver was observed during all experimental periods in fish exposed to CMW (46%, 50%, and 56% at 7, 15, and 30 days, respectively) compared with the control or treated-water groups. The highest increase in hepatic GST activity (106%) was observed only in fish exposed to CMW for 30 days. There was an increase in DNA damage in liver (50% at 7 and 15 days) and blood (79%, 77%, and 48% at 7, 15, and 30 days, respectively) after exposure to CMW. In contrast, the fish exposed to wastewater treated with chitosan microspheres exhibited DNA fragmentation indexes similar to the control group. The results obtained indicate the use of oxidative stress biomarkers as useful tools for the toxicity evaluation of coal mining effluents and also suggest that chitosan microspheres may be used as an alternative approach for remediation of coal mining wastewaters.

Investigating the Estrogenic Risk Along the River Po and Its Intermediate Section by L. Viganò; A. Mandich; E. Benfenati; R. Bertolotti; S. Bottero; E. Porazzi; E. Agradi (pp. 641-651).

Different endpoints have been used to investigate the occurrence of estrogenic risk along the Po River, particularly its middle section. An in vitro assay based on recombinant yeast could not detect estrogenic activity in bed sediments of the Italian river or in bile samples of five Cyprinid species, with the only exception being one carp (Cyprinus carpio) captured downstream of the River Lambro, a polluted tributary of the middle River Po. Chemical analyses of fish bile and water samples from the same middle section showed diffuse contamination by moderately low levels of estrogenic chemicals (estrone [E₁], 17β-estradiol, estriol [E₃], 17α-ethinylestradiol, 4-nonylphenol [NP], 4-tert-octylphenol [tOP], 4-n-octylphenol, and bisphenol A) but they were of limited help in understanding the risk present in the downstream area where intersex barbel were previously found. In contrast, the analyses of River Lambro waters showed that this tributary is a source to the middle River Po of all eight estrogens investigated. Analyses of bed sediments and macroinvertebrates from the same area consistently showed at least two levels of contamination, with the downstream stretch showing higher concentrations of natural steroids (E₁ and E₃) and xenoestrogens (NP and tOP). Accordingly, new histologic examinations undertaken on young barbel (Barbus sp.) showed intersex gonads only in the individuals captured in the downstream stretch, thereby confirming previous results. Present findings confirm the occurrence of disrupting conditions in the middle River Po and provide the first suggestions of cause-effect relationships.

Ecotoxicity of Sediments Contaminated by the Oil Spill Associated with the Tanker “Prestige” Using Juveniles of the Fish Sparus aurata by Carmen Morales-Caselles; Natalia Jiménez-Tenorio; M. Luisa González de Canales; Carmen Sarasquete; T. Ángel DelValls (pp. 652-660).

In November 2002, the oil spill from the tanker Prestige in the Galician Coast caused an ecological catastrophe in Spain. The adverse effects associated with the contaminants bound to sediments were tested using juveniles of the fish Sparus aurata (seabream). The approach evaluates sediment quality by using an integrated assessment including chemical and ecotoxicological data. Sediment samples were physicochemically characterized, and the concentration of contaminants (polycyclic aromatic hydrocarbons—(PAHs) and metals) was measured. Different biomarkers of exposure (metallothioneins and ethoxyresorufin O-deethylase activity (EROD)) and biomarkers of effect (histopathology) were analyzed along the time. A multivariate analysis approach was used to correlate concentration of contaminants and sublethal effects measured in individuals of fish. Results show that increasing concentrations of PAHs in sediments were related to increased EROD activities and histopathological lesions. This is the first evidence showing adverse effects associated with petroleum contamination of PAHs in sediments after this spill, and it demonstrates the utility of the sublethal toxicity tests for monitoring the impact of petroleum spills.

Environmental Contaminants in Fish and Their Associated Risk to Piscivorous Wildlife in the Yukon River Basin, Alaska by Jo Ellen Hinck; Christopher J. Schmitt; Kathy R. Echols; Tom W. May; Carl E. Orazio; Donald E. Tillitt (pp. 661-672).

Organochlorine chemical residues and elemental contaminants were measured in northern pike (Esox lucius), longnose sucker (Catostomus catostomus), and burbot (Lota lota) from 10 sites in the Yukon River Basin (YRB) during 2002. Contaminant concentrations were compared to historical YRB data and to toxicity thresholds for fish and piscivorous wildlife from the scientific literature. A risk analysis was conducted to screen for potential hazards to piscivorous wildlife for contaminants that exceeded literature-based toxicity thresholds. Concentrations of total DDT (sum of p,p′-homologs; 1.09–13.6 ng/g), total chlordane (0.67–7.5 ng/g), dieldrin (<0.16–0.6 ng/g), toxaphene (<11–34 ng/g), total PCBs (<20–87 ng/g), TCDD-EQ (≤1.7 pg/g), arsenic (0.03–1.95 μg/g), cadmium (<0.02–0.12 μg/g), copper (0.41–1.49 μg/g), and lead (<0.21–0.27 μg/g) did not exceed toxicity thresholds for growth and reproduction in YRB fish. Concentrations of mercury (0.08–0.65 μg/g), selenium (0.23–0.85 μg/g), and zinc (11–56 μg/g) exceeded toxicity thresholds in one or more samples and were included in the risk analysis for piscivorous wildlife. No effect hazard concentrations (NEHCs) and low effect hazard concentrations (LEHCs), derived from literature-based toxicity reference values and avian and mammalian life history parameters, were calculated for mercury, selenium, and zinc. Mercury concentrations in YRB fish exceeded the NEHCs for all bird and small mammal models, which indicated that mercury concentrations in fish may represent a risk to piscivorous wildlife throughout the YRB. Low risk to piscivorous wildlife was associated with selenium and zinc concentrations in YRB fish. Selenium and zinc concentrations exceeded the NEHCs and LEHCs for only the small bird model. These results indicate that mercury should continue to be monitored and assessed in Alaskan fish and wildlife.

Synthetic Pyrethroid Cypermethrin Induced Cellular Damage in Reproductive Tissues of Drosophila melanogaster: Hsp70 as a Marker of Cellular Damage by Indranil Mukhopadhyay; Hifzur Rahman Siddique; Virendra Kumar Bajpai; Daya Krishna Saxena; Debapratim Kar Chowdhuri (pp. 673-680).

We tested a working hypothesis of whether the synthetic pyrethroid cypermethrin, used worldwide for insecticidal purpose, causes adverse effects on reproduction in Drosophila melanogaster. Freshly eclosed first instar larvae of a transgenic strain of Drosophila melanogaster, Bg 9, transgenic for hsp70 (hsp70-lacZ), were transferred to different dietary concentrations of the test chemical (0.002, 0.02, 0.2, 0.5, and 50.0 ppm). Larval mortality was observed at the higher dosed groups (0.2, 0.5, and 50.0 ppm). Following pair mating of virgin flies emerging from the treatment groups, a significant (p < 0.05) effect on reproduction was observed in the lowest two dietary concentrations of the test chemical as compared to control. The test chemical exhibited a hazardous effect on the reproductive organs of the exposed organism as evident by Hsp70 expression and tissue damage. The impact of damage was comparatively more prominent in male flies than in females. Hsp70 expression was restricted only within the testis lobes of male, while ovary in the female fly did not exhibit any Hsp70 expression. Interestingly, the accessory glands of male flies in these treatment groups reflected intense tissue damage as evident by Trypan Blue staining. This was further corroborated by ultrastructural changes like higher vacuolization and disorganized filamentous bodies in the accessory glands of these groups. The present study indicates a profound effect on reproduction by cypermethrin and suggests the protective role of hsp70.

Impact of Atrazine on Chlorpyrifos Toxicity in Four Aquatic Vertebrates by M. N. Wacksman; J. D. Maul; M. J. Lydy (pp. 681-689).

Atrazine has been shown previously to potentiate chlorpyrifos toxicity in selected invertebrates. This study examined interactions of atrazine and chlorpyrifos in four aquatic vertebrates. Organisms were exposed to binary mixtures of atrazine and chlorpyrifos during toxicity bioassays. Inhibition of cholinesterase (ChE) enzyme activity and chlorpyrifos uptake kinetics were also examined with and without atrazine exposure. Atrazine alone did not affect organisms at concentrations up to 5000 μg/L; however, the presence of atrazine at 1000 μg/L did result in a significant increase in the acute toxicity of chlorpyrifos in Xenopus laevis. Mixed results were encountered with Pimephales promelas; some bioassays showed greater than additive toxicity, while others showed an additive response. No effect of atrazine on chlorpyrifos toxicity was observed for Lepomis macrochirus and Rana clamitans. Atrazine did not affect ChE activity or chlorpyrifos uptake rates, indicating that these toxicodynamic and toxicokinetic parameters may not be related to the mechanism of atrazine potentiation of chlorpyrifos toxicity. Based on the results of this study, it does not appear that a mixture toxicity of atrazine and chlorpyrifos at environmentally relevant concentrations presents a risk to the vertebrate organisms examined in this study.

Morphofunctional Evidence of Changes in Principal and Mitochondria-Rich Cells in the Epidermis of the Frog Rana kl. esculenta Living in a Polluted Habitat by C. Fenoglio; A. Grosso; E. Boncompagni; G. Milanesi; C. Gandini; S. Barni (pp. 690-702).

The epidermis of vertebrates is the body’s principal barrier against environment and its possible contaminants. The presence of keratins, as well as specific detoxifying molecules or enzyme activities, in the various epidermis layers is believed to be involved in providing protection from harmful environmental influences. Anuran integument is poorly hornified and thus permeable to some endogenous and exogenous compounds and thus serves as a good bioindicator of overall environmental conditions. In the present investigation, we studied the epidermis of Rana kl. esculenta adult specimens collected at two different rice fields, relatively unpolluted and heavily polluted, respectively. Environmental pollution was assayed by chemical analysis performed on both sediments and animals. We evaluated the structural aspects of the epidermis at both light and electron microscopy levels and the pattern of keratinization by immunohistochemistry. Furthermore, we studied the activities of some enzymes (acid and alkaline phosphatase, nitric oxide synthase–related nicotinamide adenine dinucleotide phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, catalase, nonspecific esterases, and succinic dehydrogenase) involved mainly in membrane transport, xenobiotics, and oxidative metabolism. Compared with controls, in polluted animals we found the following results: (1) an increase in pollutant levels (i.e., cadmium, mercury, and lead); (2) less keratinized superficial cells in the epidermis; and (3) changes in most enzyme activities in keratinocytes and mitochondria-rich cells (particularly glucose-6-phosphate dehydrogenase and esterases, both important to counteract oxidative and toxic stress). Taken as a whole, the present data indicate the morphofunctional plasticity of the frog epidermis in response to environmental contamination.

Comparative Developmental Toxicity of Nickel to Gastrophryne carolinensis, Bufo terrestris, and Xenopus laevis by Douglas J. Fort; Robert L. Rogers; John H. Thomas; William A. Hopkins; Christian Schlekat (pp. 703-710).

The early embryo-larval developmental toxicity of nickel (Ni) to 3 amphibian species, Xenopus laevis (South African clawed frog), Bufo terrestris (southern toad), and Gastrophryne carolinensis (eastern narrow-mouthed toad), was evaluated using a modified FETAX model. Studies were initiated from late blastulae stage (Nieuwkoop and Faber [NF] stage 10 or Gosner stage 12) and completed at a common embryological-based test termination point, which represented the completion of the major stages of organogenesis (NF stage 46 for Xenopus or Gosner stage 26 for the toads). Results indicated that, in terms of lethality, G. carolinensis was the most sensitive and X. laevis was the least sensitive of the species tested. The 4-d LC50 in X. laevis value was approximately 7.2- and 2.8-fold greater than the G. carolinensis and B. terrestris, respectively. In terms of malformation, X. laevis was the most sensitive and B. terrestris was the least sensitive of the species tested. The 7-d EC50 (malformation) in B. terrestris was 10.6- and 7.0-fold greater than X. laevis or G. carolinensis, respectively. The chronic value (ChV) for growth in X. laevis was nearly 4.5-fold less than the ChV for growth determined for B. terrestris. As with the malformation endpoint, X. laevis was more sensitive than the other species, which were nearly equisensitive. Overall, the present study provides new data regarding the toxicity of Ni to larval amphibian species, which may be useful in the establishment of new aquatic life criteria for Ni.

Effects of Contaminants on Reproductive Success of Aquatic Birds Nesting at Edwards Air Force Base, California by R. L. Hothem; J. J. Crayon; M. A. Law (pp. 711-719).

Contamination by organochlorine pesticides (OCs), polychlorinated biphenyls, metals, and trace elements at Edwards Air Force Base (EAFB), located in the Mojave Desert, could adversely affect nesting aquatic birds, especially at the sewage lagoons that comprise Piute Ponds. Estimates of avian reproduction, in conjunction with analyses of eggs and avian foods for contaminant residues, may indicate the potential for negative effects on avian populations. From 1996 to 1999, we conducted studies at the Piute Ponds area of EAFB to evaluate the impacts of contaminants on nesting birds. Avian reproduction was evaluated in 1999. Eggs were collected for chemical analyses in 1996 and 1999, and African clawed frogs (Xenopus laevis), a likely food source, were collected for chemical analyses in 1998. Avian species occupying the higher trophic levels—black-crowned night-heron (Nycticorax nycticorax), white-faced ibis (Plegadis chihi), and American avocet (Recurvirostra americana)—generally bioaccumulated higher concentrations of contaminants in their eggs. Reproductive success and egg hatchability of night-herons and white-faced ibises in the Piute Ponds were similar to results observed at other western colonies. Deformities were observed in only one embryo in this study, but concentrations of contaminants evaluated in this ibis embryo were considered insufficient to have caused the deformities. Because clawed frogs, a primary prey item for night-herons at Piute Ponds, had no detectable levels of any OCs, it is likely that OCs found in night-heron eggs were acquired from the wintering grounds rather than from EAFB. The presence of isomers of dichlorodiphenyltrichloroethane (DDT) in ibis eggs indicated recent exposure, but invertebrates used for food by ibises were not sampled at Piute Ponds, and conclusions about the source of OCs in ibis eggs could not be made. Concentrations of contaminants in random and failed eggs of individual species were not different, and we concluded that contaminants did not cause the observed egg failures.

Interfollicular Fibrosis in the Thyroid of the Harbour Porpoise: An Endocrine Disruption? by Krishna Das; Arndt Vossen; Kristal Tolley; Gisli Víkingsson; Kristina Thron; Gundi Müller; Wolfgang Baumgärtner; Ursula Siebert (pp. 720-729).

Previous studies have described high levels of polychlorobiphenyls (PCB), polybrominated diphenylether (PBDE), toxaphene, p,p′-dichlorodiphenyltrichloroethane (DDT), and p,p′-dichlorodiphenyldichloroethylene (DDE) in the blubber of the harbour porpoise from the North Sea raising the question of a potential endocrine disruption in this species. In the present study, the thyroids of 57 harbour porpoises from the German and Danish (North and Baltic Seas), Norwegian, and Icelandic coasts have been collected for histological and immunohistological investigations. The number of follicles and the relative distribution of follicles, connective, and solid tissues (%) were quantified in the thyroid of each individual. Then, the potential relationship between the thyroid morphometry data and previously described organic compounds (namely, PCB, PBDE, toxaphene, DDT, and DDE) was investigated using factor analysis and multiple regressions. Thyroid morphology differed strongly between sampling sites. Porpoises from the German (North and Baltic Seas) and Norwegian coasts displayed a high percentage of connective tissues between 30 and 38% revealing severe interfollicular fibrosis and a high number of large follicles (diameter >200 μm). A correlation-based principal component analysis (PCA) revealed two principal components explaining 85.9% of the total variance. The variables PCB, PBDE, DDT, and DDE compounds loaded highest on PC1 whereas toxaphene compound loaded most on PC2. Our results pointed out a relationship between PC1 (PCBs, PBDE, DDE, and DDT compounds) and interfollicular fibrosis in the harbour porpoise thyroids. Such an association is not alone sufficient for a cause–effect relationship but supports the hypothesis of a contaminant-induced thyroid fibrosis in harbour porpoises raising the question of the long-term viability in highly polluted areas.

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Archives of Environmental Contamination and Toxicology (v.51, #4)