Genotoxic effect of copper on salivary gland polytene chromosomes of Chironomus riparius Meigen 1804 (Diptera, Chironomidae)

The genotoxic action of copper (Cu) on the polytene chromosomes of Chironomus riparius was investigated by analysing structural and functional chromosome aberrations of fourth instars larvae hatched from eggs subject to acute (48 h) exposure with three environmentally relevant concentrations of aqueous Cu (0.005, 0.01, 0.05 mg/l). A dose dependent relationship was observed between Cu concentration and frequency of chromosomal aberrations. A significantly higher frequency of functional alterations, specifically decondensed centromeres and telomeres, and reduction in activity of Balbiani rings, was observed in treated material compared to control. A comparison of breakpoints resulting from treatment with chromium and lead from earlier studies with those Cu-induced identified a series of chromosomal weak points particularly vulnerable to trace metals. We also show that the appearance of structural and functional chromosome aberrations are more sensitive indicators of acute Cu toxicity in chironomid larvae than changes in external morphology.     

The effect of chronic toxicity of copper on the activity of Balbiani rings and nucleolar organizing region in the salivary gland chromosomes of Chironomus ninevah larvae

The effect of copper on the activity of Balbiani rings (BR1 and BR2) and nucleolar organizing region (NOR) in chromosome IV of the salivary gland of the 4th instar larvae of Chironomus ninevah has been investigated. Sublethal concentrations, i.e. 0.02, 0.1, 0.15 and 0.2 mg litre(-1) suppress the activity of BR1, and are statistically significant. The same concentrations reduced the activity of BR2 and NOR, but without significant differences, except 0.1 mg litre(-1) which was significant. The purpose of this work was to evaluate the effect of chronic toxicity of Cu(2+) on the expression of gene activity of midge salivary gland chromosomes Although there may be many more aquatic organisms that are more sensitive to copper, this midge was selected as a biological indicator species because of its association with benthic sediments.     

Assessing dimethoate contamination in temperate and tropical climates: potential use of biomarkers in bioassays with two chironomid species

This study was conducted to investigate the potential use of biomarkers in bioassays with chironomids to assess contamination by pesticides in temperate and tropical climates. Two species of midge were studied, the widespread Chironomus riparius and the tropical Kiefferulus calligaster (Kieffer, 1911). Preliminary studies included investigations of the effects of temperature on larval development and the influence of larval age on normal variability of cholinesterase (ChE) and glutathione S-transferase (GST) activities and protein content. In the second phase, the influence of two abiotic factors particularly important in tropical conditions (temperature and oxygen concentration) and of the organophosphorous (OP) insecticide dimethoate on biochemical and conventional endpoints was investigated. Results showed that K. calligaster is morphologically and physiologically similar to C. riparius and for both, the time of larval development decreases with the increase of temperature. Moreover, 3rd and 4th instars appeared to be the most suitable for biomarkers determinations. ChE activity seems to be valuable biomarker regarding temperature and dissolved oxygen (DO) variations, while some caution should be taken when using GST as an environmental biomarker, since it shows some dependence of these parameters. C. riparius was more sensitive to dimethoate than K. calligaster suggesting that the use of bioassays with the former species in tropical conditions may overestimate the toxicity of OP pesticides to autochthonous species. When testing sub-lethal effects of dimethoate to C. riparius, ChE activity showed to be a very sensitive parameter detecting significant effects at the lowest concentration that caused emergence delay of larvae, suggesting that it is an ecologically relevant parameter.     

Characterization of heat shock protein 40 and 90 in Chironomus riparius larvae: effects of di(2-ethylhexyl) phthalate exposure on gene expressions and mouthpart deformities

Di(2-ethylhexyl) phthalate (DEHP) is a widely distributed phthalate that organisms are frequently exposed to due to its wide range of commercial and manufacturing uses as a plasticizer. Indeed, DEHP is often found in freshwater systems that receive domestic waste water discharges. Therefore, we evaluated the effects of DEHP on the mRNA levels of heat shock protein (HSP) 40 and 90 in chironomids. In addition, we evaluated the effects of exposure to DEHP on the induction of morphological deformities in chironomids. To accomplish this, partial sequences of HSP 40 and 90 from Chironomus riparius larvae were amplified and sequenced. The inferred amino acid sequences were then aligned with those of other insect HSP 40 and 90 genes. The results of this alignment revealed that there was a high degree of similarity among the homologues. In addition, the HSP 40 and 90 mRNA expressions were significantly upregulated in response to short and long-term exposure to DEHP at concentrations of 1, 10, and 30 microgL(-1). Furthermore, the occurrence of mouthpart deformities was significantly higher in chironomids that were treated with DEHP (12-20%) than in controls (3-5%). Taken together, these results indicate that HSP 40 and 90 play important roles in the physiological changes related to metabolism and cell protection that occur in C. riparius larvae that have been exposed to DEHP.     

Low field-relevant tebufenozide concentrations affect reproduction in Chironomus riparius (Diptera: Chironomidae) in a long-term toxicity test

A few studies reporting the effects of tebufenozide, a non-steroidal ecdysone agonist that mimics natural moulting hormones in chironomids exist in the literature. However, nothing is known about its chronic or multigenerational effects on the reproduction of aquatic insects, although tebufenozide is present in aquatic ecosystems. Here, we investigated the chronic toxicity of tebufenozide in two successive generations of Chironomus riparius using nominal concentrations that ranged from 4 to 26.2 μg/L. We started the test from the first instar larvae in the parental (P) generation, quantifying life cycle parameters (emergence, sex ratio, development rate, fecundity and fertility) until the emergence in the subsequent F1 generation. Results showed a reduction in reproduction and a significant decrease in male developmental rate of midges for all treatments, in the F1 generation compared with the P generation (paired t?test; p?<?0.001). Two-way analysis of variance revealed a significant exposure?×?generation effect on male fraction with male fraction increasing (P generation) or decreasing (F1 generation) with increasing exposure. These effects on C. riparius underline the importance of conducting long-term studies with environmentally relevant concentrations to investigate population-level endpoints for endocrine disrupting chemicals.     

Measurement of dynamic mobilization of trace metals in sediments using DGT and comparison with bioaccumulation in Chironomus riparius: first results of an experimental study

Sediments in aquatic ecosystems are often contaminated as a result of anthropogenic activities. Sediments and benthic organisms have been used to monitor trace metals contamination. However, due to the high variability of contaminant bioavailability, the attempt to link metal concentration in sediments and contamination of the organisms or ecotoxicological effect often lead to disappointing results. The technique of diffusive gradients in thin films (DGT) has been proposed as a relevant tool to study metal bioavailability, for example for accumulation in plants. In the present study, laboratory microcosm experiments were conducted with six contaminated sediments to compare metal accumulation in DGT and bioaccumulation in a chironomid (Chironomus riparius) for Cu, Cd and Pb . Metal accumulation in DGT was measured over time then modelled to determine two parameters of the dynamic response of the metals to DGT deployment: the size of the particulate labile pool and the kinetic of the solid-dissolved phase exchange. The mobility of metals was found metal and sediment dependent. A significant relationship between metal accumulated in DGT and bioaccumulated in chironomids was found for Cu and Pb. However, total metals in sediments were the best predictors of bioaccumulation. Nevertheless, the knowledge of the metals dynamic enhanced our ability to explain the different biological uptake observed in sediments of similar total metal concentrations.     

Impacts of a mosquito selective pesticide, Bti, on the macroinvertebrates of a subtropical stream in Hong Kong

To test the hypothesis that Bti was specific to mosquito larvae, the granular form of Bti was tested on commonly found Hong Kong species from four naturally occuring aquatic insect orders and one species of decapod crustacean (Neocardina serra). Because data on Bti impacts on species in tropical and subtropical countries is relatively rare, the present study was conducted in Hong Kong's New Territories. Using static acute toxicity bioassays, all mosquito larvae exposed to Bti at the recommended dosage were dead except for some dark pigmented fourth instar individuals that had stopped feeding prior to emergence and as a result, did not ingest the Bti toxin. The only non-target species killed by the Bti were some chironomid species. In 1998 Bti inoculated pools along the Tai Tan River in the New Territories of Hong Kong had significantly fewer chironomids and mosquito larvae than control pools. By January, 1999, these same Bti inoculated pools still had no mosquito larvae in them, however, chironomids were no longer significantly rarer when compared to the control ponds. Thus, it would appear, that during the 1998-99 study in which Bti was added to the study sites at weekly intervals, Bti resistant chironomid species replaced Bti sensitive species in the Bti inoculated pools.     

Applying adult emergence as an endpoint in a post-exposure laboratory test using two midge species (Diptera: Chironomidae)

Several approaches have been used to evaluate biological impairment in aquatic ecosystems which can be categorized as either laboratory or field. In the recent years, the laboratory toxicity test approach has been extended to field exposures where ambient factors are allowed to influence the test response. Field exposures of laboratory test organisms require method modifications. In this paper, a novel in situ method is described which measures growth, survival and emergence of sediment inhabiting insects (Diptera: Chironomidae) that are used in standardized laboratory toxicity testing. Two standard chironomid species (Chironomus riparius and Chironomus tentans) were used to test the suitability of the approach and to compare the performance of the species. The larvae were transferred to the laboratory for emergence after 7 days in situ exposure which was compared to laboratory responses. Growth, survival and emergence were significantly lower in the in situ pre-exposure than in the laboratory. Also, emergence success was significantly lower in one reference sediment (LMR) than in the other test sediments in both in situ and the laboratory treatment. These lower response levels likely resulted from sediment characteristics and artifacts related to the exposure in the in situ chamber. Feeding and water quality within the exposure chamber appear to be factors that may differ markedly from the laboratory exposure and may affect organism responses. C. riparius developed (growth, emergence time) faster than C. tentans in all treatments, otherwise the species responded similarly. C. riparius may be a better alternative for the chronic in situ exposures because of shorter exposure times and reduced feeding requirements.     

Induction of mouthpart deformities in Chironomus riparius larvae exposed to 4-n-nonylphenol

Chironomid mouthpart deformities have often been associated with sediment contamination and are, therefore, currently used to assess sediment quality. Deformities were only occasionally induced in laboratory bioassays. Mouthpart deformities results from a physiological disturbance during larval molting. In the past few years it has been shown that some chemicals can exert negative effects on both vertebrates and invertebrates at the level of endocrine regulation. As insect molting is hormonally regulated, we wanted to test the hypothesis that deformities are induced due to a hormonal disruption in the developmental process. The aim of the present study was to test whether the endocrine disrupter, 4-n-nonylphenol (4NP), induces mouthpart deformities in chironomids. A laboratory bioassay was performed exposing Chironomus riparius larvae to 10, 50 and 100 micrograms l-1 4NP. Survival of the larvae was not affected by the tested concentrations, but the frequency of mentum deformities increased significantly (P < 0.01) after exposure to 4NP.     

Dynamics of metal adaptation in riverine chironomids

The ability of the non-biting midge Chironomus riparius to survive and reproduce in metal polluted lowland rivers facilitates the opportunity to study micro-evolutionary processes in situ. However, due to larval drift, adapted midge populations are subject to regular immigration of non-adapted specimens from clean upstream river reaches. To examine the influence of non-adapted genes in adapted midge populations on the level of metal adaptation, an upstream and downstream chironomid population were crossbred on eight separate occasions in the laboratory to mimic gene flow. Several life-history characteristics, indicating adaptation to metals, were followed seasonally in the parental strains as well as in the reciprocal crossings. Such crossings were done over a 14-month period and maternal effects were found to be absent, indicating a major genetic component for the increased metal tolerance in the exposed midge populations. Furthermore, results confirmed the presence of adaptation to metals in exposed chironomids. However, a rapid loss of metal adaptation in the first generation hybrid offspring was clearly demonstrated. Consequently, the large temporal variation in metal adaptation in midge populations from the river can be explained by the earlier reported seasonal variations in selection pressure and immigration rates from non-adapted sub-populations.     

Physicochemical characteristics of the Dombrovska pit lake (Ukraine) formed in an opencast potassium salt mine and the genome response of Chironomus salinarius Kieffer (Chironomidae,Diptera) to these conditions

This study focuses on the Dombrovska pit lake, near the city of Kalush in Ukraine, which is a former potassium salt mine filled with brine and freshwater. The water level is still increasing and as a result the salinity is decreasing. We analyzed the benthic fauna communities and the genome instability by assessing the rearrangements in the polytene chromosomes of Chironomus salinarius and the physicochemical parameters of the near-bottom water (pH, conductivity, mineralization, major ions, NO₃⁻, NH₄⁺, metals Cd, Pb, Cu, Mn, and Fe) and sediment (pH, organic matter and metals Cd, Pb, Cu, Zn, Mn, and Fe) at four sites. The water mineralization ranged from 17.3 to 26.2 g dm⁻³ which are classified as mesohaline and polyhaline waters, respectively. The biodiversity of the benthic fauna was low, and the dominant species was C. salinarius. The density of C. salinarius varied spatially and changed from 637 ind./m² at a depth of 5 m to 8167 ind./m² at a depth of 2.5 m. The genome instability was analyzed by examining the structural and functional changes in the salivary gland chromosomes of C. salinarius. The exposure of C. salinarius damaged the chromosomes and the activities of key structures, such as the Balbiani ring and nucleolar organizer, were partially or completely suppressed.

     

Carbaryl-A selective genotoxicant

Mutagenic effects of carbaryl, a contact insecticide with slight systemic properties, have been investigated employing histidine reversion assay in Salmonella typhimurium strains and in vivo chromosomal aberrations in root meristems of Allium cepa. A detailed investigation revealed that carbaryl did not enhance significantly the frequency of histidine revertants in any of the strains of Salmonella i.e. frameshift mutagen tester (TA98), base pair substitution tester strain (TA1535) and ochre mutant strain (TA102). The supplementation with S9 mix did not have any appreciable effect. S14 prepared from wheat seedlings also did not enhance the reversion frequency significantly. However, carbaryl induced both clastogenic and physiological types of chromosomal aberration. The spectrum of chromosomal aberrations included c-mitosis, stickiness, vagrant chromosomes, polyploidy multi-polarity, delayed anaphases, end to end joining of chromosomes, chromosome breaks, ring chromosomes and anaphase bridges. The frequency of chromosomal aberrations was reduced by transferring the carbaryl treated bulbs to distilled water for 24 and 48 h. Similarly, recovery in the mitotic index was noticed by such transfer. The differences between the two assays may be attributed to differences in the metabolism of the test organisms.     

Short-term exposure to sub-lethal doses of lindane affects developmental parameters in Chironomus riparius Meigen, but has no effect on larval glutathione-S-transferase activity

Chironomus riparius Meigen were exposed to 0, 0.01, 0.1, 0.5, 0.75 and 1.0 ppm lindane for 48 h as fourth instar larvae. Exposure had no effect on glutathione-S-transferase (GST) activity in larvae snap-frozen immediately following exposure. In contrast, exposure had longer-term consequences affecting developmental parameters. Concentrations above 0.5 ppm lindane affected larval behaviour, reduced adult body size and fecundity and delayed emergence times. The lack of significant change in GST activity when life history characters were affected by high concentrations of lindane, suggests that in C. riparius, GST is not a sensitive biomarker of pesticide exposure or effect.     

Development of rearing and testing protocols for a new freshwater sediment test species: the gastropod Valvata piscinalis

This paper aimed at proposing rearing and testing protocols for Valvata piscinalis, a new potential species for sediment toxicity testing. Such tests were developed since this species reliably represents the bio/ecological characteristics of other gastropods. It may thus be representative of their sensitivity to chemicals. V. piscinalis was successfully cultured in our laboratory for six generations. Cultures provided a high productivity for a low working time and low costs. The tests conditions we proposed seemed to be relevant for the development of reliable tests with this species. Indeed, hatching probability of egg-capsules, as well as embryo, newborn and juvenile survival rates, were close to 100%. Moreover, growth rates and fecundity were significantly higher than in field and in other laboratory studies. Partial life-cycle tests on clean sediments were achieved for various feeding levels to determine survival, growth and reproduction patterns, ad libitum feeding level and life cycle parameters values. Ad libitum feeding levels for newborn, juveniles and adults were 0.1, 0.4 and 0.8 mg Tetramin/individual/working day. Growth tests with zinc-spiked sediments provided a no-effect concentration and a lowest effect concentration of respectively 200 and 624 mg zinc/kg dry sediment. Other growth tests on spiked sediments we ran at our laboratory with second, third and fourth instars larvae of Chironomus riparius pointed out that V. piscinalis was more sensible to zinc than the chironomid, which is a routine test species in ecotoxicology. According to these results, V. piscinalis is a promising candidate species for sediment toxicity testing.     

The fate of trace metals during the metamorphosis of chironomids (diptera, chironomidae)

The fate of the trace metals zinc, cadmium and copper during the metamorphosis of chironomids was studied under field and experimental conditions. Field observations demonstrated a significant decrease in the trace metal body burden of larvae and imagines of Stictochironomus histrio Fabricius. No such difference were noted between successive developmental stages of Chironomus anthracinus Zett. The exuviae of both species contained small amounts of trace metals. Experiments, in which larvae of S. histrio and C. riparius Meigen were individually exposed, showed that larvae of both species accumulated substantial amounts of zinc and cadmium. For these metals, a transfer to pupae and imagines occurred. However, pupae had a lower body burden than larvae, and imagines a lower body burden than the pupae. Copper was also accumulated in the larvae, but it was excreted almost completely before the pupal stage. Elimination pathways of the trace metals during metamorphosis and the ecotoxicological consequences of trace metal transfer are discussed.     

Effects of phytosterols on zebrafish reproduction in multigeneration test

Zebrafish from mixed sex populations were exposed continuously across three generations to two phytosterol preparations both containing -sitosterol. The phytosterols were isolated from wood and soy beans. Blood vitellogenin levels and sex ratio changes were used as intermediate indicators of the reproduction failures. Both sterol preparations caused vitellogenin induction in the exposed fish. The wood sterol changed the sex ratio of the exposed fish. In generation F1, the predominant sex was male, and in generation F2 it was female. The soy sterol in the used test concentration was lethal to the exposed fish in generation F1. This multigeneration test evidenced that phytosterols containing -sitosterol disrupt the reproduction system of zebrafish by changing the sex ratios and by inducing the vitellogenin production in the exposed fish.     

Fluoxetine effects assessment on the life cycle of aquatic invertebrates

Fluoxetine is a serotonin re-uptake inhibitor, generally used as an antidepressant. It is suspected to provoke substantial effects in the aquatic environment. This study reports the effects of fluoxetine on the life cycle of four invertebrate species, Daphnia magna, Hyalella azteca and the snail Potamopyrgus antipodarum exposed to fluoxetine spiked-water and the midge Chironomus riparius exposed to fluoxetine-spiked sediments. For D. magna, a multi-generational study was performed with exposition of newborns from exposed organisms. Effects of fluoxetine could be found at low measured concentrations (around 10mugl(-1)), especially for parthenogenetic reproduction of D. magna and P. antipodarum. For daphnids, newborns length was impacted by fluoxetine and the second generation of exposed individuals showed much more pronounced effects than the first one, with a NOEC of 8.9mugl(-1). For P. antipodarum, significant decrease of reproduction was found for concentrations around 10mugl(-1). In contrast, we found no effect on the reproduction of H. azteca but a significant effect on growth, which resulted in a NOEC of 33mugl(-1), expressed in nominal concentration. No effect on C. riparius could be found for measured concentrations up to 59.5mgkg(-1). General mechanistic energy-based models showed poor relevance for data analysis, which suggests that fluoxetine targets specific mechanisms of reproduction.     

The development of genetically inherited resistance to zinc in laboratory-selected generations of the earthworm Eisenia fetida

The capacity of species to adapt both physiologically and genetically to contaminants may allow populations to persist in polluted environments. Such 'adaptation' can have important implications for risk assessment, since it may mean that prediction based on extrapolation of toxicity studies with na?ve populations may prove invalid for long-term contaminated sites. To investigate the evolution of zinc resistance in Eisenia fetida, worms from a previously unexposed population (parent) were selected and reared over two generations (F1, F2) while exposed to zinc in the laboratory. Relative sensitivities of unexposed and selected generations were then compared by exposing parent, F1 and F2 individuals to zinc in contact filter papers tests. Calculation of effect concentrations from this work indicated differences in sensitivity to zinc for successive generations, with higher toxicity values (LC(50), LC(90), LC(99)) found for the selected worms. The increases in resistance found for F1 and F2 worms were confirmed in a discriminating dose study. In addition to comparing the sensitivities of the parent, F1 and F2 generations for zinc, toxicity tests were also conducted with copper to assess if there was evidence of cross-resistance between the two metals. Results indicated similar increases in resistance to copper to those found for zinc. Mechanisms underlying the increased metal resistance were studied in toxicokinetic experiments. Results indicated no clear trends between the three generations indicating that physiological responses, other than differences in kinetic parameters, are responsible for the increased resistance found in the selected worms.     

Photochemically induced formation of the "Al13" tridecameric polycation in the presence of Fe(III) and organic acids

We found that light-induced Fe(III) reduction associated with the oxidation of a simple hydroxy-carboxylic acid (lactate) caused the formation of the AlO4Al12(OH)24(H2O)12 polycation ("Al13"). Initial conditions were a lactate:Al:Fe ratio of 1:0.76:0.11 in a partially neutralized solution. Base was added rapidly and no Al13 was detected in samples kept in the dark. With exposure to light, Fe(III) reduction was rapid and Fe(II) reached a maximum within 1 day. After the maximum, steady-state Fe(II) declined from 54% to 43% over eight days. During this same time period, the lactate concentration fell to 2% of the original, pH rose from 4.05 to 4.46, and the Al13 detectable by 27Al NMR increased to 2.3 mmol l(-1) (51% of the total solution Al). The formation of Al13 is attributed to the pH rise resulting from the removal of the organic acid buffer. Similar photo-induced chemical changes occur in natural waters and may promote the formation of Al13, conditions permitting.