Laboratory and In situ Selenium Bioaccumulation Assessment in the Benthic Macroinvertebrates Hyalella azteca and Chironomus dilutus.

Selenium (Se) bioaccumulation and toxicity in aquatic vertebrates have been thoroughly investigated. Limited information is available on Se bioaccumulation at the base of aquatic food webs. In this study, we evaluated Se bioaccumulation in two benthic macroinvertebrates (BMI), Hyalella azteca and Chironomus dilutus raised in the laboratory and caged in-situ to a Canadian boreal lake e (i.e., McClean Lake) that receives continuous low-level inputs of Se (< 1 µg/L) from a uranium mill. Additional Se bioaccumulation assays were conducted in the laboratory with these BMI to (i) confirm field results, (ii) compare Se bioaccumulation in lab-read and native H. azteca populations and (iii) identify the major Se exposure pathway (surface water, top 1 cm and top 2-3 cm sediment layers) leading to Se bioaccumulation in H. azteca. Field and laboratory studies indicated overall comparable Se bioaccumulation and trophic transfer factors (TTFs) in co-exposed H. azteca (whole-body Se 0.9-3.1 µg/g d.w; TTFs 0.6-6.3) and C. dilutus (whole-body Se at 0.7-3.2 µg Se/g d.w.; TTFs 0.7-3.4). Native and lab-reared H. azteca populations exposed to sediment and periphyton from McClean Lake exhibited similar Se uptake and bioaccumulation (NLR, p = 0.003; 4.1 ± 0.8 µg Se/g d.w), demonstrating that lab-reared organisms are good surrogates to assess on-site Se bioaccumulation potential. The greater Se concentrations in H. azteca exposed to the top 1-3 cm sediment layer relative to waterborne exposure, corroborates the importance of the sediment-detrital pathway leading to greater Se bioaccumulation potential to higher trophic levels via BMI.

Biofragmentation of Polystyrene Microplastics: A Silent Process Performed by Chironomus sancticaroli Larvae.

Polystyrene (PS) is one of the main synthetic polymers produced around the world, and it is present in the composition of a wide variety of single-use objects. When released into the environment, these materials are degraded by environmental factors, resulting in microplastics. We investigated the ability of Chironomus sancticaroli (Diptera, Chironomidae) to promote the fragmentation of PS microspheres (24.5 ± 2.9 µm) and the toxic effects associated with exposure to this polymer. C. sancticaroli larvae were exposed to 3 different concentrations of PS (67.5, 135, and 270 particles g-1 of dry sediment) for 144 h. Significant lethality was observed only at the highest concentration. A significant reduction in PS particle size as well as evidence of deterioration on the surface of the spheres, such as grooves and cracks, was observed. In addition, changes in oxidative stress biomarkers (SOD, CAT, MDA, and GST) were also observed. This is the first study to report the ability of Chironomus sp. to promote the biofragmentation of microplastics. The information obtained demonstrates that the macroinvertebrate community can play a key role in the degradation of plastic particles present in the sediment of freshwater environments and can also be threatened by such particle pollution.

Oxidative stress is an essential factor for the induction of anhydrobiosis in the desiccation-tolerant midge, Polypedilum vanderplanki (Diptera, Chironomidae).

The sleeping chironomid (Polypedilum vanderplanki) is the only insect capable of surviving complete desiccation in an ametabolic state called anhydrobiosis. Here, we focused on the role of oxidative stress and we observed the production of reactive oxygen species (ROS) in desiccating larvae and in those exposed to salinity stress. Oxidative stress occurs to some extent in desiccating larvae, inducing carbonylation of proteins. Oxidative stress overcomes the antioxidant defenses of the larvae during the first hour following rehydration of anhydrobiotic larvae. It facilitates the oxidation of DNA and cell membrane lipids; however, these damages are quickly repaired after a few hours. In addition to its deleterious effects, we demonstrated that artificial exposure to oxidative stress could induce a response similar to desiccation stress, at the transcriptome and protein levels. Furthermore, the response of anhydrobiosis-related genes to desiccation and salinity stress was inhibited by antioxidant treatment. Thus, we conclude that oxidative stress is an essential trigger for inducing the expression of protective genes during the onset of anhydrobiosis in desiccating of P. vanderplanki larvae.

Genome-wide analyses of Glutathione S-transferase gene family and expression profiling under deltamethrin exposure in non-biting midge Propsilocerus akamusi.

Glutathione S-transferases (GSTs) are major enzymes in detoxification phase II, and have been functioned in resistance to various insecticides or oxidative stress. Herein, we selected the non-biting midge, Propsilocerus akamusi, widespread in Asian aquatic ecosystems, to uncover the gene location, structure, and phylogenetics relationship of GSTs at genome scale first time. Thirty-three cytosolic and four microsomal GST genes were identified and located on the four chromosomes. The cytosolic GSTs involved in the eight subclasses and five GST genes were unclassified. The expansion of GST genes in P. akamusi experienced duplication events on the delta, theta, xi, iota, and unclassified subclasses. The RNA-Seq analyses and RT-qPCR validation showed that the expression of PaGSTt2 gene is significantly elevated, with deltamethrin concentration increasing. The tertiary structure of PaGSTt2 enzyme was reconstructed, which was different from the other theta gene in the active site. In addition, the GST genes of six chironomids were first described based on the assembled genomes to explore the difference of those in the adaptation to kinds of environments. The GST frame for P. akmusi and its expression profiles provide valuable resources to understand their role in insecticide resistance of this species, as well as those of other biting midges.

Gene family expansions in Antarctic winged midge as a strategy for adaptation to cold environments.

Parochlus steinenii is the only flying insect native to Antarctica. To elucidate the molecular mechanisms underlying its adaptation to cold environments, we conducted comparative genomic analyses of P. steinenii and closely related lineages. In an analysis of gene family evolution, 68 rapidly evolving gene families, involved in the innate immune system, unfolded protein response, DNA packaging, protein folding, and unsaturated fatty acid biosynthesis were detected. Some gene families were P. steinenii-specific and showed phylogenetic instability. Acyl-CoA delta desaturase and heat shock cognate protein 70 (Hsc70) were representative gene families, showing signatures of positive selection with multiple gene duplication events. Acyl-CoA delta desaturases may play pivotal roles in membrane fluidity, and expanded Hsc70 genes may function as chaperones or thermal sensors in cold environments. These findings suggest that multiple gene family expansions contributed to the adaptation of P. steinenii to cold environments.

Accumulation of nylon microplastics and polybrominated diphenyl ethers and effects on gut microbial community of Chironomus sancticaroli.

Microplastics (MP) are emerging contaminants with the capacity to bind and transport hydrophobic organic compounds of environmental concern, such as polybrominated diphenyl ethers (PBDEs). The aim of this study was to investigate the ingestion of nylon (polyamide) MP alone and when associated with PBDEs and their effects on Chironomus sancticaroli larvae survival and microbiome structure. Survival, PBDE uptake and microbial community composition were measured in fourth instar larvae exposed for 96 h to BDEs- 47, 99, 100 and 153 in the presence and absence of 1% w/w MP in sediment. Microbiome community structures were determined through high throughput sequencing of 16S small subunit ribosomal RNA gene (16S rRNA). Initial experiments showed that larvae ingested MP faster at 0.5% w/w MP, while depuration was more efficient at 1% w/w MP, although retention of MP was seen even after 168 h depuration. No mortality was observed as a result of PBDEs and MP exposure. MP had a negative effect on PBDE concentration within larvae (η2 = 0.94) and a negative effect on sediment concentrations (η2 = 0.48). In all samples, microbial communities were dominated by Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Gammaproteobacteria. Bacterial alpha diversity was not significantly affected by PBDEs or MP exposure. However, the abundance of discrete bacterial taxa was more sensitive to MP (X2 = 45.81, p = 0.02), and PBDE exposure. Our results highlight that C. sancticaroli showed no acute response to MPs and PBDEs, but that MPs influenced bacterial microbiome structure even after only short-term acute exposure.

Desiccation-tolerance and globular proteins adsorb similar amounts of water.

When exposed to desiccation stress, extremotolerant organisms from all domains of life produce protective disordered proteins with the potential to inform the design of excipients for formulating biologics and industrial enzymes. However, the mechanism(s) of desiccation protection remain largely unknown. To investigate the role of water sorption in desiccation protection, we use thermogravimetric analysis to study water adsorption by two desiccation-tolerance proteins, cytosolic abundant heat soluble protein D from tardigrades and late embryogenesis abundant protein 4 from the anhydrobiotic midge Polypedilum vanderplanki, and, as a control, the globular B1 domain of staphylococcal protein G. All samples adsorb similar amounts of water, suggesting that modulated water retention is not responsible for dehydration protection by desiccation-tolerance proteins.

No Adverse Effects of Stacked Bacillus thuringiensis Maize on the Midge Chironomus riparius.

Material from genetically engineered maize producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) may enter aquatic ecosystems and expose nontarget organisms. We investigated the effects on life table parameters of the midge Chironomus riparius (Diptera: Chironomidae) of SmartStax maize leaves, which contain six different Cry proteins targeting Lepidoptera and Coleoptera pests, in two plant backgrounds. For midge development and emergence, 95% confidence intervals for the means of six conventional maize lines (Rheintaler, Tasty Sweet, ES-Eurojet, Planoxx, EXP 258, and EXP 262), were used to capture the natural range of variation. For reproduction, lowest and highest means were used. The natural range of variation allows one to judge whether observed effects between Bt maize and the closest non-Bt comparator are likely to be of biological relevance. No adverse effects on C. riparius were observed with any Bt maize line compared with the respective non-Bt counterpart. Development time was shorter when females were fed Bt maize than when they were fed non-Bt maize, but this effect was not considered adverse. Development time, emergence ratio, sex ratio, and larvae/egg rope measured for Bt maize were within the natural range of variation. Fecundity for the Bt lines was equal to or higher than that for the conventional lines. Future risk assessment studies may consider plant background effects and the natural range of variation to judge the relevance of observed differences between particular genetically engineered and non-genetically engineered plants. Environ Toxicol Chem 2022;41:1078-1088. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Warming Decreases Bioconversion of Polyunsaturated Fatty Acids in Chironomid Larvae Maintained on Cyanobacterium Microcystis.

Cyanobacteria dominance and warming have been suggested to decrease the production of polyunsaturated fatty acids (PUFA) in freshwater ecosystems. Physiological adaptations of poikilothermic animals to higher temperatures may further decrease PUFA levels in aquatic food webs. We conducted diet manipulation experiments to investigate the combined effects of dietary PUFA and warming on the proportions of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) in Chironomus riparius. The experimental diet consisted of a nontoxic cyanobacterium Microcystis, which contained C20 PUFA: 20:3n-3, 20:4n-3, and 20:3n-6, but no EPA or ARA. Additionally, we used TetraMin® fish flakes as a control treatment. A temperature increase from 20 °C to 25 °C decreased the proportion of n-3 C20 PUFA and the n-3/n-6 ratio in Microcystis. Diet manipulation experiments indicated that Chironomus desaturated dietary C20 precursors to EPA and ARA, but warming decreased this bioconversion and resulted in lower levels of EPA and ARA in Chironomus. Warming did not alter the proportions of EPA and ARA in Chironomus larvae if these PUFA were readily available in the diet (TetraMin® control treatment). In conclusion, warming and cyanobacteria dominance may decrease the production and trophic transfer of physiologically important PUFA in freshwaters by (1) decreasing the n-3/n-6 ratio and the abundance of n-3 C20 precursors in Microcystis, and (2) decreasing the bioconversion of n-3 and n-6 C20 precursors to EPA and ARA in chironomids. These changes may have cascading effects throughout the food web and decrease the content of EPA in fish, potentially affecting its availability to humans.

Expression of aquaporins in response to distinct dehydration stresses that confer stress tolerance in the Antarctic midge Belgica antarctica.

Larvae of the Antarctic midge Belgica antarctica Jacobs (Diptera: Chironomidae) are highly tolerant of diverse environmental stresses, including freezing, severe desiccation, and osmotic extremes. Furthermore, dehydration confers subsequent desiccation and freeze tolerance. While a role for aquaporins-channels for water and other solutes-has been proposed in these dehydration processes, the types of aquaporins involved in dehydration-driven stress tolerance remain unknown. In the present study, we investigated expression of six aquaporins (Drip, Prip, Eglp1, Eglp2, Aqp12L, and Bib) in larvae of B. antarctica subjected to three different dehydration conditions: desiccation, cryoprotective dehydration, and osmotic dehydration. The expression of Drip and Prip was up-regulated under desiccation and cryoprotective dehydration, suggesting a role for these aquaporins in efficient water loss under these dehydration conditions. Conversely, expression of Drip and Prip was down-regulated under osmotic dehydration, suggesting that their expression is suppressed in larvae to combat dehydration. Larval water content was similarly decreased under all three dehydration conditions. Differences in responses of the aquaporins to the three forms of dehydration suggests distinct water management strategies associated with different forms of dehydration stress.

Intrinsic stochasticity and the emergence of collective behaviours in insect swarms.

Intrinsic stochasticity associated with finite population size is fundamental to the emergence of collective behaviours in insect swarms. It has been assumed that this intrinsic stochasticity is purely additive (position independent) in quiescent (unperturbed) swarms. Here, I identify the hallmarks of intrinsic multiplicative (position dependent) stochasticity and show that they are evident in quiescent laboratory swarms of the non-biting midge Chironomus riparius. In accordance with theoretical expectations, the smallest well-documented laboratory swarms (containing between 14 and 46 individuals) are found to have q-Gaussian density profiles with [Formula: see text] 1, whereas larger laboratory swarms have Gaussian ([Formula: see text]1) density profiles. I show that these newly identified states are analogous to interstellar clouds and thereby extend a long-standing analogy between insect swarms and self-gravitating systems. Smaller laboratory swarms have been observed and are predicted to be gas-like, filling the available space rather than occupying just a small proportion of it. The new results unify laboratory swarms with wild swarms. Unlike laboratory swarms, wild swarms must contend with environmental (extrinsic) noise and have density profiles that are accurately represented by q-Gaussians with [Formula: see text] 1. Finally, it is shown how intrinsic multiplicative noise allows for the nucleation of swarms away from prominent visual features (basins of attraction) known as swarm markers.

Effects of anthropogenic activities on microplastics in deposit-feeders (Diptera: Chironomidae) in an urban river of Taiwan.

The presence of microplastics (MPs) in the environment has generated global concerns. However, the explicit assessment of the effect of multiple anthropogenic activities on the existence of MPs in the freshwater system is scarcely reported. This study quantified anthropogenic activities and analyzed their relationship with MPs on a freshwater organism: the midge larvae (Diptera: Chironomidae). The study took place in an urban river and consisted of comparing the abundance and types of MPs. Our results highlight that, while industrial area was the most important variable contributing to the total MP concentration in midge larvae, residential area also influenced the concentration of microfibers in midge larvae. The impact of a residential area on the relative abundance of microfibers in each sample site was diluted by the proximity to an industrial area. In conclusion, we suggest that industrial areas are a potential source of MP pollution in river sediment, and midge larvae can be a good indicator of the MP concentrations in urban river systems. Quantifying anthropogenic activities can help discern their effects on MP concentration in a river system and promote management strategies.

A multigenerational approach can detect early Cd pollution in Chironomus riparius.

Cadmium (Cd) is a non-essential highly toxic metal and its presence in the environment has been a concern over the years. On the present study we adopt the spiked water exposure scenario to study early Cd contamination across five generations of the model organism Chironomus riparius. Animals were, at the beginning of each generation, submitted to 0, 1, 3.2, 10, 32 and 100 µg/L of Cd. Classical endpoints like total emergence, EmT50, fertility and the integrative fitness measure, population growth rate (PGR), were calculated at each generation. Results could demonstrate that exposure to brief and low Cd concentrations can affect all the measured endpoints and, therefore, initial Cd pollution in previously unpolluted sites can be detected after just five consecutive generations. Importantly, at 100 µg/L of Cd fertility was greatly impaired after three generations. Also, PGR calculation is a sensitive tool for monitoring early pollution of Cd. Yet, no adaptation to Cd over five generations could be observed on the present experimental setup.

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.

Ibuprofen as an emerging pollutant on non-target aquatic invertebrates: Effects on Chironomus riparius.

The concern about pharmaceuticals has been increased over the last decade due to their burgeoning consumption. Ibuprofen has an extensive presence in surface water with risks for the aquatic biota. This study focuses on the effects of ibuprofen at environmental concentrations on the survival, transcriptional level, and enzymatic activity for 24, 96 h on Chironomus riparius. Ibuprofen developed a substantial effect on survival by all the conditions. mRNA levels of EcR, Dronc, and Met (endocrine system), hsp70, hsp24, and hsp27 (stress response), and Proph and Def (immune system) were modified, joined to increased GST and PO activity. The results confirmed alterations on the development of C. riparius, as well as two essential mechanisms, involved in protection against external toxicological challenge. Ibuprofen poses an incipient risk to C. riparius and could at an organismal level by compromising their survival, development, and ability to respond to adverse conditions on the future populations.

Application of Dispersive Liquid-Liquid Microextraction Followed by High-Performance Liquid Chromatography/Tandem Mass Spectrometry Analysis to Determine Tetrabromobisphenol A in Complex Matrices.

An accurate and sensitive ultrasound-dispersive liquid-liquid microextraction technique followed by high-performance liquid chromatography separation coupled with electrospray ionization tandem mass spectrometry detection method to determine the presence of tetrabromobisphenol A (TBBPA) in complex environmental matrices is proposed. The miniaturized procedure was used to extract and quantify the analyte in domestic sewage, anaerobic sludge, and the aquatic test organism species Daphnia magna and Chironomus sancticaroli, which are standardized organisms for ecotoxicity bioassays. Limits of detection of 2 ng L-1 (domestic sewage), 2 ng g-1 (anaerobic sludge), 0.25 ng g-1 (D. magna), and 5 ng g-1 (C. tentans) were obtained. The presence of TBBPA was determined in domestic sewage and anaerobic sludge from an anaerobic batch bioreactor at a concentration of 0.2 ± 0.03 µg L-1 and 507 ± 79 ng g-1 , respectively. In D. magna and C. sancticaroli exposed to TBBPA in an acute toxicity bioassay, the micropollutant accumulated at 3.74 and 8.87 µg g-1 , respectively. The proposed method is a simple and cost-effective tool to determine TBBPA environmental occurrence and biomagnification potential compared with conventional extraction methods. To the best of our knowledge, this is the first liquid-liquid miniaturized extraction method to be applied to D. magna and C. sancticaroli. Environ Toxicol Chem 2020;39:2147-2157. © 2020 SETAC.

Fatty acid metabolism and modifications in Chironomus riparius.

A priori knowledge of fatty acid modifications in consumers is essential for studies using fatty acids as biomarkers. We investigated fatty acid metabolism and possible modification pathways in benthic invertebrate Chironomus riparius larvae (Diptera). We conducted diet manipulation experiments using natural food sources (two chlorophyte algae, a diatom and a non-toxic cyanobacterium). We also did a diet-switch experiment on two different resources, fish food flakes TetraMin® and cyanobacterium Spirulina, to study fatty acid turnover in Chironomus. Results of the diet manipulation experiments indicate that Chironomus larvae have a strong tendency to biosynthesize 20:5n-3 and 20:4n-6 from precursor fatty acids, and that the dietary availability of polyunsaturated fatty acids (PUFA) does not control larval growth. Fatty acid modifications explain why low dietary availability of PUFA did not significantly limit growth. This has ecologically relevant implications on the role of benthic chironomids in conveying energy to upper trophic level consumers. A diet-switch experiment showed that the turnover rate of fatty acids in Chironomus is relatively fast--a few days. The compositional differences of algal diets were large enough to separate Chironomus larvae into distinct groups even if significant modification of PUFA was observed. In summary, fatty acids are excellent dietary biomarkers for Chironomus, if modifications of PUFA are considered, and will provide high-resolution data on resource use. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Effects of arthropod inquilines on growth and reproductive effort among metacommunities of the purple pitcher plant (Sarracenia purpurea var. montana).

Many plant species harbor communities of symbionts that release nutrients used by their host plants. However, the importance of these nutrients to plant growth and reproductive effort is not well understood. Here, we evaluate the relationship between the communities that colonize pitcher plant phytotelmata and the pitcher plants' vegetative growth and flower production to better understand the symbiotic role played by phytotelma communities. We focus on the mountain variety purple pitcher plant (Sarracenia purpurea var. montana), which occurs in small and isolated populations in Western North Carolina. We found that greater symbiont community diversity is associated with higher flower production the following season. We then examined geographic variation in communities and found that smaller plant populations supported less diverse symbiont communities. We relate our observations to patterns of community diversity predicted by community ecology theory.

Developmental and sexual divergence in the olfactory system of the marine insect Clunio marinus.

An animal's fitness strongly depends on successful feeding, avoidance of predators and reproduction. All of these behaviours commonly involve chemosensation. As a consequence, when species' ecological niches and life histories differ, their chemosensory abilities need to be adapted accordingly. The intertidal insect Clunio marinus (Diptera: Chironomidae) has tuned its olfactory system to two highly divergent niches. The long-lived larvae forage in a marine environment. During the few hours of terrestrial adult life, males have to find the female pupae floating on the water surface, free the cryptic females from their pupal skin, copulate and carry the females to the oviposition sites. In order to explore the possibility for divergent olfactory adaptations within the same species, we investigated the chemosensory system of C. marinus larvae, adult males and adult females at the morphological and molecular level. The larvae have a well-developed olfactory system, but olfactory gene expression only partially overlaps with that of adults, likely reflecting their marine vs. terrestrial lifestyles. The olfactory system of the short-lived adults is simple, displaying no glomeruli in the antennal lobes. There is strong sexual dimorphism, the female olfactory system being particularly reduced in terms of number of antennal annuli and sensilla, olfactory brain centre size and gene expression. We found hints for a pheromone detection system in males, including large trichoid sensilla and expression of specific olfactory receptors and odorant binding proteins. Taken together, this makes C. marinus an excellent model to study within-species evolution and adaptation of chemosensory systems.

Laboratory and Field-Based Assessment of the Effects of Sediment Capping Materials on Zinc Flux, Bioavailability, and Toxicity.

A former mining site has been the subject of extensive remediation and restoration, with a significant focus on disconnecting mine spoils from groundwater and managing the quantity and quality of runoff. A remaining task is ensuring concentrations of zinc (Zn) in the stream outflow of a pit lake are reduced below water quality standards. The efficacy of multiple capping materials for decreasing Zn dissolution from sediments was conducted under natural and reasonable worst-case conditions (pH = 5.5). Capping materials included AquaBlok™, limestone, and limestone-bone char. Field exposures were conducted in limnocorrals that isolated overlying water columns above the sediment and capping treatments. Simultaneous in situ and ex situ toxicity tests were conducted using Daphnia magna, Hyalella azteca, and Chironomus dilutus. In situ caged organisms were protected from temperature shock (warm epilimnetic waters) by deploying within a Toxicity Assessment Container System (TACS). Organisms were exposed to surficial sediments, caps, and hypolimnetic overlying waters for 4 d. Ex situ testing was conducted in core tube mesocosms containing sediments and caps at similar temperatures (15-19 °C). Results demonstrated the usefulness of TACS deployment in stratified lake systems. There were no differences in responses between treatments involving sediment capping materials in both in situ and ex situ tests. The lack of differences was likely due to dissolved Zn in surface water being below the hardness-adjusted threshold effects levels (164 µg L-1 ). This field- and laboratory-based weight-of-evidence study provided site-specific data to support the selection of an effective remedy, with reduced uncertainty compared to laboratory and chemistry-only approaches. Environ Toxicol Chem 2019;39:240-249. © 2019 SETAC.