In situ bioassay chambers and procedures for assessment of sediment toxicity with Chironomus riparius

The purpose of this study was to develop an in situ sediment bioassay chamber and respective procedures, suitable for performing toxicity bioassays with benthic invertebrates, using the midge Chironomus riparius. It was also our objective to compare the responses obtained under controlled conditions (laboratory 10-day larval growth and survival test) with those obtained in situ. Clean sand and a formulated sediment were incorporated in the in situ bioassay, along with local sediments, as a way of minimizing natural variability due to physicochemical differences among sediments or due to interactions with indigenous organisms. Recovery of organisms was good (80-100% in the control and reference site), indicating that the developed chamber and protocol were suitable for exposing and retrieving C. riparius in situ. Results also showed differences between responses obtained with formulated and natural sediment in situ, as well as between laboratory and in situ.     

In situ exposures using caged organisms: a multi-compartment approach to detect aquatic toxicity and bioaccumulation

An in situ toxicity and bioaccumulation assessment approach is described to assess stressor exposure and effects in surface waters (low and high flow), the sediment-water interface, surficial sediments and pore waters (including groundwater upwellings). This approach can be used for exposing species, representing major functional and taxonomic groups. Pimephales promelas, Daphnia magna, Ceriodaphnia dubia, Hyalella azteca, Hyalella sp., Chironomus tentans, Lumbriculus variegatus, Hydra attenuatta, Hexagenia sp. and Baetis tibialis were successfully used to measure effects on survival, growth, feeding, and/or uptake. Stressors identified included chemical toxicants, suspended solids, photo-induced toxicity, indigenous predators, and flow. Responses varied between laboratory and in situ exposures in many cases and were attributed to differing exposure dynamics and sample-processing artifacts. These in situ exposure approaches provide unique assessment information that is complementary to traditional laboratory-based toxicity and bioaccumulation testing and reduce the uncertainties of extrapolating from the laboratory to field responses.     

Sediment toxicity and stormwater runoff in a contaminated receiving system: consideration of different bioassays in the laboratory and field.

Several field and laboratory assays were employed below an urban storm sewer outfall to define the relationship between stormwater runoff and contaminant effects. Specifically, two bioassays that measure feeding rate as a toxicological endpoint were employed in the field and in the laboratory, along with bioassays measuring survival and growth of test organisms. In 7 to 10 d in situ exposures, amphipod leaf disc processing, growth and survival were monitored. Different exposure scenarios were investigated by varying the mesh size (74 microns or 250 microns mesh) and method of deployment (water column, sediment surface, or containing sediment) of in situ exposure chambers. Hyalella azteca, Daphnia magna, and Pimephales promelas survival were monitored in 48 h in situ exposures. Feeding inhibition was investigated via enzyme inhibition of H. azteca and D. magna and via leaf disc processing measurements of the detritivore H. azteca. Additionally, we investigated the extent of phototoxicity at this site via field exposures in sun and shade and laboratory exposures with and without UV light. The measurement of detritivore leaf disc processing, and thus its usefulness as an endpoint, was hindered by individual variability in the amount of leaf consumed and by leaf weight gain during the summer field exposures. For D. magna, enzyme inhibition measured in a laboratory exposure did not reveal the toxicity observed in field exposures. For H. azteca, enzyme inhibition measured in the laboratory indicated toxicity similar to that observed in short term chronic in situ exposures. Enzyme inhibition also did not detect differences in toxicity due to variations in flow conditions. There were no statistically significant effects of any exposure on P. promelas survival or H. azteca growth, and there were no statistically significant effects due to mesh size or sun exposure. Survival of H. azteca was the most sensitive and the least variable endpoint. Effects on survival were noted in the same treatments over short-term chronic exposures in the laboratory and in situ. Significant differences in survival were noted due to the method of deployment under low flow conditions. In situ chambers containing sediment resulted in greater mortality in the 10 d low flow in situ experiments. Under high flow conditions, significant reductions in survival and leaf disc processing were noted under all methods of deployment at the two impacted sites over a 7 d exposure. Also under high flow conditions, significantly greater mortality of H. azteca was reported at the downstream field site when sediment was included in the chamber at deployment. These results suggest that significant toxicity at this site is due to accumulation of contaminants in the sediment and the mobilization of these contaminants during a storm event. In situ exposures detected toxicity not observed in laboratory exposures. These results suggest that a combination of laboratory and field bioassays is most useful in defining field effects.     

Species-specific responses to metals in organically enriched river water, with emphasis on effects of humic acids

Invertebrate communities in polluted rivers are often exposed to a wide variety of compounds. Due to complex interactions, 'pollution tolerant' species are not necessarily the most tolerant species to toxicants tested under standard laboratory conditions. It was hypothesized that the distribution of species in polluted rivers is not only dependent on the tolerance of species to toxicants, but also on species-specific capacities to modify or compensate for negative effects of toxicants. To test this hypothesis, species-specific responses to metals in organically enriched river water were studied under controlled conditions. The zebra mussel Dreissena polymorpha and the midge Chironomus riparius were exposed to metal-polluted water from the River Dommel. Additionally, the (interactive) effects of metals and humic acids (HA) on both species were evaluated. In spite of a lower tolerance of Chironomus riparius to metals in laboratory studies, the midge was the most tolerant of the two test species to metal-polluted site water. The results indicated that the sensitivities of the two test species determined in laboratory tests were inversely related to their sensitivities to polluted river water. In accordance with these results, midge larvae were protected from copper (Cu) toxicity by HA, while metal toxicity was not reduced (Cu) or even amplified (cadmium) by HA for the zebra mussel. Thus, the presence of (naturally occurring) HA in site water may partly account for discrepancies between responses of species to bioassays and toxicity tests. It is suggested that these differences in responses to metals in site water are strongly influenced by species-specific preferences for organic compounds (like HA). It is concluded that the response to organic compounds present in site water largely determines whether a species is classified as 'pollution tolerant' or 'pollution sensitive'.     

An automated overlying water-renewal system for sediment toxicity studies

An automated water-renewal toxicity test system is described for exposing benthic invertebrates to whole sediments. The system will intermittently deliver laboratory or on-site water for overlying water replacement in sediment exposures. A range of cycle rates can be used to produce different volume additions of overlying water per day to exposure chambers. The system can be used with six different treatments and eight replicates per treatment producing 48 exposure chambers. Three formulated sediments with variable organic carbon (1.5%, 7.5%) and sand (14%, 63%) content were prepared to test the system exposing amphipods, Hyalella azteca and midges, Chironomus tentans in 10 day whole sediment tests. Intermittent water flow was used with a 90 min cycle time to create two volume additions of laboratory water per 24 h in exposure chambers (180 ml sediment, 320 ml water). Overlying water quality conditions, and survival and growth of both species were consistent and within acceptable limits for the testing requirements of the U.S. EPA guidelines for sediments with freshwater invertebrates.     

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.     

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.     

Modelling of the life cycle of Chironomus species using an energy-based model

Little is known about the differences between the species of the genus Chironomus relatively to their life cycle strategies. This knowledge is however crucial to fully understand the response of the Chironomus community to field perturbations. Here, we proposed to study four Chironomus species by using an energy-based model to describe growth, emergence and reproduction. We used data from the literature for two species (Chironomus plumosus and Chironomus tentans) and data from our experiments for two other species (Chironomus prasinus and Chironomus riparius). We showed that our model is able to accurately describe the life-history attributes for all the species tested, which suggests that Chironomus species have the same fundamental characteristics (low maintenance energetic costs, isomorphism), which makes possible the building of a common modelling framework to assess effects of toxicants at individual and population level. The species showed a few differences relatively to the parameters of the models with possible consequences when assessing effects of chemicals on Chironomus community. For instance, due to differences in growth parameters, C. riparius population dynamics should be more sensitive to effects on individual growth than C. prasinus or C. plumosus ones.     

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.     

Sediment toxicity testing using large water-sediment ratios: an alternative to water renewal

Deterioration of overlying water quality during toxicity tests with benthic invertebrates is a serious problem with some sediments. One solution is periodic renewal of overlying water. However, this is either labour intensive or requires construction and maintenance of special equipment. Furthermore, water renewal has the potential for flushing toxic chemicals out of the test chamber and establishes nonequilibrium conditions between the water and sediment. An alternative is testing under static conditions using atypical test vessels (e.g. Imhoff settling cones) with a large water volume (1 l) overlaying a much smaller sediment volume (e.g. 15 ml). This results in dramatic improvement of overlying water quality compared to standard static toxicity tests. Compared to water renewal, the test method is much simpler, all toxic substances leached from the sediment are retained in the test vessel, and contaminant concentrations in water and sediment have more time to equilibrate. Chronic sediment toxicity tests (10-28 days) have been conducted successfully under these conditions with Chironomus riparius, Hexagenia sp., Hyalella azteca and Tubifex tubifex.     

Life cycle responses of the midge Chironomus riparius to polycyclic aromatic compound exposure

During acute exposure, polycyclic aromatic compounds (PACs) act mainly by narcosis, but during chronic exposure the same compounds may exert sublethal life cycle effects. The aim of this study was therefore to evaluate the chronic effects of sediment spiked PACs on the emergence of the midge Chironomus riparius. Three isomer pairs were selected, and 28-day LC50 values and 50% emergence times (EMt50) were determined. Concentration-response relationships were observed for phenanthrene, acridine, phenanthridine and acridone. Anthracene and phenanthridone had no effect on total emergence, but did cause a delay in emergence. Calculated porewater LC50 values correlated well with logKow values, suggesting narcosis as mode of action. In contrast, effect concentrations for delay in emergence (EMt50) deviated from narcosis, suggesting a specific mode of action during chronic exposure. It is concluded that emergence is a powerful endpoint to detect life cycle effects of PACs on a key sediment inhabiting invertebrate.     

Impact of a flood disaster on sediment toxicity in a major river system--the Elbe flood 2002 as a case study

The ecotoxicological implications of a flooding disaster were investigated with the exceptional Elbe flood in August 2002 as an example. Sediment samples were taken shortly after the flood at 37 sites. For toxicity assessment the midge Chironomus riparius (Insecta) and the mudsnail Potamopyrgus antipodarum (Gastropoda) were exposed to the sediment samples for 28 days. For a subset of 19 sampling sites, the contamination level and the biological response of both species were also recorded before the flood in 2000. The direct comparison of biological responses at identical sites revealed significant differences for samples taken before and immediately after the flood. After flood sediments of the river Elbe caused both higher emergence rates in the midge and higher numbers of embryos in the mudsnail. Contrary to expectations the toxicity of the sediments decreased after the flood, probably because of a dilution of toxic substances along the river Elbe and a reduction in bioavailability of pollutants as a result of increasing TOC values after the flood.     

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.     

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.     

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.     

Assessing sediment toxicity and arsenite concentration with bacterial and traditional methods

Three sediment samples LP (pool where logs are stored), LF (brook through landfill area), KN (Kaskesniemi) which is in Lake Pyh?selk? downstream from the mill, were taken from an old sawmill area and one from the unpolluted Lake H?yti?inen. The arsenite concentration was measured by GFAAS and two arsenite biosensing bacterial strains Pseudomonas fluorescens OS8 (pTPT31) and Escherichia coli MC1061 (pTOO31). The toxicity of sediment and pore water samples was determined by using luminescent bacteria (Flash test) and, further, whole sediment toxicity was measured using 10 days growth test and 50 days emergency test with midges (Chironomus riparius). With the flash test a lowered EC50 value was found only in sediment LF (EC50=0.17 v/v%). The Flash test indicated that all sediment samples taken from the sawmill area were highly toxic to bacteria, whereas growth and the emergence of chironomids showed no effects in other samples than LF. The midges tolerate well the contaminated environment. In contrast, bioavailability of arsenite of sediment samples KN and LF was quite high determined using the biosensor-strains in a direct contact assay. The bioavailable fraction of sediment LP was 6-10% out of the total arsenite concentration obtained with GFAAS (0.46-0.77 microg g-1 dw). The results show that the choice of analysis method grossly affects the outcome without any of the method giving an incorrect result. Different methods measure different parameters of a toxic sample and can thus be used to complement each other.     

Effects of low dissolved oxygen on organisms used in freshwater sediment toxicity tests

Minimum dissolved oxygen requirements are part of standard guidelines for toxicity testing of freshwater sediments with several benthic invertebrates, but the data underlying these requirements are somewhat sparse. We exposed three common test organisms to ranges of dissolved oxygen concentrations to determine their responses in 10-d exposures, relative to published guidelines for sediment toxicity tests. The oligochaete, Lumbriculus variegatus, showed 100% survival in all exposures down to the lowest concentration tested, 0.7 mg O(2)l(-1). Midge (Chironomus dilutus) larva showed a more pronounced response; while survival was less than 90% only below 1.0mg O(2)l(-1), the biomass endpoint showed EC(50), EC(20), and EC(10) values of 1.00 (0.91-1.10), 1.41 (1.16-1.71), and 1.67 (1.25-2.24) mg O(2)l(-1). The amphipod, Hyalella azteca, showed no adverse effects at concentrations as low as 2.12 mg O(2)l(-1). The combination of these data with other literature data suggest that DO minima in current North American 10-d sediment test guidelines are reasonable.     

Waterborne and sediment toxicity of fluoxetine to select organisms

Ecological risk assessments of pharmaceuticals are currently difficult because little-to-no aquatic hazard and exposure information exists in the peer-reviewed literature for most therapeutics. Recently several studies have identified fluoxetine, a widely prescribed antidepressant, in municipal effluents. To evaluate the potential aquatic toxicity of fluoxetine, single species laboratory toxicity tests were performed to assess hazard to aquatic biota. Average LC(50) values for Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas were 0.756 (234 microg/l), 2.65 (820 microg/l), and 2.28 microM (705 microg/l), respectively. Pseudokirchneriella subcapitata growth and C. dubia fecundity were decreased by 0.044 (14 microg/l) and 0.72 microM (223 microg/l) fluoxetine treatments, respectively. Oryias latipes survival was not affected by fluoxteine exposure up to a concentration of 28.9 microM (8.9 mg/l). An LC(50) of 15.2 mg/kg was estimated for Chironomus tentans. Hyalella azteca survival was not affected up to 43 mg/kg fluoxetine sediment exposure. Growth lowest observed effect concentrations for C. tentans and H. azteca were 1.3 and 5.6 mg/kg, respectively. Our findings indicate that lowest measured fluoxetine effect levels are an order of magnitude higher than highest reported municipal effluent concentrations.     

Isonychia spp. and macroinvertebrate community responses to stressors in streams utilizing the benthic in situ toxicity identification evaluation (BiTIE) method

Exposures of caged organisms in situ have proven to be a useful way to improve exposure realism and link to stressor effects in aquatic assessments of hazard or risk. A novel cage system, the benthic in situ toxicity identification evaluation (BiTIE), was developed for benthic macroinvertebrates (surrogate species, resident populations and communities) to separate low and high flow effects, and major chemical classes of stressors in streams. Three resin types were used to separate the chemical stressors in the streams Honey Creek and Little Beavercreek, Ohio, USA: Dowex Optipore (non-polar organics), zeolite (ammonia), and polywool (control). Isonychia spp. sensitivity was compared to Chironomus tentans, and no significant differences were found (p>0.05). Isonychia spp. growth (length) showed a stressor response in the zeolite treatments, and community testing revealed improved metric responses in the Dowex treatments. The BiTIE chamber system demonstrated stressor-response relationships using sublethal and multimetric endpoints.     

Testing sediment biological effects with the freshwater amphipod Hyalella azteca: the gap between laboratory and nature

The freshwater amphipod, Hyalella azteca, is widely used in laboratory sediment toxicity and bioaccumulation tests. However, its responses in the laboratory are probably very different from those in the field. A review of the literature indicates that in its natural habitat this species complex is primarily epibenthic, derives little nutrition from the sediments, and responds primarily to contaminants in the overlying water column (including water and food), not sediment or porewater. In laboratory sediment toxicity tests H. azteca is deprived of natural food sources such as algal communities on or above the sediments, and is subjected to constant light without any cover except that afforded by burial into the sediments. Under these constraining laboratory conditions, H. azteca has been reported to respond to sediment or porewater contamination. In nature, contamination of overlying water from sediment is less likely than in the laboratory because of the large, generally non-static sink of natural surface water. H. azteca does not appear to be the most appropriate test species for direct assessments of the bioavailability and toxicity of sediment contaminants, though it is probably appropriate for testing the toxicity of surface waters. Toxic and non-toxic responses will be highly conservative, though the latter are probably the most persuasive given the exposure constraints. Thus H. azteca is probably a suitable surrogate species for determining sediments that are likely not toxic to field populations; however, it is not suitable for determining sediments that are likely toxic to field populations.