Binding properties to nicotinic acetylcholine receptors can explain differential toxicity of neonicotinoid insecticides in Chironomidae.

Neonicotinoids are neuroactive insecticides commonly detected in freshwater ecosystems. Recent studies have indicated that these compounds are markedly toxic to Chironomidae, a widespread family of ecologically important aquatic insects. However, despite their sensitivity, the pharmacological mechanisms driving neonicotinoid toxicity have yet to be characterized in these insect species. Here, we used a combination of saturation and competition binding studies to characterize neonicotinoid binding properties to nicotinic acetylcholine receptors (nAChR) in two different Chironomidae (Chironomus riparius and Chironomus dilutus) at two different life stages (larval and adult). Using radiolabeled imidacloprid ([3H]-IMI), we characterized and compared receptor density (Bmax), imidacloprid binding affinity (KD), and receptor binding affinity (Ki) to three different neonicotinoid competitors (imidacloprid, clothianidin, and thiamethoxam). We then compared receptor density and binding affinity parameters derived for Chironomidae to data previously generated for other dipterans and agricultural pests. We found that there were limited differences in neonicotinoid binding between C. riparius and C. dilutus, with both organisms demonstrating high affinities for imidacloprid (KD = 0.22-0.87 nM) and high receptor densities (Bmax = 0.92-6.53 pmol/mg). However, there were significant differences between life-stages, with larvae expressing higher densities of nicotinic acetylcholine receptors and higher imidacloprid affinities than adults. Moreover, there were compound-specific differences in receptor affinity, with larval stages displaying relative affinities (Ki) that generally correlated with acute neonicotinoid toxicity (e.g. clothianidin ≥ imidacloprid >>> thiamethoxam). Finally, compared to other dipterans and agricultural pests, Chironomidae display very high densities of high affinity nicotinic acetylcholine receptors, which likely contribute to their sensitivity. Results indicated that receptor-level differences in neonicotinoid binding may be responsible for ecotoxicological differences amongst distinct insect species, and they likely play a role in life stage-, and compound-level toxicity differences previously observed for Chironomidae. Overall, this study highlights the value of understanding the toxicological mechanisms of action of neonicotinoids in sensitive, non-target aquatic insects, to better predict adverse effects associated with unintentional neonicotinoid exposure.

Neonicotinoid insecticide mixtures: Evaluation of laboratory-based toxicity predictions under semi-controlled field conditions.

Neonicotinoid insecticide mixtures are frequently detected in aquatic environments in agricultural regions. Recent laboratory studies have indicated that neonicotinoid mixtures can elicit greater-than-additive toxicity in sensitive aquatic insects (e.g. Chironomus dilutus). However, this has yet to be validated under field conditions. In this study, we compared the chronic (28- and 56-day) toxicity of three neonicotinoids (imidacloprid, clothianidin, and thiamethoxam) and their mixtures to natural aquatic insect communities. Using experimental in-situ enclosures (limnocorrals), we exposed wetland insects to single-compounds and binary mixtures at equitoxic concentrations (1 toxic unit under the principle of Concentration Addition). We assessed the composition of all emerged insect taxa and the cumulative Chironomidae emergence and biomass over time. In treated limnocorrals, there were subtle shifts in community composition, with greater mean proportions of emergent Trichoptera and Odonata. Cumulative emergence and biomass increased over time and there was a significant interaction between time and treatment. At 28 days, cumulative Chironomidae emergence and biomass were not significantly different between neonicotinoid treatments and controls. However, cumulative emergence in the imidacloprid, clothianidin, and clothianidin-thiamethoxam treatments were 42%, 20%, and 44% lower than predicted from applied doses. At 56 days, effects on cumulative emergence and biomass were significant for imidacloprid, clothianidin, and the clothianidin-thiamethoxam mixture. Contrary to laboratory predictions, mixtures were not more toxic than single compounds under semi-controlled field settings. Furthermore, only clothianidin significantly shifted sex-ratios towards female-dominated populations. Results showed that the responses of natural Chironomidae populations to neonicotinoids and their mixtures cannot be adequately predicted from laboratory-derived single-species models, and although occasional overdosing may have influenced the magnitude of effects, reductions in Chironomidae emergence and biomass can occur at average neonicotinoid concentrations below some current water quality guidelines. Therefore, neonicotinoid guidelines should be revised to ensure that Chironomidae and other sensitive aquatic insects inhabiting agricultural wetlands are adequately protected.

Can chronic exposure to imidacloprid, clothianidin, and thiamethoxam mixtures exert greater than additive toxicity in Chironomus dilutus?

Widespread agricultural use of neonicotinoid insecticides has resulted in frequent detection of mixtures of these compounds in global surface waters. Recent evidence suggests that neonicotinoid mixtures can elicit synergistic toxicity in aquatic insects under acute exposure conditions, however this has not been validated for longer exposures more commonly encountered in the environment. Therefore, we aimed to characterize the chronic (28-day) toxicity of imidacloprid, clothianidin, and thiamethoxam mixtures under different doses and mixture ratios to determine if the assumption of synergistic toxicity would hold under more environmentally realistic exposure settings. The sensitive aquatic insect Chironomus dilutus was used as a representative test species, and successful emergence was used as a chronic endpoint. Applying the MIXTOX modeling approach, predictive parametric models were fitted using single-compound toxicity data and statistically compared to observed toxicity in subsequent mixture tests. Imidacloprid-clothianidin, clothianidin-thiamethoxam and imidacloprid-clothianidin-thiamethoxam mixtures did not significantly deviate from concentration-additive toxicity. However, the cumulative toxicity of the imidacloprid-thiamethoxam mixture deviated from the concentration-additive reference model, displaying dose-ratio dependent synergism and resulting in up to a 10% greater reduction in emergence from that predicted by concentration addition. Furthermore, exposure to select neonicotinoid mixtures above 1.0 toxic unit tended to shift sex-ratios toward more male-dominated populations. Results indicate that, similar to acute exposures, the general assumption of joint additivity cannot adequately describe chronic cumulative toxicity of all neonicotinoid mixtures. Indeed, our observations of weak synergism and sex-ratio shifts elicited by some mixture combinations should be considered in water quality guideline development and environmental risk assessment practices for neonicotinoid insecticides, and explored in further investigations of the effects of neonicotinoid mixtures on aquatic communities.

Reconstructing long-term trends in municipal sewage discharge into a small lake in northern Manitoba, Canada.

Ross Lake lies within the City of Flin Flon (Manitoba, Canada), a mining community originally formed by the Hudson Bay Mining and Smelting Company (now Hudbay Minerals Inc.) in 1927. At the time of this investigation, a continuous effluent stream from Hudbay Minerals (approximately 80 years) and a discontinuous and unknown amount of raw and minimally treated municipal sewage (>20 years, likely ending in 1951) was discharged into the north basin of the lake. Maximum concentrations of fecal sterols, such as coprostanol and terrestrial phytosterols, such as: ß-sitosterol, campesterol, stigmastanol were measured in vertical sections of sediment cores, collected from Ross Lake, in the 15-16-cm section, which likely corresponds to the 1930s. Concentrations of coprostanol increased from <1 µg g(-1) in older sediments, to 252.3 µg g(-1) organic carbon at the peak. Observed changes in concentrations of sterols, in combination with radiometric dating and changes to sediment physicochemical characteristics, support the conclusion that sediments of a depth of less than 17.5-cm depth were deposited during the post-industrial era from approximately 1930 onwards. Ratios of coprostanol to cholesterol>1, peaking at 3.6 are consistent with anecdotal information that municipal sewage was discharged into Ross Lake during the early years of urbanization, prior to changes in treatment of sewage and discharge practices that began in 1951. Finally, historical concentrations of terrestrial phytosterols followed trends similar to those of coprostanol and cholesterol and may possibly be the result of an increase in the flux of terrestrial organic matter into Ross Lake as the result of regional deforestation due to logging and fire.

Endocrine disruption and oxidative stress in larvae of Chironomus dilutus following short-term exposure to fresh or aged oil sands process-affected water.

Understanding the toxicity of oil sands process-affected water (OSPW) is a significant issue associated with the production of oil from the Alberta oil sands. OSPW is acutely and chronically toxic to organisms, including larvae of Chironomus dilutus. In this study, fresh OSPW ('WIP-OSPW') was collected from the West In-Pit settling pond and aged OSPW ('FE5-OSPW') was collected from the FE5 experimental reclamation pond, both of which are located on the Syncrude Canada Ltd. lease site near Fort McMurray, Alberta, Canada. Larvae of C. dilutus were exposed to a freshwater control, WIP-OSPW, or FE5-OSPW for 4 or 7 days and survival, growth, and markers of oxidative stress and endocrine disruption were assessed. Survival was not significantly different among treatment groups. Compared to masses of larvae exposed to freshwater, masses of larvae exposed to WIP-OSPW were 49% lesser on day 4 and 62% lesser on day 7. However, organisms exposed to FE5-OSPW did not have significantly lesser masses than controls. Abundances of transcripts of glutathione-s-transferase (gst), catalase (cat), and glutathione peroxidase (gpx), which are important for the response to oxidative stress, were significantly altered in larvae exposed to WIP-OSPW, but not FE5-OSPW, relative to controls. Peroxidation of lipids was greater in larvae exposed to WIP-OSPW, but not FE5-OSPW. Exposure to fresh OSPW might have caused endocrine disruption because abundances of transcripts of the steroid hormone receptors, ultraspiricle protein (usp), ecysteroid receptor (esr), and estrogen related receptor (err) were greater in larvae exposed to WIP-OSPW for 7 days, but not FE5-OSPW. These results suggest that lesser growth of larvae of C. dilutus exposed to fresh OSPW might be due to oxidative stress and disruption of endocrine processes, and that aging of OSPW attenuates these adverse effects.

Food web structure in oil sands reclaimed wetlands.

Boreal wetlands play an important role in global carbon balance. However, their ecosystem function is threatened by direct anthropogenic disturbance and climate change. Oil sands surface mining in the boreal regions of Western Canada denudes tracts of land of organic materials, leaves large areas in need of reclamation, and generates considerable quantities of extraction process-affected materials. Knowledge and validation of reclamation techniques that lead to self-sustaining wetlands has lagged behind development of protocols for reclaiming terrestrial systems. It is important to know whether wetlands reclaimed with oil sands process materials can be restored to levels equivalent to their original ecosystem function. We approached this question by assessing carbon flows and food web structure in naturally formed and oil sands-affected wetlands constructed in 1970-2004 in the postmining landscape. We evaluated whether a prescribed reclamation strategy, involving organic matter amendment, accelerated reclaimed wetland development, leading to wetlands that were more similar to their natural marsh counterparts than wetlands that were not supplemented with organic matter. We measured compartment standing stocks for bacterioplankton, microbial biofilm, macrophytes, detritus, and zoobenthos; concentrations of dissolved organic carbon and residual naphthenic acids; and microbial production, gas fluxes, and aquatic-terrestrial exports (i.e., aquatic insect emergence). The total biomass of several biotic compartments differed significantly between oil sands and reference wetlands. Submerged macrophyte biomass, macroinvertebrate trophic diversity, and predator biomass and richness were lower in oil sands-affected wetlands than in reference wetlands. There was insufficient evidence to conclude that wetland age and wetland amendment with peat-mineral mix mitigate effects of oil sands waste materials on the fully aquatic biota. Although high variability was observed within most compartments, our data show that 20-year-old wetlands containing oil sands material have not yet reached the same level of function as their reference counterparts.

Effectiveness of ozonation treatment in eliminating toxicity of oil sands process-affected water to Chironomus dilutus.

Water soluble organic compounds (OCs), including naphthenic acids (NAs), are potentially toxic constituents of oil sands process-affected water (OSPW) that is generated during extraction of bitumen from Alberta oil sands. Ozonation can decrease concentrations of OCs in OSPW. However, effects of ozonated-OSPW on multicellular organisms are unknown. A 10-day and a chronic exposure of Chironomus dilutus to OSPW were conducted to assess effects on survival, growth, development, and behavior. Two separate batches of OSPW were treated with 30 or 80 mg ozone (O(3))/L. Wet body masses of larvae exposed to OSPW were 64 to 77% less than their respective controls (p < 0.001). However, both levels of ozonation significantly attenuated effects of OSPW on growth. Similarly, chronic exposure to untreated OSPW resulted in significantly less pupation than in the controls, with 31% and 71% less pupation of larvae exposed to the two batches of OSPW (p < 0.05). Emergence was significantly less for larvae exposed to OSPW, with 13% and 8% of larvae emerging, compared to 81% in controls (p < 0.0001). Both levels of ozonation of OSPW attenuated effects on emergence. These results suggest that OCs degraded by ozonation causes toxicity of OSPW toward C. dilutus, and that ozonation attenuates toxicity of OSPW.

Sublethal toxicity of two wastewater treatment polymers to lake trout fry (Salvelinus namaycush).

Lake trout fry (Salvelinus namaycush) were exposed in laboratory experiments to two wastewater treatment polymers, one anionic (MagnaFloc 156) and one cationic (MagnaFloc 368; Ciba Speciality Chemical), to determine if these chemicals which are used and discharged by mining operations in Canada's North pose a significant hazard to juvenile fishes. The cationic polymer was substantially more toxic to lake trout fry than the anionic polymer, with 96-h LC50 estimates of 2.08 and >600 mg/l, respectively. Separate 30-d exposure experiments yielded no observed and lowest observed effect concentrations, respectively, of 0.25 and 0.5mg/l for MagnaFloc 368, and 75 and 150 mg/l for MagnaFloc 156. In both cases, behavioural responses, especially startle response, were the most sensitive test endpoints. Histopathological assessment revealed that gill pathology appeared within a few days of exposure to both polymers, apparently as a result of localized hypoxia. Acute (4 d) effects included cloudy swelling of epithelial cells, increased gill vascularization, and thickening and shortening of the gill lamella. Chronic (30 d) polymer exposure produced only slightly greater pathological effects than acute exposure, with comparable responses observed only at >1.0mg/l MagnaFloc 368 and 150 mg/l MagnaFloc 156, suggesting that the fish displayed some level of both behavioural and physiological adaptation to the respiratory stress imposed by the two polymers.

Evaluation of bioaccumulation and photo-induced toxicity of fluoranthene in larval and adult life-stages of Chironomus tentans.

Laboratory sediment tests were conducted to evaluate the bioaccumulation and photo-induced toxicity of fluoranthene in larval and adult life-stages of the midge, Chironomus tentans. In the first of 2 experiments, fourth-instar and adult C. tentans exposed to spiked sediments (204 microg fluoranthene/g dry weight) were collected for determination of fluoranthene tissue concentrations and toxicity after ultraviolet (UV) radiation treatment in the absence of sediment (water-only). Fluoranthene tissue concentrations in larvae collected after a 72-hour exposure period were 7 times greater than concentrations in adults collected on emergence from the same spiked sediments. Fluoranthene-exposed adults were tolerant of UV exposure (100% survival after 7 hours), whereas larvae were sensitive (0% survival after 1 hour). In the second experiment, C. tentans larvae were exposed for 96 hours to 2 sediment treatments (170 and 54 microg fluoranthene/g dry weight), after which fluoranthene tissue concentrations were determined and UV exposures conducted under water-only versus sediment conditions. Exposure to UV radiation, in conjunction with sediment, provided larvae with significant protection from photo-induced toxicity compared with the water-only exposure. Adults that emerged from the 2 sediment treatments were also analyzed for fluoranthene tissue residues and exposed to UV radiation. Fluoranthene tissue residues were higher in adult female than in adult male midges, but exuviae from both sexes contained higher fluoranthene concentrations than whole-body tissue, thus demonstrating that the molting process is a possible detoxification mechanism. Consistent with the greater tissue residue concentrations, female midges demonstrated a significant decrease in survival under UV exposure compared with male midges. These studies indicated that both the burrowing behavior of the larvae and the elimination of fluoranthene in molted exuviae are protective mechanisms against photo-enhanced PAH toxicity in this species.

Assessment of the probabilistic ecological risk assessment-toxic equivalent combination approach for evaluating pesticide mixture toxicity to zooplankton in outdoor microcosms.

The probabilistic ecological risk assessment-toxic equivalent (PERA-TE) combination approach was recently introduced in response to the increased demand for risk assessment approaches that can accommodate mixtures. The effectiveness and validity of the PERA-TE approach was assessed using two types of pesticide mixtures tested in outdoor microcosms. The first type of mixture consisted of pesticides with similar modes of action (the organophosphorus insecticides chlorpyrifos and diazinon) and the second of pesticides with different modes of action (chlorpyrifos, endosulfan, and trifluralin). To assess the toxicity of, and potential interaction within, each type of mixture, theoretically equitoxic TE mixtures were prepared in different proportional ratios. The TE mixtures were based on the 10th centile of acute toxicity effects distributions (data obtained from the literature) and a factor of the sum of the 90th centile field concentrations extrapolated from exposure distributions based on North American surface water monitoring data. Changes in zooplankton population abundances were used as the effect measure. The binary organophosphorus mixtures were equitoxic and conformed to the concentration addition model. The observed response trends of zooplankton exposed to the mixture of chemicals with different modes of action were a result of the susceptibility of individual taxa to the dominating pesticide in each mixture. Overall, the PERA-TE approach was not effective in predicting the toxicity and interaction of all mixture types and should be limited to assessing mixtures of chemicals with similar modes of action.

An estimation of radiation doses to benthic invertebrates from sediments collected near a Canadian uranium mine.

A new method is described for calculating radiation doses to benthic invertebrates from radionuclide concentrations in freshwater sediment. Both internal and external radiation doses were estimated for all 14 principal radionuclides of the uranium-238 decay series. Sediments were collected from three sites downstream of a uranium mining operation in northern Saskatchewan, Canada. Sediments from two sites, located approximately 1.6 and 4.4 km downstream from mining operations, yielded absorbed doses to both larval midges, Chironomus tentans, and adult amphipods, Hyalella azteca, of 59-60 and 19 mGy/year, respectively, compared to 3.2 mGy/year for a nearby control site. External beta radiation from protactinium-234 (234Pa) and alpha radiation from uranium (U) contributed most of the dose at the impacted sites, whereas polonium-210 (210Po) was most important at the control site. If a weighting factor of 20 was employed for the greater biological effect of alpha vs. beta and gamma radiation, then total equivalent doses rose to 540-560 mGy/year at the site closest to uranium operations. Such equivalent doses are above the 360-mGy/year no-observed-effect level for reproductive effects in vertebrates from gamma radiation exposure. Data are not available to determine the effect of such doses on benthic organisms, but they are high enough to warrant concern. Detrimental effects have been observed in H. azteca at similar uranium concentration in laboratory toxicity tests, but it remains unclear whether the radiotoxicity or the chemotoxicity of uranium is responsible for these effects.

Effects of Diflubenzuron on Benthic Macroinvertebrates in Littoral Enclosures

Two applications of the insect growth regulator diflubenzuron were made to replicate littoral enclosures at nominal concentrations of 0.7, 2.5, 7.0, and 30 &mgr;g/L. Assessment of the effects of this insecticide on benthic macroinvertebrate community structure was accomplished by measuring changes in abundance and taxonomic richness. Chironomidae and Ephemeroptera were the most sensitive groups sampled, with no observed effect concentrations of 2.5 and 0.7 &mgr;g/L, respectively. No adverse effects were observed on Mollusca or Oligochaeta at any of the test concentrations. Taxonomic richness was noticably reduced at 7.0 and 30 &mgr;g/L on all post-application sampling dates, producing changes in community structure that persisted for >/=57 days.

Persistence and lateral movement of 2,4-dichlorophenoxy acetic acid and picloram on power line rights-of-way.

Soil persistence and lateral movement of 2,4-D (2,4-dichlorophenoxy acetic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) were examined following their application as a stem-foliage spray for brush control on two power line rights-of-way. Ditches to collect runoff water were located 3, 10, 20, and 30 m downslope from the treated areas. Runoff water and soil samples were collected after 0.14, 0.43, 0.57, 1, 2, 4, 7, 8, 11, 15, 16, and 48 weeks and were analyzed for picloram and 2,4-D residues. Only 3 of 85 soil samples downslope from the target areas contained residues of 2,4-D, and only 1 of 85 down slope samples contained a detectable residue of picloram. Of 56 runoff water samples, only 11 contained 2,4-D residues and only 1 contained residues of picloram. The greatest distances down-slope at which residues were detected in runoff water were 20 and 10 m for 2,4-D and picloram, respectively. No residues of either herbicide were recovered in soil or water at 15 weeks or 48 weeks after spraying. Despite normal rainfall frequency and amounts in the first several weeks after spraying in mid-June, significant runoff of either herbicide was not evident at either study site.