In situ bioassays with Chironomus riparius larvae to biomonitor metal pollution in rivers and to evaluate the efficiency of restoration measures in mine areas

In this study we evaluate the ability of an in situ bioassay with Chironomus riparius larvae, using larval development and growth as endpoints, to biomonitor water quality and to assess the biological recovery of metal contaminated freshwater ecosystems of mine areas that are subject of restoration measures. The bioassay was carried out in streams located near an abandoned goldmine in North Portugal, throughout an environmental rehabilitation of the mine (2002-2004). During this period, a decrease in the inhibition of larval growth in the metal contaminated stream was observed. The bioassay was also performed in streams located near an active tungsten mine in Central Portugal. Larval growth and development were highly inhibited in the stream that receives acid drainage from the tungsten mine and treated water from the AMD treatment station. The results indicate that the bioassay can be used to evaluate the efficiency of environmental restoration measures in mining areas.     

Acute toxicity tests with Daphnia magna, Americamysis bahia, Chironomus riparius and Gammarus pulex and implications of new EU requirements for the aquatic effect assessment of insecticides

Threshold concentrations for treatment related effects of 31 insecticides, as derived from aquatic micro-/mesocosm tests, were used to calibrate the predictive value of the European Tier-1 acute effect assessment on basis of laboratory toxicity tests with Daphnia magna, Chironomus spp., Americamysis bahia and Gammarus pulex. The acute Tier-1 effect assessment on basis of Daphnia (EC(50)/100) overall was protective for organophosphates, carbamates and most pyrethroids but not for neonicotinoids and the majority of insect growth regulators (IGRs) in the database. By including the 28-day water-spiked Chironomus riparius test, the effect assessment improves but selecting the lowest value on basis of the 48-h Daphnia test (EC50/100) and the 28-day Chironomus test (NOEC/10) is not fully protective for 4 out of 23 insecticide cases. An assessment on basis of G. pulex (EC(50)/100) is sufficiently protective for 15 out of 19 insecticide cases. The Tier-1 procedure on basis of acute toxicity data (EC(50)/100) for the combination of Daphnia and A. bahia and/or Chironomus (new EU dossier requirements currently under discussion) overall is protective to pulsed insecticide exposures in micro-/mesocosms. For IGRs that affect moulting, the effect assessment on basis of the 48-h Chironomus test (EC(50)/100) may not always be protective enough to replace that of the water-spiked 28-day C. riparius test (NOEC/10) because of latency of effects.     

Comparison of test procedures for sediment toxicity evaluation with Vibrio fischeri bacteria

The use of bacterial luminescence assays is particularly effective in the contaminated sediment evaluation. In the present study, a comparison was made of different baterial luminescence assays including acute toxicity of elutriates with Microtox® and LUMIStox®; chronic toxicity of elutriates with LUMIStox®; and, acute toxicity of solvent extracts with both the tests and the Solid Phase test with Microtox. The toxicity assay procedures were utilised for the Po River sediment toxicity evaluation, testing samples with different physical and chemical characteristics. The results evidentiated that each sediment test procedures provided indipendent and complementary ecotoxicological responces usefull for a sediment classification of the most polluted samples.     

Contaminated sediments and bioassay responses of three macroinvertebrates, the midge larva Chironomus riparius, the water louse Asellus aquaticus and the mayfly nymph Ephoron virgo

Bioassays are widely used to estimate ecological risks of contaminated sediments. We compared the results of three whole sediment bioassays, using the midge larva Chironomus riparius, the water louse Asellus aquaticus, and the mayfly nymph Ephoron virgo. We used sediments from sixteen locations in the Dutch Rhine-Meuse Delta that differed in level of contamination. Previously developed protocols for each bioassay were followed, which differed in sediment pretreatment, replication, and food availability. The Chironomus bioassay was conducted in situ, whereas the other two were conducted in the laboratory. The measured endpoints, survival and growth, were related to contaminant levels in the sediment and to food quantity in water and sediment.

Only the response of A. aquaticus in the bioassay was correlated with sediment contamination. Food availability in overlying water was much more important for C. riparius and E. virgo, thereby masking potential sediment contaminant effects. We conclude that growth of A. aquaticus was depressed by sediment contamination, whereas growth of E. virgo and C. riparius was stimulated by seston food quantity. We discuss that the trophic state of the ecosystem largely affects the ecological risks of contaminated sediments.     

Combined effects of copper and food on the midge Chironomus riparius in whole-sediment bioassays

Effects observed in whole-sediment bioassays must be seen as the joint effect of all sediment characteristics. In whole-sediment bioassays, however, adverse effects on test organisms are usually attributed to the presence of contaminants and effects of food are often ignored. The aim of this study was to analyze the response of the midge Chironomus riparius to sediment spiked with different combinations of food and copper. The responses of C. riparius to these spiked sediments were assessed in 10-day whole-sediment bioassays. Decreases in survival, dry weight, and length of C. riparius were observed with increasing copper concentrations. However, an increase in the amount of food resulted in an increase of larval dry weight and length until copper concentrations reached a critical threshold of 200 mg/kg. In addition, an increase in the amount of food resulted in a decrease of accumulated copper in the larvae. The present study demonstrated that the combination of copper and food in the sediment determines the performance of C. riparius in whole-sediment bioassays. The dependency of C. riparius on high feeding levels, which mask toxic effects, questions its suitability as a test organism for whole-sediment bioassays. Because benthic communities in polluted ecosystems are often exposed to varying levels of both food and toxicants it is concluded that the trophic state of the ecosystem may alter the ecological risk of sediment-bound toxicants to opportunistic benthic invertebrates such as C. riparius.     

Use of whole-body and subcellular Cu residues of Lumbriculus variegatus to predict waterborne Cu toxicity to both L. variegatus and Chironomus riparius in fresh water

We tested the use of whole-body and subcellular Cu residues (biologically-active (BAM) and inactive compartments (BIM)), of the oligochaete Lumbriculus variegatus to predict Cu toxicity in fresh water. The critical whole-body residue associated with 50% mortality (CBR₅₀) was constant (38.2-55.6 μg g⁻¹ fresh wt.) across water hardness (38-117 mg L⁻¹ as CaCO₃) and exposure times during the chronic exposure. The critical subcellular residue (CSR₅₀) in metal-rich granules (part of BIM) associated with 50% mortality was approximately 5 μg g⁻¹ fresh wt., indicating that Cu bioavailability is correlated with toxicity:subcellular residue is a better predictor of Cu toxicity than whole-body residue. There was a strong correlation between the whole-body residue of L. variegatus (biomonitor) and survival of Chironomus riparius (relatively sensitive species) in a hard water Cu co-exposure. The CBR₅₀ in L. variegatus for predicting mortality of C. riparius was 29.1-45.7 μg g⁻¹ fresh wt., which was consistent within the experimental period; therefore use of Cu residue in an accumulator species to predict bioavailability of Cu to a sensitive species is a promising approach.     

In situ assays with tropical cladocerans to evaluate edge-of-field pesticide runoff toxicity

Tropical regions’ economy is usually based on agriculture, which involves an intensive use, and even frequent overuse, of pesticides. Nevertheless, not much research has been done on the impact of pesticides on tropical aquatic ecosystems, which are often contaminated by runoff-related pesticide inputs due to unpredictable and torrential rainfalls. This study aimed to: (i) adapt and evaluate a short-term sublethal in situ assay using post-exposure feeding as an endpoint, to the tropical cladoceran species Diaphanosoma brachyurum (collected at the Pedra do Cavalo dam in the Paraguaçu River basin, Bahia, Brazil), and, (ii) assess the role of the standard species Daphnia magna as an adequate laboratory surrogate. Lethal and sublethal (post-exposure feeding) responses were assessed for the two species. To evaluate these responses under environmentally realistic exposure conditions, a runoff event was simulated in an agricultural area previously contaminated with different deltamethrin concentrations. The resultant runoff water was used to set up microcosms with different dilutions, simulating the entrance of runoff water in an adjacent lentic system. An in situ assay with D. brachyurum was performed inside the microcosms, allowing to discriminate the effects due to deltamethrin from those due to other potential stressors associated with the experimental design (e.g. organism handling, load of suspended particles, microcosm design). Water samples were collected from microcosms to conduct a laboratory assay with D. magna. The in situ methodologies revealed to be suitable to conduct assays with D. brachyurum under tropical conditions, since all exposed organisms were successfully retrieved from the chambers. Furthermore, none of the potential stressors associated with the experimental design influenced the daphnids’ performance. The tropical cladoceran species, exposed under more realistic conditions, revealed to be more sensitive than the laboratory standard species: lethal effects were only observed for D. brachyurum and sublethal effects were noticed at a lower deltamethrin concentration for this species than for D. magna.     

Comparative metabolism of phenanthridine by carp (Cyprinus carpio) and midge larvae (Chironomus riparius)

Abiotic transformation of azaarenes in the environment has been analysed extensively, but metabolism is less well described. To further elucidate preliminary observations of interspecific differences in azaarene metabolism by aquatic organisms, phenanthridine biotransformation by midge larvae and carp was studied. In both experiments, 6(5H)-phenanthridinone (phenanthridone) was found as an important metabolite. The fish were clearly capable of metabolising phenanthridine, but in the midge experiment the metabolite was principally formed by bacteria growing on the food and not by the midges. Phenanthridone itself was further degraded to non-observed compounds in both experiments, due to bacteria and midges acting together in the midge experiment, and by carp in the fish experiment. Internal concentrations of phenanthridine and phenanthridone were non-detectable in the midge larvae, but concentrations of both compounds in carp organs suggested a major role of bile and liver. Since phenanthridone did not account for all phenanthridine loss, it was suggested that, apart from phenanthridone degradation, other metabolic pathways may play a role. This study clearly demonstrates the importance of interspecies differences in metabolism, which should not be neglected in risk assessment.     

Sampling method, storage and pretreatment of sediment affect AVS concentrations with consequences for bioassay responses

Sediment treatment and sediment storage may alter sediment toxicity, and consequently biotic response. Purpose of our study was to combine these three aspects (treatment-toxicity-biotic response) in one integrated approach. We used Acid Volatile Sulfide (AVS) concentrations as a proxy of the disturbance of the sediment. AVS and Simultaneously Extracted Metal (SEM) concentrations were compared to bioassay responses with the freshwater benthic macroinvertebrate Asellus aquaticus. Storage conditions and sediment treatment affected AVS but not SEM levels. AVS can be used as a proxy for sediment disturbance. The best way to pretreat the sediment for use in a bioassay in order to maintain initial AVS conditions was to sample the sediment with an Ekman grab, immediately store it in a jar without headspace, and freeze it as soon as possible. In a survey using seven different sediments, bioassay responses of A. aquaticus were correlated with SEM and AVS characteristics.     

Comparative toxic and genotoxic effects of chloroacetanilides,formamidines and their degradation products on Vibrio fischeri and Chironomus riparius

Toxic and genotoxic effects of alachlor, metolachlor, amitraz, chlordimeform, their respective environmentally stable degradation products 2,6-diethylaniline, 2-ethyl-4-methylaniline, 2,4-dimethylaniline, and two other related compounds, 3,4-dichloroaniline and aniline were compared. Acute toxicity tests with Chironomus riparius (96 h) and Vibrio fischeri (Microtox) and genotoxicity tests with a dark mutant of V. fischeri (Mutato) were carried out. Our results demonstrate that toxicity and genotoxicity of the pesticides are retained upon degradation to their alkyl-aniline metabolites. In the case of the herbicides alachlor and metolachlor, the toxicity to V. fischeri was enhanced upon degradation. Narcosis alone explains toxicity of the compounds to the midge, but not so for the bacteria suggesting a disparity in the selectivity of the test systems. All compounds showed direct genotoxicity in the Vibrio test. but amitraz and its metabolite were genotoxic at concentrations 10(3)-10(5) lower than all the other compounds. The observations indicate that stable aniline degradation products of the pesticides may contribute considerably to environmental risks of pesticides application and that genotoxic effects may arise upon degradation of pesticides.     

Cyst-based toxicity tests XVI--sensitivity comparison of the solid phase Heterocypris incongruens microbiotest with the Hyalella azteca and Chironomus riparius contact assays on freshwater sediments from Peninsula Harbour (Ontario,Canada)

In view of the complexity and costs of "traditional" whole sediment assays, a "culture/maintenance free" direct contact microbiotest has been developed with the freshwater ostracod Heterocypris incongruens. The new Toxkit assay (named Ostracodtoxkit) has been applied to 33 sediment samples from Peninsula Harbour, located in Lake Superior of the Great Lakes water basin in Ontario, Canada. The microbiotest was applied in parallel to direct contact tests with the amphipod Hyalella azteca and the midge larva Chironomus riparius, to compare its relative sensitivity with that of the two "conventional" assays. The study was undertaken in the framework of remediation action plans for specific areas of concern, to enable decision making by the Canadian authorities for the restoration of impacted aquatic environments. Most sediments were found non-toxic (<20% mortality) to both the conventional test species and the ostracod. For the large majority of samples, a very good correspondence was found between the two crustacean test species for the intensity of the toxic signal "mortality", as reflected by a 0.71 (p<0.05) correlation coefficient. Growth inhibition, which is determined in the ostracod microbiotest as a sublethal effect criterion, allowed the earmarking of some sediment samples, which were apparently more toxic for the amphipod than to the ostracod. For 20% of the samples, substantially higher mortality scores were noted with the ostracod assay than with the midge larvae tests and the overall correlation coefficient between these two tests was lower (r=0.60,p<0.05). The results obtained in the present study corroborate those of previous research on sediments collected from various rivers in Flanders, Belgium, and confirm the potential of the new ostracod microbiotest as a reliable and sensitive low cost alternative for traditional whole sediment assays.     

Biological responses of midge (Chironomus riparius) and lamprey (Lampetra fluviatilis) larvae in ecotoxicity assessment of PCDD/F-, PCB- and Hg-contaminated river sediments

Environmental Science and Pollution Research - We evaluated the utility of chironomid and lamprey larval responses in ecotoxicity assessment of polychlorinated dibenzo-p-dioxins, dibenzofurans...     

The toxicity and bioavailability of atrazine and molinate to Chironomus tepperi larvae in laboratory and river water in the presence and absence of sediment

Acute (10 day) semi-static toxicity tests in which the midge, Chironomus tepperi, were exposed to atrazine and molinate were conducted in laboratory water and in river water, in the absence and presence of sediment. The bioavailability measured as median lethal concentrations (LC50) and 95% fiducial limits (FLs) of atrazine to C. tepperi in laboratory water in the absence and presence of sediment were 16.6 (14.3-19.4) and 21.0 (18.2-24.1) mg l(-1), respectively while the corresponding values in river water were 16.7 (14.7-19.0) and 22.7 (20.3-25.4) mg l(-1), respectively. For molinate, the LC50 and FL values in laboratory water in the absence and presence of sediment were 8.8 (6.8-11.4) and 14.3 (12.4-16.4) mg l(-1), respectively and the corresponding values in river water were 9.3 (7.6-11.3) and 14.5 (12.4-16.9) mg l(-1), respectively. Atrazine has low toxicity (LC50 > 10 mg l(-1)) while molinate has moderate toxicity (1 mg l(-1) < LC50 < 10 mg l(-1)) to C. tepperi. River water did not significantly (P > 0.05) reduce the bioavailability of either chemical to C. tepperi. However, the presence of sediment did significantly (P < 0.05) reduce the bioavailability of both atrazine and molinate to C. tepperi.     

In situ monitoring of urban air in Southern Italy with the Tradescantia micronucleus bioassay and semipermeable membrane devices (SPMDs)

This study was designed to assess toxic and genotoxic compounds in the urban air of Caserta, South Italy using cuttings from the plant Tradescantia #4430. In situ monitoring of gaseous pollutans was made at 17 sampling points in two seasons of the year. Genotoxicity was evaluated by recording the micronuclei in meiotic pollen mother cells (Trad-MCN assay). In addition, the passive sampler semipermeable membrane devices (SPMDs) were deployed at the sampling points with a significant increase in micronuclei frequency. SPMDs concentrated priority organic pollutants were identified by high performance liquid chromatography and gas chromatography, while toxicity and mutagenesis were assessed on the bacterium Vibrio fischeri using the Microtox and Mutatox systems respectively. Significant toxic and mutagenic effects were observed at different points on the town grid and SPMDs effectively concentrated trace contaminants. The relationship between what was present in the air sampled by SPMDs and the micronuclei frequency was also explored.     

Effects of two diluents in the Microtox toxicity bioassay with marine sediments

This paper compares the use of two different diluents, EPA synthetic seawater (salinity 31 per thousand ) and NaCl standard diluent (salinity 35 per thousand ), in the Microtox toxicity bioassay performed on elutriate and solid phase derived from marine sediments. The study was performed comparing three series of data obtained by the use of the two diluents.In the first series the intensity of the natural light output of Vibrio fischeri was considered; in the second series pH value, sulphite and ammonia present in the control and in the diluent after the treatment with the sediment samples; in the last series, the measured toxicity in marine sediments was considered. The light output intensity measured with respect to time, gives information about the bacterial activity due to the different osmotic conditions. pH values joint with ammonia and sulphite content, give information about the effect of the bacterial metabolic activities and of the different interaction between each diluent with the sediment sample. At last, the comparison of the two diluents on real samples show how the different osmotic and hydrogenionic conditions determine different toxicity responses. The results show that the EPA diluent allows more suitable environment for the metabolic activities of bacteria depending on lower stressing conditions than those present when the standard diluent is used. Moreover, the use of EPA diluent reduces the risk of false positive response in the execution of the toxicity bioassay.     

Evaluation of the toxicity of marine sediments and dredge spoils with the Microtox bioassay

The Microtox^R bioassay was used to evaluate the toxicity of sediment and dredge spoil elutriates from several potentially-contaminated sites in Mobile and Pascagoula Bays. Elutriates were prepared using either local seawater or distilled deionized water (osmotically adjusted with NaCl prior to testing), and Microtox^R assays were performed with the elutriates and three reference toxicants. There were marked differences in the toxicity of several elutriates and reference toxicants in the two different waters, with the seawater generally resulting in the same or lesser toxicity than the osmotically-adjusted distilled deionized water.     

Exposure of Chironomus riparius larvae (diptera) to lead, mercury and beta-sitosterol: effects on mouthpart deformation and moulting

Mouthpart deformation in chironomid larvae is induced by exposure to chemical contaminants and is becoming an established bio-indicator in sediment assessment programmes. However, concentration-response relationships with causal agents have only been established occasionally and with varying success. In this laboratory study, instar II and III larvae were exposed to sub-lethal concentrations of lead, mercury and beta-sitosterol. A significant deformation response was induced in the pecten with lead and mercury. Deformation frequencies of the mentum after metal exposures were not significantly different from the control. Moulting was retarded by both metals and was well correlated with mouthpart deformation. The beta-sitosterol is an endocrine disruptor, which was used to test the hypothetical cause-effect relation between disruption of ecdyson functioning and chironomid deformation. In the present study, exposure to sublethal concentrations of beta-sitosterol did not result in any effect on deformation or moulting. As such, the proposed hypothesis of endocrine disruptors as primary causal agents of chironomid deformation could not be substantiated. Acetone, which was used as a solvent to apply beta-sitosterol caused a significant increase of mentum deformation. The ground filtration paper used as substrate seemed to induce deformities as well. Substrate contamination, acetone and (especially) inbreeding were most probably responsible for the high deformation frequencies in the control conditions.     

Exposure of Chironomus riparius larvae to uranium: effects on survival, development time, growth, and mouthpart deformities

Among non-biologically essential metals, data concerning uranium effects on freshwater benthic macroinvertebrates are scarce. The effects of uranium on survival, development time, growth and mouthpart deformities of midge Chironomus riparius were investigated. A 10-day static laboratory bioassay was performed exposing first instar larvae to artificial sediment spiked with four concentrations of uranium (2.97; 6.07; 11.44; 23.84 microg U g(-1) dry wt). As uranium was released from the sediment to the overlying water during this bioassay, both the sediment and the water column act as contamination pathways in giving rise to the observed effects. Significant negative effects on survival, development time, and growth were detected at 6.07, 6.07 and 2.97 microg U g(-1) dry wt, respectively. An LC20 of 2.49 microg U g(-1) dry wt (95% CI=1.48-4.27), and an LC50 of 5.30 microg U g(-1) dry wt (95% CI=3.94-7.25) were estimated. With respect to effects of uranium on larvae mouthpart deformities, we found that the lower the concentrations, the higher the deformity rates. These results highlight the potential impact of uranium at population level in environmentally realistic concentrations.