Single and Combined Effects of Sediment-Associated PAHs on Three Species of Freshwater Macroinvertebrates

Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous pollutants of sediments. Sediment quality criteria often use toxicity data for individual PAHs. However, PAHs always occur in field sediments as a complex mixture of compounds. In this study, the toxicity of phenanthrene (P), fluoranthene (FLA) and benzo(k)fluoranthene (B), alone or in combination, was assessed using monospecific sediment tests of acute toxicity (between 24h and 14 days). The test sediments were spiked formulated sediments. Toxicity of PAHs was determined on a freshwater pelagic organism, Daphnia magna, on an epibenthic organism, Hyalella azteca, and on a benthic organism, Chironomus riparius. The phototoxicity of fluoranthene and phenanthrene was verified. Benzo(k)fluoranthene was not phototoxic and presented no toxicity for the three organisms tested up to 300mg/kg, concentration rarely found in the environment. For all PAHs, the toxicity towards Daphnia magna was two-fold higher in the sediment toxicity tests than in the water-only standard tests, probably due to suspended contaminated particles and contact of daphnids with sediment. For phenanthrene, the LC50s found were 50, 15 and 20mg/kg dry weight sediment, respectively for Daphnia magna, Hyalella azteca and Chironomus riparius. The bioassays on fluoranthene yielded LC50s of 10, 5 and 15mg/kg dw respectively for Daphnia magna, Hyalella azteca and Chironomus riparius. LC50s of the PAHs mixture (1/3 of each compound by weight) were around 10mg/kg dry weight sediment for Hyalella azteca and 20mg/kg for Chironomus riparius. The effects of PAHs in the mixture seemed to be synergistic. This study gives new data about PAH toxicity and showed that the additivity of PAH effects could be inadequate to assess the environmental risk.     

Hyalella azteca 10-day sediment toxicity test: Comparison of growth measurement endpoints

Hyalella azteca are presently used in toxicity tests designed to evaluate adverse biological effects associated with exposure to contaminated sediments. Biological endpoints examined in a sediment test include changes in survival, as well as sublethal effects such as changes in growth, and reproduction. Length and weight, two methods used routinely to measure growth, were compared using H. azteca recovered from a 10-day sediment toxicity test. Length of individual organisms was measured using an image analyzer and dry weight of organisms was determined using an electronic microbalance. Statistical analysis was conducted using one-way analysis of variance to compare treatment sites. Length measures were shown to have greater power, a small minimum detectable difference, and smaller amount of underlying variability. The results indicate that length, measured using an image analyzer, was a more sensitive means to detect significant changes in growth and can be conducted with a greater degree of statistical confidence than use of weight measures. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 243–248, 1998     

A caridean grass shrimp (Palaemonetes pugio Holthius) as an indicator of sediment quality in Florida coastal areas affected by point and nonpoint source contamination

Grass shrimp are one of the more widely distributed estuarine benthic organisms along the Gulf of Mexico and Atlantic coasts, but they were used infrequently in contaminated sediment assessments. Early‐life stages of caridean grass shrimp, Palaemonetes pugio (Holthius), were used in this baseline survey as indicators of sediment pore water quality for several Florida coastal areas affected by nonpoint and point sources of contamination. Static toxicity tests were conducted using 24 well culture plates assessing embryo and larval survival after 12 days exposure to pore water (interstitial water). Acute toxicity was observed in 28% of pore waters (15 of 53 samples) suggesting that sediment contamination was not widespread in the study area based on the response of this species. When toxicity was observed, grass shrimp pore water toxicity test was usually a more sensitive indicator of contamination than solid‐phase tests conducted with the epibenthic invertebrate, Mysidopsis bahia. P. pugio embryos were more sensitive than larval form and response of different brood stocks usually were similar (P>0.05) when exposed to same pore waters. Based on all considerations, this early‐life stage toxicity test shows promise being useful detecting pore water toxicity. This conclusion is based on availability of a test methodology which requires minimal space and effort, and the test species widespread geographical distribution. However, there are several remaining research needs, including the important issue of its sensitivity relative to that for other pore water test species as well as for species used in solid‐phase toxicity tests. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 234–242, 2000     

Screening for sediment toxicity in the Rio Santiago basin: A baseline study

Toxicity testing of sediment samples from the Rio Santiago and its main tributaries was performed using a battery of tests that included the ECHA dipstick biocide monitor, the MetPad test, the lettuce seed germination and the root elongation test, the nematode test, spot plate test, and the SOS Chromotest. Assessment was carried out on organic extracts, pore water, and direct sediment. Results of bioassays showed toxicity sources from tributaries and canals flowing into the river and areas being degraded by insults from industrial spills. © by John Wiley & Sons, Inc.     

Transfer modelling and toxicity evaluation of the effluent from an installation of cleansing and uranium recovery using a battery of bioassays

On July 7, 2008, a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) led to the spillage of uranium in a stream. The acute toxicity of the effluent was evaluated, and compared to the toxicity of uranium nitrate in bioassays using several organisms: Chlamydomonas reinhardtii, Daphnia magna, Chironomus riparius and Danio rerio. A sediment bioassay was also performed on C. riparius using water and sediment sampled along the river. Results showed that effluent EC₅₀ 72 h was 0.65 mg U/l for algae and LC₅₀ 48 h was 1.67 mg U/l for daphnia, while values obtained for uranium nitrate were higher. The LC₅₀ 96 h of effluent to C. riparius was 22.7 mg U/l, similar to value for uranium nitrate; the sediment collected was not toxic to C. riparius larvae. The LOEC of effluent and uranium nitrate on HT₅₀ of D. rerio were similar (0.03 mg U/l), but larvae were more sensitive to uranium nitrate than to effluent. Our results suggest that other substances contained in the effluent could potentially be toxic to wildlife in association with uranium. In parallel, the modelling of the transfers based on uranium measurements in the surface water was used to fill data gaps and assess the impact along the river. These results provided an estimate of exposure conditions that occurred along the river. This approach allowed us to see that the risk to ecosystem during this incident was certainly low and concerned a short period of time, but it could have existed at least for some species.     

Influence of chloride on the chronic toxicity of sodium nitrate to <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis> and <Emphasis Type="Italic">Hyalella azteca</Emphasis>

While it has been well established that increasing chloride concentration in water reduces the toxicity of nitrite to freshwater species, little work has been done to investigate the effect of chloride on nitrate toxicity. We conducted acute and chronic nitrate (as sodium nitrate) toxicity tests with the cladoceran Ceriodaphnia dubia and the amphipod Hyalella azteca (chronic tests only) over a range of chloride concentrations spanning natural chloride levels found in surface waters representative of watersheds of the Great Lakes Region. Chronic nitrate toxicity test results with both crustaceans were variable, with H. azteca appearing to be one of the more sensitive invertebrate species tested and C. dubia being less sensitive. While the variability in results for H. azteca were to an extent related to chloride concentration in test water that was distinctly not the case for C. dubia. We concluded that the chloride dependent toxicity of nitrate is not universal among freshwater crustaceans. An additional sodium chloride chronic toxicity test with the US Lab strain of H. azteca in the present study suggested that when present as predominantly sodium chloride and with relatively low concentrations of other ions, there is a narrow range of chloride concentrations over which this strain is most fit, and within which toxicity test data are reliable.     

Toxicity evaluation using bioassays in rural developing district 063 Hidalgo,Mexico

One of the oldest and biggest irrigation districts that use wastewater in Mexico is the Rural Developing District 063 (RDD-063). The water supplies of this district are the industrial and domestic discharges of Mexico City and its metropolitan area. RDD-063 production of grains, vegetables, and fruits as well as cattle products supplies the Mexico City market, so it is important to evaluate the chemical risks to which this population is exposed. Fourteen sampling sites were selected for collection of water, soil, and sediment samples in March, July, and October 1993. Bioassays with Daphnia magna, Spirillum volutans, and Panagrellus redivivus were used to estimate acute toxicity and genotoxicity in water samples, sediment pore water, and Milli-Q water extracts of soil. Acute toxicity effects were detected in March and October, while in July genotoxicity responses were found. The results demonstrate that RDD-063 had serious ecological damage, and its waters, soils, and sediments represent a hazard to animal and human health. Bioconcentration and persistence of toxic substances in drinking and irrigation water is a major problem that requires a long-term solution. © 1996 by John Wiley & Sons, Inc.     

Sediment contamination and biological effects in a Chesapeake Bay marina

Complementary measures of sediment toxicity, sediment chemistry and benthic community structure were evaluated at stations within and on the outside perimeter of an enclosed marina on the Bohemia River, a northeastern tributary to Chesapeake Bay. Sediment concentrations of polynuclear aromatic hydrocarbons, copper and tributyltin (TBT) were elevated at stations inside the marina basin. A 28 day partial life-cycle test with the amphipod Hyalella azteca indicated no significant lethal effects associated with test sediments. However, amphipods exposed to sediments collected from three stations inside the marina basin were significantly larger than amphipods from control sediments, possibly as the result of hormesis. Sediment pore water from two out of the three stations eliciting enhanced amphipod growth caused a reduction in light emission by luminescent bacteria in the Microtox^® assay. Furthermore, sediments from these two stations contained the greatest measured concentrations of copper and TBT. Benthic infaunal communities that typically reflect environmental degradation were found exclusively at stations within the marina basin. The area of environmental impact appears to be localized to the enclosed basin, as the marina design limits flushing and, hence, contaminant export.     

Assessing bioavailability and toxicity of permethrin and DDT in sediment using matrix solid phase microextraction

Matrix solid phase microextraction (matrix-SPME) was evaluated as a surrogate for the absorbed dose in organisms to estimate bioavailability and toxicity of permethrin and dichlorodiphenyltrichloroethane (DDT) in laboratory-spiked sediment. Sediments were incubated for 7, 28, and 90 days at room temperature to characterize the effect of aging on bioavailability and toxicity. Sediment toxicity was assessed using two freshwater invertebrates, the midge Chironomus dilutus and amphipod Hyalella azteca. Disposable polydimethylsiloxane fibers were used to estimate the absorbed dose in organisms and to examine bioavailability and toxicity. The equilibrium fiber concentrations substantially decreased with an increase in sediment aging time, indicating a reduction in bioavailability. Based on median lethal fiber concentrations (fiber LC50), toxicity of permethrin was not significantly different among the different aging times. Due to the substantial degradation of DDT to dichlorodiphenyldichloroethane (DDD) in sediment, sediment toxicity to C. dilutus increased, while it decreased for H. azteca with extended aging times. A toxic unit-based fiber LC50 value represented the DDT mixture (DDT and DDD) toxicity for both species. Significant linear relationships were found between organism body residues and the equilibrium fiber concentrations for each compound, across aging times. The study suggested that the matrix-SPME fibers mimicked bioaccumulation in the organisms, and enabled estimation of body residues, and could potentially be used in environmental risk assessment across matrices (e.g. sediment and water) to measure bioavailability and toxicity of hydrophobic pesticides.     

Chronic toxicity of tire and road wear particles to water- and sediment-dwelling organisms

Tire and road wear particles (TRWP) consist of a complex mixture of rubber, and pavement released from tires during use on road surfaces. Subsequent transport of the TRWP into freshwater sediments has raised some concern about the potential adverse effects on aquatic organisms. Previous studies have shown some potential for toxicity for tread particles, however, toxicity studies of TRWP collected from a road simulator system revealed no acute toxicity to green algae, daphnids, or fathead minnows at concentrations up to 10,000 mg/kg under conditions representative of receiving water bodies. In this study, the chronic toxicity of TRWP was evaluated in four aquatic species. Test animals were exposed to whole sediment spiked with TRWP at concentrations up to 10,000 mg/kg sediment or elutriates from spiked sediment. Exposure to TRWP spiked sediment caused mild growth inhibition in Chironomus dilutus but had no adverse effect on growth or reproduction in Hyalella azteca. Exposure to TRWP elutriates resulted in slightly diminished survival in larval Pimephales promelas but had no adverse effect on growth or reproduction in Ceriodaphnia dubia. No other endpoints in these species were affected. These results, together with previous studies demonstrating no acute toxicity of TRWP, indicate that under typical exposure conditions TRWP in sediments pose a low risk of toxicity to aquatic organisms.     

Ecotoxicity assessment of the aquatic environment around Lake Kojima,Japan

To reduce the impact of chemical substances on the aquatic ecosystem, it is essential to understand their ecotoxicological properties in the natural aquatic environment. Consequently, we conducted an ecotoxicological study on the aquatic environment around Lake Kojima, a man-made lake located in the southwest of Japan. Lake Kojima receives its chemical inputs mainly from two rivers that flow through various agricultural and industrial areas. For ecotoxicity screening, surface water and sediment samples were collected 4 times in 1993 from 16 preselected sites. Then, the solutes in the filtered surface water were concentrated by ODS resin, and the organic chemicals in the suspended solids (SS) and sediments were extracted by acetone. A battery of five ecotoxicity tests (agar plate test using bacteria and yeast, algal growth inhibition test, Daphnia magna immobilization test, and root elongation test using lettuce seeds) was used to assess these extracts. The results show that the surface water extracts had a lethal effect on D. magna, the SS extracts suppressed algal growth, and the sediment extracts were inhibitory to the growth of yeast. A significant inhibitory effect by the sediment extracts from 4 lake sites and 3 river sites was detected by these ecotoxicity tests. Attempts also were made to identify the putative ecotoxic chemicals in the collected samples. Elementary sulfur was identified as one of the major toxicants in the sediment extracts that were inhibitory to the yeast growth. Moreover, samples of surface water around Lake Kojima, collected weekly from June to September in 1994, were found to contain three pesticides and were toxic to D. magna. But the concentration of the pesticides detected was too low to cause daphnia immobilization. It is believed that the toxicity of the water extracts was mainly due to the combined toxic effect of natural and man-made components. © 1996 by John Wiley & Sons, Inc.     

Preliminary risk assessment of the wet landscape option for reclamation of oil sands mine tailings: Bioassays with mature fine tailings pore water

Chemical and biological assays have been carried out on the “pore water” that results from the settling of the tailings that accompany bitumen recovery from the Athabasca oil sands. Examination of the nonacidic extracts of pore water by gas chromatography–mass spectroscopy allowed the identification of numerous two‐ to three‐ring polycyclic aromatic compounds (PACs), to a total concentration of 2.6 μg/L of pore water. The PACs were biodegraded by microflora naturally present in the pore water. Acute toxicity was associated principally with the acidic fraction (naphthenic acids) of pore water extracts according to the Microtox assay; other work has shown that acute toxicity dissipates fairly rapidly. Both individual PACs and concentrated pore water extracts showed minimal levels of binding to the rat Ah receptor and induced minimal ethoxyresorufin‐O‐deethylase activity in primary rat hepatocytes, showing an insignificant risk of inducing monooxygenase activity. Taken together with previous work showing negligible mutagenic activity of these extracts, we conclude that it should be possible to develop tailing slurries into biologically productive artificial lakes. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 197–208, 2001     

Concentration of dioxin and screening level ecotoxicity of pore water from bottom sediments in relation to organic carbon contents

The information about concentrations of dioxin in pore water, ecotoxicity and DOC and TOC content can be key factor for the prediction of the fate of dioxins in the aquatic environment as well as an ecological risk assessment. The aims of the study were to assess the concentration of PCDDs/PCDFs and ecotoxicity of pore water and to compare above results in relation to the dissolved organic carbon (DOC) and total organic carbon (TOC) content. The concentration of dioxins was assessed using an enzyme-linked immunoassay test, while the ecotoxicity of pore water was determined using a crustacean Daphnia magna and bacteria Aliivibrio fischeri. The studies were conducted on two different dammed reservoirs Rożnów (catchment basin of an agricultural character) and Rybnik (catchment basin of an industrial character) located in southern Poland. The concentration of dioxins in pore water was between 8.56 to 90.92 ng EQ/L, with a significantly higher concentration in the pore water from the Rożnów Reservoir than the Rybnik Reservoir. The DOC content in pore water was from 30.29 to 63.02 mg/L (Rożnów Reservoir) and from 35.46 to 60.53 mg/L (Rybnik Reservoir). Higher toxic responses were recorded for A. fischeri than for D. magna. Moreover a significantly higher toxicity for both tested organisms was indicated in pore water from the Rożnów Reservoir. Besides of TOC and DOC, the fine fractions of the sediments were particularly important in the concentration of dioxin in pore water. The other pore water parameters, such as pH and EC can influence the toxicity of water for organisms. The result indicate complex relationships between the PCDD/F, ecotoxicity and DOC, TOC concentration in pore water and confirms that these parameters are important in terms of water environmental contamination.

     

Biomonitoring toxicity of natural sediments using juvenile Hyalella curvispina (Amphipoda) as test species: evaluation of early effect endpoints

The utility of early effect endpoints as biomarkers of ecotoxicity of natural sediments in water–sediment static system was investigated. The particular goal was to evaluate the ecotoxicity of the sediment samples from La Choza stream, located in upper basin of the Reconquista river, the second most polluted river of Argentina. Native juveniles Hyalella curvispina were used as test organisms evaluating survival, growth, oxidative stress parameters (SOD; CAT, TBARS) and the electron transport system (ETS) activity as early toxic effect. This study used methodologies and techniques that allow the assessment of sediment pollution with a native species as test organism and provided data to discuss the viability of sublethal endpoints as tools for freshwater sediment assessment. In spring and in summer two ten-day series of whole-sediment assays were conducted simultaneously: (a) standard assays and (b) biomarkers assays. A control sediment was ran simultaneously in which no––effect on survival was measured. In summer there was a significant increase in length and biomass in both exposed and control groups. In spring an inhibitory effect on growth and an increase in oxidative damage with a concomitant rise in antioxidant defenses, was observed in animals exposed to La Choza sediment. ETS measurement indicated a significant depression of metabolic activity of amphipods exposed to contaminated sediments. The measured biomarkers represent the first record for juvenile H. curvispina exposed to polluted natural sediments under standardized laboratory conditions. The used bioanalytical tools demonstrated higher sensitivity and a more accurate assessment of the effects than those obtained by the standard tests of survival and growth. We propose their adoption in biomonitoring of freshwater sediment toxicity.     

Toxicity of elemental sulfur in sediments

Elemental sulfur occurs naturally in marine and limnic sediments. Elemental sulfur, brought in solution in aqueous media by using organic solvents such as methanol as carrier solvent, was toxic in a bacterial luminescence test, known as the Microtox test. Previously, it has been shown that the toxicity in the luminescence test of whole sediments also was correlated to i.a. elemental sulfur using multivariate statistical analysis. Organic solvent extracts of sediments obtained in receiving waters of effluents from a pulp and paper mill was toxic in the luminescence test, and using a toxicity evaluation procedure, the toxic substance was identified as octameric cyclic sulfur, S₈. The substance dominated the toxicity in extracts of both a contaminated sediment and a sediment from a control area. Since the toxicity in the Microtox test of aqueous solutions of S₈ decreased upon storage, a conversion process of the toxic form was indicated. Acute toxicity of S₈ was not limited to the luminescent bacteria in the Microtox test, but was observed in tests with fish larvae if tested with the transient form of elemental sulfur. Tests of acute toxicity with zebra fish and perch larvae were responsive to elemental sulfur. Probably, the toxic form of elemental sulfur is the single cyclic octamer, that due to low aqueous solubility, binding to particulate sediment material or aggregation is converted into a nontoxic form. Acute toxic effects may occur in sulfur containing sediments of varying redox potentials or where elemental sulfur deposits are turbated. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 217–224, 1998     

Teratogenic effects of organic extracts from the Pearl River sediments on Xenopus laevis embryos

Toxicity of organic extracts from the Pearl River sediments was investigated with Xenopus laevis embryos. The effects of sediment organic extracts on the mortality, body length and malformation of X. laevis embryos were tested by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The 96-h LC₅₀ values for X. laevis embryos ranged from 62 to 137 g/L (g extracted sediment per L), and the toxicity effect on body length of larvae was not significant under 20 g/L. However, the teratogenic effects produced by sediment organic extracts were diverse, including edema, hypopigmentation, cardiac and ocular malformations, abdomen recurved and curved spine. The percentage of malformations increased with increasing sediment organic extracts, and even reached almost 100% at 10 and 20 g/L in Guangzhou district. A gradient of pollution in the Pearl River sediments was discerned from the teratogenic toxicity. Guangzhou district showed higher teratogenic toxicity compared with Panyu and Nansha districts as a possible consequence of high levels of PAHs, PCBs, OCPs and NP in the sediments. The teratogenic effects of organic extracts from the Pearl River sediments were successfully assessed which indicated the feasibility of teratogenic potential studies of sediments using X. laevis embryos.     

Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439?C455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8?C10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present.     

A test battery of bacterial toxicity assays and comparison with LD50 values

The toxicity of some mycotoxins, lactones and dicarboxylic anhydrides, insecticides, herbicides, and fungicides can be detected by means of bacterial assays. In addition, the order of magnitude of the toxicity can also be evaluated. Fifty-eight chemicals as well as extracts of peanuts, apple juices, and grains have been investigated in a test battery of three different kinds of assays. In cup plate diffusion assays a minimal amount of approximately 0. 1 mg/mL of mycotoxin may be detected by means of growth inhibition assays with Bacillus thuringiensis on one hand, and pigment synthesis inhibition assays with mutants of Serratia marcescens on the other hand. Swarming inhibition assays with the motile strains Azospirillum brasilense and Proteus mirabilis show the same sensitivity. Comparisons of our test battery with other bioassays, as well as with mammalian toxicity tests (LD₅₀ values), reveal correlations between these kinds of assays in regard to the test substances.     

Effects of copper on energy metabolism and larval development in the midge Chironomus riparius

When spiked in sediments, copper is known to reduce growth of Chironomus riparius larvae and the production of eggs by adult females. The aim of this work was to better understand the origin of these phenomena by studying the effects of copper using developmental and energetic biomarkers, such as changes in larval weight and age and changes in the levels of sugars and lipids. Four-day-old C. riparius larvae were exposed to nominal concentrations of copper of 0, 6.5, 12.5, 25 and 50 mg/kg of dry sediment (silica) in 0.6 l beakers. They were fed ad libitum and exposures were stopped at 7 and 9 days after the beginning of the tests. The larvae were weighed, sexed and aged. For each sex, the larvae belonging to the phases the most frequently found in the beakers were selected for dissection and measurement of energy reserves. The increase in the concentration of copper resulted in an increasing delay in larval growth in both sexes. Desynchronized development was observed, as shown by the increase in the number of individuals that remained in the third instar or early phases of the fourth instar, as well as by a reduction in age of males. Concerning energy reserves, the levels of sugars (glycogen, trehalose and glucose) in the dissected larvae remained almost constant among levels of exposure. In contrast, at the highest copper concentration (50 mg/kg), triglyceride levels suffered a slight reduction whereas the level of free glycerol significantly increased. It is concluded that selection of C. riparius larvae for both sex and age improves the relevance of some energy-yielding substrates as indicators of adverse physiological effects of copper.     

Evaluation of the genotoxicity of environmental contaminants in sediments to rainbow trout hepatocytes

Rainbow trout hepatocytes were used as an in vitro bioassay to assess the genotoxic potential of single chemicals and marine sediment extracts. Freshly prepared trout hepatocytes were exposed to either benzo[a]pyrene, N-methyl-N′-nitro-N-nitrosoguanidine, β-naphtoflavone, or organic extracts of marine sediments for 24 h at 15°C. Genotoxicity was assayed using the nick translation assay, which makes use of a nonradioactive nucleotide (biotin-dUTP), and the DNA alkaline precipitation assay followed by fluorometric detection of DNA strands. Exposure to benzo[a]pyrene or methyl-N′-nitro-N-nitrosoguanidine, known indirect-and direct-acting genotoxins respectively, produced genotoxicity to rainbow trout hepatocytes with both assays. β-Naphtoflavone displayed genotoxic activity in trout hepatocytes. Sediment extracts and reference sediment extracts displayed high toxicity and genotoxicity to trout hepatocytes. Chemical analyses showed that these sediments contained significant amounts of organochlorine pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons. Cell toxicity was correlated with total levels of organochlorine pesticides and polychlorinated biphenyls but not total levels of polycyclic aromatic hydrocarbons. No positive correlation was found with the nick translation assay between total levels of chemicals and genotoxicity in marine sediments. Genotoxicity obtained with the alkaline precipitation assay was correlated with levels of the organochlorine pesticide DDT. However, more tests would be required to further substantiate possible links with other specific chemicals. © by John Wiley & Sons, Inc.