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Archives of Environmental Contamination and Toxicology (v.34, #1)

Toxicity of Inorganic Compounds in the Spirotox Test: A Miniaturized Version of the Spirostomum ambiguum Test by G. Nałe¸cz-Jawecki; J. Sawicki (pp. 1-5).

The Spirostomum ambiguum toxicity test has been intensively studied in the Department of Environmental Health Sciences, Warsaw University of Medicine for the last 5 years. The purpose of the present work was to develop and evaluate a miniaturized microplate version of the test, called the Spirotox test, and to estimate the toxicity of selected inorganic compounds to the Spirostomum ambiguum. The test was carried out in conventional 24-well (6 × 4) polystyrene multiwell plate. Preliminary test was one control and 11 toxicant concentrations with two duplicates. Definitive test was one control and five toxicant concentrations with three duplicates per concentration. Dilution of the sample was made directly in the plate. Toxicity series of heavy metals based on 24-h LC50 may be established as follows: Cu > Ag > Hg > Cr > Cd > Zn > Ni > Pb > Co > Mn. The series may be divided into four classes: extremely toxic: below 0.1 ppm (Cu, Ag, Hg); very toxic: 0.1–1.0 ppm (Cr, Cd, Zn, Ni); toxic: 1.0–10 ppm (Pb, Co); and low toxic: above 10 ppm (Mn). Anions were much less toxic to S. ambiguum than cations. Using the same classification, only cyanide (CN) was toxic, other anions were low toxic. Toxicity series based on 24-h LC50 may be established as follows: CN > SeO₃ > Cr₂O₇ > NO₂ > S₂O₃ >WO₄ > BO₃.

Liquid-Gas Partitioning of the Gasoline Oxygenate Methyl tert-Butyl Ether (MTBE) Under Laboratory Conditions and Its Effect on Growth of Selected Algae by J. M. Rousch; M. R. Sommerfeld (pp. 6-11).

The partitioning of the widely used gasoline additive methyl tert-butyl ether (MTBE) between liquid growth media and gaseous phase was measured daily under laboratory conditions to determine how closely dissolved MTBE concentrations matched nominal concentrations. Total (gaseous and dissolved) MTBE averaged across 6 days for 29.6, 503.2, and 1005.7 mg L⁻¹ MTBE treatments were 89.9, 90.3, and 73.0% of nominal, respectively, and mean dissolved MTBE in these same treatments were 74.6, 73.8, and 69.6% of total MTBE, respectively. This suggests that dissolved MTBE concentrations can vary substantially from nominal. The effect of MTBE on the growth of selected algae was also evaluated under laboratory conditions. Three unicellular algae, Selenastrum capricornutum (Chlorophyta), Navicula pelliculosa (Bacillariophyta), and Synechococcus leopoliensis (=Anacystic nidulans, Cyanophyta = Cyanobacteria), representative of three taxonomic groups, were used as test organisms. Toxicity tests were acute and increase in cell number was used as an indicator of growth. Algal species were exposed by injection of MTBE into sealed vessels containing defined liquid growth media. The growth of N. pelliculosa and S. leopoliensis was negatively affected at nominal 2400 mg L⁻¹ MTBE, whereas the growth of S. capricornutum was negatively affected at nominal 4800 mg L⁻¹ MTBE and positively affected at nominal 600 mg L⁻¹ MTBE. The differential sensitivity of the growth of these representative species suggests that MTBE may alter algal community composition in the natural environment.

Influence of Water Hardness on Accumulation and Elimination of Cadmium in Two Aquatic Mosses Under Laboratory Conditions by C. Gagnon; G. Vaillancourt; L. Pazdernik (pp. 12-20).

This study investigated the effect of water hardness on the accumulation and elimination of cadmium (Cd) by two aquatic mosses, Fontinalis dalecarlica and Platyhypnidium riparioides, under laboratory conditions. The two mosses were exposed to nominal Cd concentrations of 0, 0.8, 2, and 10 μg · L⁻¹, which includes the concentration range generally found in nature. The influence of three levels of water hardness (very soft: 11.7 mg · L⁻¹; soft: 44.2 mg · L⁻¹; and hard water: 92.3 mg · L⁻¹ as CaCO₃) was measured while maintaining the alkalinity and pH constant during the 28-day exposure. The Cd accumulation by the aquatic mosses was rapid, showing the potential of accumulation and the sensitivity of this biomonitor. Even if the actual Cd concentration in the water was low (concentration <0.15 μg · L⁻¹ to 6.82 μg · L⁻¹ of Cd), the uptake of Cd was very fast and mostly linear. This study was conducted in water hardness comparable to that found in the Canadian shield (hardness was <100 mg · L⁻¹ as CaCO₃). When the actual Cd concentration in the water was as high as 6.82 μg · L⁻¹, the uptake of Cd was mostly linear and the steady state condition was not reach. Accumulation rates of Cd were significantly different when the mosses were in very soft (11.7 mg · L⁻¹) as compared to hard water (92.3 mg · L⁻¹ as CaCO₃). The elimination of Cd followed a very slow process for the two species studied. The elimination rates of Cd from the mosses were not influenced by water hardness.

Alteration of [¹⁴C]-Testosterone Metabolism After Chronic Exposure of Daphnia magna to Tributyltin by E. Oberdörster; D. Rittschof; G. A. LeBlanc (pp. 21-25).

Tributyltin (TBT) is a marine biocide that has been shown to alter the activity of cytochrome P450 monooxygenases and elicit toxicity indicative of androgenization in some species. The present study was conducted to determine whether TBT altered P450-, reductase-, and transferase-mediated testosterone metabolic processes in Daphnia magna at sublethal exposure concentrations. Two generations of daphnids were continuously exposed for 21 days to nominal TBT concentrations ranging from 0.31 to 2.5 μg/L TBT. The highest TBT concentration (2.5 μg/L) was lethal to 60% of the exposed organisms. Lower TBT concentrations elicited no adverse effects on molting or reproduction of the daphnids. No differences were observed in the response of the first- and second-generation daphnids to the toxicity of TBT. The ability of daphnids to metabolize [¹⁴C]-testosterone in vivo was assessed following exposure of each generation to TBT. Production of hydroxylated, reduced/dehydrogenated, and glucose-conjugated metabolites of testosterone were all elevated following exposure of both generations to 1.25 μg/L TBT. These findings indicate that, under these conditions, TBT elicits no discernible effects on molting and reproduction of daphnids at sublethal concentrations, and testosterone metabolism is enhanced at concentrations approaching those that are lethal to organisms. Alterations of steroid metabolism by xenobiotics can be used as a more sensitive indicator of sublethal exposure in daphnids than reproductive endpoints.

Application of a Benthic Euryhaline Amphipod, Corophium sp., as a Sediment Toxicity Testing Organism for Both Freshwater and Estuarine Systems by R. V. Hyne; D. A. Everett (pp. 26-33).

The use of an as-yet-undescribed euryhaline Corophium sp. amphipod as a sediment toxicity testing organism was assessed. The species was found to be ubiquitous in many tidal areas of the Hawkesbury River catchment. The salinity of habitat sites ranged from 0.1 to 24 ppt, sediment total organic carbon (TOC) ranged from 0.4% to 3.5%, and the fines content (< 63 μm particle size) of the sediment ranged from 4.3% to 47.6%. Monitored populations ranged from a density of 59 to 6622 individuals per m², with freshwater sites with a sediment fines content greater than 20% having the highest population densities. The sensitivity of the Corophium sp. was assessed by using copper chloride and ammonium chloride as reference toxicants in a 96-h static water-only test and a 10-day static sediment test. The LC50 for copper in freshwater-only exposures was 80 to 86 μg/L, using adult animals collected from the field. In contrast, the LC50 for copper in freshwater sediment and the sediment pore water were 840 mg/kg (dry weight) and 99 μg/L, respectively. The LC50 for ammonia (total) in freshwater-only at pH 7 was 5.5 mg/L. In contrast, the LC50 for ammonia (total) in freshwater sediment and the sediment pore water were 110 mg/kg (dry weight) and 6 mg/L, respectively. Laboratory cultures of 5‰ to 15‰ salinity were optimal for supporting the release of juveniles. Juveniles collected from laboratory cultures had a LC50 for copper in 5‰ and 10‰ salinity of 9 μg/L and 28.5 μg/L, respectively, in water-only exposures. The juveniles would be suitable for use in the development of a chronic sediment toxicity test with growth as the endpoint.

Biotic Factors Modifying Acute Toxicity of Aqueous Cadmium to Estuarine Amphipod Leptocheirus plumulosus by B. L. McGee; D. A. Wright; D. J. Fisher (pp. 34-40).

A 96-h exposure to aqueous cadmium (Cd) is the recommended reference toxicity test for 10-day sediment bioassays with the estuarine amphipod, Leptocheirus plumulosus (US EPA 1994). This water-only test was used to assess the influence of organism size, sex, and nutritional status on the sensitivity of laboratory-cultured L. plumulosus to Cd. In addition, the response of field-collected amphipods was compared to similarly sized laboratory animals to assess potential seasonal changes in Cd sensitivity. Lipid content of test organisms was measured in these seasonal experiments and those evaluating effects of nutritional status because of its potential as an indicator of physiological condition. LC₅₀ values of laboratory animals size-sorted on nested 500-, 710-, and 1000-μm mesh sieves, increased with size class: 0.36, 0.65, and 0.88 mg Cd/L, respectively. Gravid females were less sensitive than males or mature females to aqueous Cd. Studies on the influence of the molt cycle on Cd toxicity indicated enhanced sensitivity of immediate postmolt animals that may explain some of the observed differences in Cd tolerance. Nutritional effects were investigated by comparing the sensitivity of fed and starved laboratory-reared amphipods. Starved juveniles and adults were significantly smaller than their fed counterparts and exhibited a 28–43% reduction in lipid content, respectively. However, comparison of LC₅₀ values indicated no significant differences in sensitivity to Cd between starved and fed juveniles (0.23 vs 0.30 mg Cd/L) or adults (0.37 vs 0.52 mg Cd/L). Field-collected amphipods were typically more sensitive to Cd than laboratory animals, regardless of the season, although their lipid content varied, ranging from 6.6% in August to 13.7% in November. Results are discussed with respect to the use and interpretation of toxicity tests with this species.

The Influence of Fresh Water Pollutants and Interaction with Asellus aquaticus (L.) on the Feeding Activity of Gammarus pulex (L.) by S. J. Blockwell; E. J. Taylor; I. Jones; D. Pascoe (pp. 41-47).

The feeding response of juvenile amphipod Gammarus pulex (L.) was investigated following exposure to freshwater pollutants. The method employed is nondestructive, provides a rapid indication of the status of groups of individuals, and is based on a time-response analysis of the consumption of the eggs of Artemia salina and the determination of median feeding times or FT50s. The feeding activity of juvenile G. pulex was found to be a sensitive response criterion for use in assessing the sublethal toxicity of copper, lindane, and 3,4-dichloroaniline (3,4-DCA). Reductions in gammarid feeding activity were identified following 96 hours exposure at 12.1 μg/L copper or 8.4 μg/L lindane and 240 hours exposure at 918 μg/L 3,4-DCA. However, a significant increase was observed in the feeding rate of gammarids that had been exposed for 240 h at 0.09 μg/L lindane in comparison with control values. The increase in feeding rate may be interpreted as a possible stimulatory effect associated with the toxicant action of lindane. Increases in gammarid feeding activity were not determined during the experiments conducted with either copper or 3,4-DCA. A sustained reduction in G. pulex feeding rates may cause growth inhibition and impaired reproduction which have previously been identified as sublethal responses of other freshwater organisms exposed to comparable concentrations of lindane, 3,4-DCA, or copper. The feeding bioassay was also used as a tool in an investigation of species interactions in toxicant systems. The feeding responses of G. pulex, which had been maintained in the presence of Asellus aquaticus (as interacting pairs) and exposed to a range of concentrations of lindane or 3,4-DCA, were recorded and compared. The findings illustrate the complex nature of test systems that integrate the stresses of toxicant and competition. In the lindane test system a reduction in gammarid feeding activity was observed following a 96-h exposure with A. aquaticus at 3.8 and 6.0 μg/L lindane (mean measured concentrations). After a 240-h exposure period a decrease in feeding rate was recorded only for gammarids that had been exposed to 6.5 μg/L lindane, however exposure to very low concentrations of lindane (0.1 and 0.9 μg/L) resulted in a significant increase in gammarid feeding activity. In the experiment conducted with 3,4-DCA the calculation of median feeding times or FT50s of gammarids that had been exposed for 96 and 240 h in the toxicant treatment groups with A. aquaticus was largely precluded (in most groups less than 50% of the A. salina eggs were eaten). However, control group FT50 values were determined on each occasion the bioassay was performed, indicating that a substantial reduction in gammarid feeding activity had occurred in the majority of the 3,4-DCA treatment groups.

Trace Metals and Antioxidant Enzymes in Gills and Digestive Gland of the Mediterranean Mussel Mytilus galloprovincialis by F. Regoli (pp. 48-63).

A seasonal variability of trace metal concentrations and antioxidant enzymes was observed in gills and digestive gland of the Mediterranean mussel Mytilus galloprovincialis from both a polluted and a nonpolluted population. Trace metals (As, Cu, Fe, Mn, Pb, and Zn) exhibited, in both organs, maximum values in later winter–early spring followed by a progressive decrease during the summer. While in the gills this behavior probably reflects a different bioavailability of metals, in the digestive gland it is influenced mainly by the progressive infiltration of the organ by gonadic tissues during gametogenesis. Metals, as other pollutants, are known to influence the oxidative status of these organisms and antioxidant enzymes have been often proposed as biomarkers of exposure to contaminants. In this respect, it was of interest to compare the variations of these biochemical parameters with those of metal levels in two mussel populations from a polluted and a nonpolluted site, respectively. The biochemical parameters examined included the level of glutathione and the activity of the following glutathione dependent and antioxidant enzymes: glyoxalase I, EC 4.4.1.5; glyoxalase II, EC3.1.2.6; glutathione S-transferases, EC 2.5.1.18; glutathione reductase, EC 1.6.4.2; Se-dependent, EC 1.11.1.9 and Se-independent, EC 2.5.1.18 glutathione peroxidases; catalase, EC 1.11.1.6; superoxide dismutase, EC 1.15.1.1. Seasonal variations of trace metals did not appear to influence those of biochemical parameters, which generally showed an opposite trend with higher enzymatic activities in summer when trace metal concentrations were lower. The effects of metals on antioxidant enzymes were more evident when the two mussel populations were compared. In particular, organisms from the polluted site showed lower levels of glutathione and higher enzymatic activities of glyoxalase I even though the magnitude of these differences was not constant during the year. Moreover, native mussels from both the polluted and control populations exhibited limited differences in the activities of glutathione reductase, glutathione peroxidases, catalase, and superoxide dismutase, suggesting the possibility of some biochemical adaptation in organisms from chronically polluted environments.

Cadmium Accumulation in Liver and Kidneys and Hepatic Metallothionein and Glutathione Levels in Rana ridibunda, After Exposure to CdCl₂ by A. K. Vogiatzis; N. S. Loumbourdis (pp. 64-68).

Adult female Rana ridibunda were exposed to 200 ppm (mg/l) of cadmium (Cd as CdCl₂) dissolved in water for 4, 10, and 30 days. The 96-h LC₅₀ value for Cd was determined to 534 ppm. The concentration of Cd in the liver and kidneys and the effect of Cd on the concentrations of hepatic metallothionein (Mts) and glutathione (GSH), were estimated. Cd accumulated in the liver and kidneys in a time-dependent pattern, with the kidneys accumulating the heavy metal at higher rates after the tenth day of exposure. There was a strong positive correlation of Cd concentration between these two organs. The concentration of Mts and GSH increased with the increase of Cd concentration in the liver, following a time- and Cd-dependence pattern. The concentration of Mts and GSH was positively correlated with the concentration of Cd in the liver. Mts concentration was positively correlated with GSH concentration.

Biomarker Responses in Whitefish (Coregonus lavaretus L. s.l.) Experimentally Exposed in a Large Lake Receiving Effluents from Pulp and Paper Industry by M. R. Soimasuo; A. E. Karels; H. Leppänen; R. Santti; A. O. J. Oikari (pp. 69-80).

Physiological and biochemical biomarker responses were studied in juvenile whitefish (Coregonus lavaretus L. s.l.) exposed experimentally to effluent from the forest industry. The large study area (609 km²), Southern Lake Saimaa, in Southeast Finland, receives 330,000 m³ d⁻¹ of biologically and 55,000 m³ d⁻¹ of chemically treated effluents, discharged from two integrated elementary chlorine free (ECF) bleached kraft pulp and paper mills, from one ECF pulp mill, and from one mill producing unbleached pulp and cardboard. The assessment of exposure to effluent discharged from the mills was based on lake water chlorophenolics (CPs) and resin acids (RAs) measured in samples collected from the 22 experimental sites along the area. Despite the low levels of effluent constituents in the lake, they were still accumulated in detectable levels in fish bile, indicating an exposure to the bioactive compounds of effluents. In comparison to the reference area, a two- to four-fold increase in ethoxyresorufin O-deethylase (EROD) activity was observed in whitefish exposed in the vicinity (1–6 km) of all the mills. However, cytochrome P450 1A1 (CYP1A1) gene expression was increased in only one of the receiving areas, indicating higher sensitivity of the EROD activity in the present study. There were no statistically significant correlations between EROD activity and the ambient water concentrations of the CPs, the RAs, or effluent dilution expressed by water sodium concentration. Neither bile chlorophenolics nor bile resin acids showed a significant correlation with EROD. No significant changes in circulating reproductive steroids, 17β-estradiol and testosterone, in juvenile whitefish were observed. The vitellogenin gene was expressed in the vicinity of the pulp mill discharging the most wood-derived compounds, i.e. resin acids and wood-sterols, including β-sitosterol. No differences were observed in plasma immunoglobulin M, glucose, or lactate concentrations between the effluent sources.

Concentrations and Hazard Assessment of Organochlorine Contaminants and Mercury in Smallmouth Bass from a Remote Lake in the Upper Peninsula of Michigan by K. S. Henry; K. Kannan; B. W. Nagy; N. R. Kevern; M. J. Zabik; J. P. Giesy (pp. 81-86).

Concentrations of PCBs, DDTs, toxaphene, chlordanes, dieldrin, and mercury were determined in smallmouth bass (Micropterus dolomieui) collected from Fumee Lake, a remote lake in the Upper Peninsula of Michigan. An ecological hazard assessment was conducted to determine potential impacts of contaminants on bald eagles and mink eating fish from this lake. Concentrations of organochlorines, except toxaphene, and mercury in smallmouth bass were similar to those found in fish from Lake Superior, where atmospheric inputs are the primary sources. Bioaccumulation was indicated by a positive correlation between fish weight and contaminant concentrations for organochlorines, while mercury concentrations did not appear to correspond predictably to body weight. Concentrations of mercury and PCBs in smallmouth bass were sufficiently great to be of concern regarding their consumption by eagles or mink.

Heavy Metal, Organochlorine Pesticide, and PCB Residues in Eggs and Feathers of Herons Breeding in Northern Italy by M. Fasola; P. A. Movalli; C. Gandini (pp. 87-93).

We report on organochlorine pesticide and PCB concentrations in eggs of the little egret, Egretta garzetta, and the black-crowned night-heron, Nycticorax nycticorax, collected in 1993–1994, and on mercury, cadmium, and lead concentrations in feathers of 20-day-old nestlings collected from the same nests in 1994, from heronries near Pavia, northern Italy. Organochlorine pesticide and PCB residues were lower than those commonly associated with mortality and reduced reproductive success. As population levels of the species studied are not declining, these contaminants appear to have no significant adverse effect on reproduction in the heronries studied. DDE levels have decreased markedly in heron eggs since 1978. However, the presence of both DDT and β-HCH, albeit at low levels, is notable, given that these compounds were banned in Italy in 1978 and 1988, respectively. Relatively high levels of Hg, Cd, and Pb in feathers suggest birds in their colonies are exposed to these contaminants, although both Cd and Pb may relate more to external than to internal contamination.

Evaluation of Early Embryonic Loss Induced by Tributyltin Chloride in Rats: Phase- and Dose-Dependent Antifertility Effects by A. Harazono; M. Ema; Y. Ogawa (pp. 94-99).

In our previous study, tributyltin chloride (TBTCl) on days 0–7 of pregnancy was found to produce implantation failure in rats. The objective of the present study was to determine the susceptible period for the antifertility effect of TBTCl in rats. Inseminated females were orally administered TBTCl at 8.1, 16.3, or 32.5 mg/kg on days 0–3 of pregnancy, or at 8.1, 16.3, 32.5, or 65.1 mg/kg on days 4–7 of pregnancy. Pregnancy outcome was determined on day 20 of pregnancy. Dosing with TBTCl on days 0–3 of pregnancy at 16.3 mg/kg and higher produced a significant increase in the rate of implantation failure. Dosing with TBTCl on days 4–7 of pregnancy caused a significant increase in the incidence of postimplantation loss at 16.3 mg/kg and higher in females with implantations. No increase in the incidence of fetal malformations was found in any TBTCl-treated groups. It could be concluded that the susceptibility to and manifestation of the antifertility effects of TBTCl vary with the gestational stage at the time of administration.

Concentration of Mercury in Hair of Indigenous Mothers and Infants from the Amazon Basin by A. C. Barbosa; S. R. L. Silva; J. G. Dórea (pp. 100-105).

Hair mercury concentration, as an indicator of mercury body load, was studied in 251 samples of indigenous women and children living in selected areas of the Amazonian region. The mothers or women of child-bearing age, either non-Indians or Indians, and their children were sampled along the Madeira River and in the Kayapó reservation (Fresco River), respectively. Among the sampled individuals there were mothers with infants less than 2 years old. Total mercury in hair was determined by cold vapor atomic absorption spectrometry after alkaline digestion. The distribution of hair mercury concentration greater than 10 μg/g occurred in 67.4% of non-Indian women and 25% of Indian women; overall only 1% of non-Indian women had concentrations of hair mercury above 50 μg/g. In women of child-bearing age, the median and range of hair mercury concentration was 14.08 μg/g, and 0.8–94.7 μg/g for non-Indians, and 8.30 μg/g, and 0.8–13.3 μg/g for Indians. The correlation between maternal hair mercury and mercury in hair of infants (less than 2 years of age) still breast-feeding, was statistically significant only for non-Indians (r = 0.555 p < 0.001). The correlation between length of breast-feeding and mercury concentration in infant's hair was significant for Indian children (r = 0.512; p = 0.029) but not for non-Indian children (r = 0.025; p = 0.832). A subsampling of 30 mothers had segmented hair analysis that showed a mean decrease of 20% in body burden during pregnancy, thus indicating the extent of placental transference of mercury to fetuses.

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Archives of Environmental Contamination and Toxicology (v.34, #1)