Cross-Platform Comparison of Amino Acid Metabolic Profiling in Three Model Organisms Used in Environmental Metabolomics.

Environmental metabolomics is a promising approach to study pollutant impacts to target organisms in both terrestrial and aquatic environments. To this end, both nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based methods are used to profile amino acids in different environmental metabolomic studies. However, these two methods have not been compared directly which is an important consideration for broader comparisons in the environmental metabolomics field. We compared the quantification of 18 amino acids in the tissue extracts of Daphnia magna, a common model organism used in both ecotoxicology and ecology, using both 1H NMR spectroscopy and liquid chromatography with tandem MS (LC-MS/MS). 1H NMR quantification of amino acids agreed with the LC-MS/MS quantification for 17 of 18 amino acids measured. We also tested both quantitative methods in a D. magna sub-lethal exposure study to copper and lithium. Again, both NMR and LC-MS/MS measurements showed agreement. We extended our analyses with extracts from the earthworm Eisenia fetida and the plant model Nicotiana tabacum. The concentrations of amino acids by both 1H NMR and LC-MS/MS, agreed and demonstrated the robustness of both techniques for quantitative metabolomics. These findings demonstrate the compatibility of these two analytical platforms for amino acid profiling in environmentally relevant model organisms and emphasizes that data from either method is robust for comparisons across studies to further build the knowledge base related to pollutant exposure impacts and toxic responses of diverse environmental organisms.

Fish tissue accumulation and proteomic response to microcystins is species-dependent.

Cyanotoxins including microcystins are increasing globally, escalating health risks to humans and wildlife. Freshwater fish can accumulate and retain microcystins in tissues; however, uptake and depuration studies thus far have not exposed fish to microcystins in its intracellular state (i.e., cell-bound or conserved within cyanobacteria), which is a primary route of exposure in the field, nor have they investigated sublethal molecular-level effects in tissues, limiting our knowledge of proteins responsible for microcystin toxicity pathways in pre-to-postsenescent stages of a harmful algal bloom. We address these gaps with a 2-wk study (1 wk of 'uptake' exposure to intracellular microcystins (0-40 µg L-1) produced by Microcystis aeruginosa followed by 1 wk of 'depuration' in clean water) using Rainbow Trout (Oncorhynchus mykiss) and Lake Trout (Salvelinus namaycush). Liver and muscle samples were collected throughout uptake and depuration phases for targeted microcystin quantification and nontargeted proteomics. For both species, microcystins accumulated at a higher concentration in the liver than muscle, and activated cellular responses related to oxidative stress, apoptosis, DNA repair, and carcinogenicity. However, intraspecific proteomic effects between Rainbow Trout and Lake Trout differed, and interspecific accumulation and retention of microcystins in tissues within each species also differed. We demonstrate that fish do not respond the same to cyanobacterial toxicity within and among species despite being reared in the same environment and diet.

No evidence of spherical microplastics (10-300 µm) translocation in adult rainbow trout (Oncorhynchus mykiss) after a two-week dietary exposure.

The consumption of fish contaminated with microplastics is often cited as a pathway for human exposure. However, because their guts are generally removed before consumption, exposure may be low compared to other routes such as shellfish, drinking water and dust. Still, microplastics have been found to translocate from the gut to other tissues, making exposure from eating fish fillets or other seafood products a potential concern. To better understand fish as an exposure route for microplastics in humans, we tested hypotheses about whether translocation occurs and if efficiency of translocation is dependent on particle size. We investigated the amount and distribution of fluorescent polyethylene microspheres (10-300 µm) in the gut, liver, fillets and gonads of adult rainbow trout after a two-week dietary exposure. Fish were fed food pellets dosed with up to ~9,800 microspheres per gram of food. Total exposures over the entire experiment ranged from ~80,000-850,000 microspheres per fish. We did not find any particles in the fillets, liver, or gonads of any fish, suggesting that translocation of spherical microplastics of this size range does not occur in adult rainbow trout. The quantity of microplastics found in the gut was also low or absent after a 24-hour depuration period, indicating effective excretion in this laboratory population. This research suggests that the consumption of fish fillets may not be a significant exposure pathway for microspheres >10 µm in size to contaminate humans. Future studies should test for different sizes, morphologies and species to further our understanding.

Assessing the toxicity of cell-bound microcystins on freshwater pelagic and benthic invertebrates.

Cyanobacterial harmful algal blooms dominated by Microcystis frequently produce microcystins, a family of toxins capable of inflicting harm to pelagic and benthic freshwater invertebrates. Research on the effect of microcystins on invertebrates is inconclusive; from one perspective, studies suggest invertebrates can coexist in toxic blooms; however, studies have also measured negative food-associated effects from microcystins. To test the latter perspective, we examined the reproduction, growth, and survival of laboratory-cultured Ceriodaphnia dubia, Daphnia magna, and Hexagenia spp. exposed to cell-bound microcystins through a series of life-cycle bioassays. Test organisms were exposed to a concentration gradient ranging from 0.5 µg L-1 to 300 µg L-1 microcystins, which corresponds to values typically found in freshwaters during bloom season. Lethal concentrations in C. dubia (LC50 = 5.53 µg L-1) and D. magna (LC50 = 85.72 µg L-1) exposed to microcystins were among the lowest recorded to date, and reproductive effects were observed at concentrations as low as 2.5 µg L-1. Length of D. magna was significantly impacted in microcystin treatments great than 2.5 µg L-1. No lethality or growth impairments were observed in Hexagenia. This information will improve our understanding of the risks posed by microcystins to food webs in freshwaters.

An efficient and affordable laboratory method to produce and sustain high concentrations of microcystins by Microcystis aeruginosa.

Microcystis aeruginosa is a cosmopolitan cyanobacteria that continues to jeopardize freshwater ecosystem services by releasing the hepatotoxin microcystin, which can, in some cases, cause death to aquatic fauna and even humans. Currently, our abilities to understand the mechanisms of microcystin toxicology are limited by the lack of a method for producing high concentrations, which are central to large-scale and long-term research in natural systems. Here we present an efficient and affordable laboratory method to produce high concentrations of microcystins by a toxigenic strain of M. aeruginosa. Through batch culture studies, we yielded microcystins at concentrations that are environmentally relevant to freshwaters around the world (1-300 µg L-1), maintained these concentrations without resupplying fresh medium (further reducing costs), and utilized rate equations to model the relationship between the environmental conditions in the cultures and changes occurring within the M. aeruginosa cells. Our assessment suggests that steady production of microcystins depends on the availability of carbon throughout the experiment. Hence, we recommend the use of tissue culture treated flasks with a vented cap to ensure the production of microcystins is uninterrupted. This method demonstrates that microcystins can be produced in the laboratory at concentrations relevant to freshwater ecosystems. •The method demonstrates M. aeruginosa CPCC 300 is a reliable strain of freshwater cyanobacteria that can yield microcystins at environmentally relevant concentrations.•Validation showed M. aeruginosa CPCC 300 is resilient in carbon-limited situations and may respond to stress by shifting the ratio of microcystin congeners.•Cell culture flasks with vented caps -filled no more than 50 % of the flask volume to allow for sufficient air exchange- are an excellent and cost-effective approach to maintaining cell growth and producing microcystins at a range between 300 to 1200 µg L-1.

Acute Effects of Binary Mixtures of Imidacloprid and Tebuconazole on 4 Freshwater Invertebrates.

Receiving waters from agricultural areas can contain multiple pesticides such as the neonicotinoid imidacloprid and the fungicide tebuconazole, leading to the potential for aquatic life to be exposed to such mixtures. In the present study, the effects of tebuconazole were tested alone and in binary mixtures with imidacloprid on 4 aquatic invertebrates: Chironomus dilutus, Hyalella azteca, Lumbriculus variegatus, and Neocloeon triangulifer. Acute (96-h) median lethal concentrations (LC50s) were derived for individual compounds and used to design a binary mixture study to determine cumulative effects. The LC50s showed that imidacloprid was more potent than tebuconazole by 1 to 3 orders of magnitude for the 4 species. Lethality data from mixture experiments were analyzed using MIXTOX to determine deviations from independent action, followed by the model deviation ratio (MDR) technique to determine the biological significance and reproducibility of observed mixture effects. MIXTOX showed that the cumulative toxicities of imidacloprid-tebuconazole differed between the species: for C. dilutus there was no deviation from independent action; however, for H. azteca the mixture was antagonistic (specifically dose ratio-dependent), and for N. triangulifer it was synergistic. The MDR method showed that only observations with H. azteca significantly deviated from independent action. Because of the lack of evidence of a clear deviation from independent action and the much greater potency of imidacloprid, the weight of evidence indicates that the presence of tebuconazole is unlikely to appreciably increase the hazard from imidacloprid exposure to aquatic invertebrates. Environ Toxicol Chem 2019;00:1-17. © 2019 SETAC.

Relative chronic sensitivity of neonicotinoid insecticides to Ceriodaphnia dubia and Daphnia magna.

Neonicotinoid insecticides are a group of plant protectants frequently detected in surface waters at low concentrations. Aquatic invertebrates therefore have the potential to be exposed chronically to low concentrations of neonicotinoids. The cladocerans Daphnia magna and Ceriodaphnia dubia are among the most commonly used invertebrate test species in aquatic toxicology. Both species are known to be acutely insensitive to neonicotinoids, and while chronic toxicity has been characterized for D. magna, little research has been conducted with C. dubia. In the present study we conducted 7-d static-renewal life cycle tests for 6 neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam) with C. dubia, and a 21-d test with imidacloprid with D. magna. 7-d LC50s for C. dubia ranged from 8.42 mg L-1 for imidacloprid to > 100 mg L-1 for clothianidin; 7-d reproduction EC50s were 2.98 for thiacloprid, to > 67 mg L-1 for dinotefuran. D. magna were less sensitive than C. dubia to imidacloprid, by 4-fold for lethality and 1.5-fold for reproduction; however, acute-to-chronic ratios (ACRs) were similar. ACRs, based on 48-h acute LC50s and 7- or 21-d chronic reproduction EC10s, ranged from 5.4 for acetamiprid to 53.0 for imidacloprid (mean 36.6, CV = 51%). Chronic toxicity values for both species were orders of magnitude greater than concentrations reported in the environment, and thus hazard to these cladocerans is negligible.

Chronic toxicity of 6 neonicotinoid insecticides to Chironomus dilutus and Neocloeon triangulifer.

Neonicotinoid insecticides are frequently detected in surface waters near agricultural areas, leading to a potential for chronic exposure to sensitive aquatic species. The midge Chironomus dilutus and the mayfly Neocloeon triangulifer have been shown to be acutely sensitive to neonicotinoids. Previous studies have established chronic effects of some neonicotinoids on C. dilutus, but reproduction has not been studied. Toxic effects have not been assessed using N. triangulifer. We present the results of chronic, static-renewal tests for 6 neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam) with C. dilutus (≤56-d in length) and N. triangulifer (≤32-d in length). Emergence was generally the most sensitive endpoint for both species across all neonicotinoids. Effect concentrations, 10% (EC10s; emergence) were 0.03 to 1.1 µg L-1 for acetamiprid, clothianidin, imidacloprid, and thiacloprid. Dinotefuran and thiamethoxam were less potent, with EC10s (C. dilutus) or median effect concentrations (EC50s; N. triangulifer) of 2.2 to 11.2 µg L-1 . Hazard was assessed through comparison of neonicotinoid environmental concentrations from agricultural surface waters in Ontario (Canada) with either the 5th percentile hazard concentration (for imidacloprid) or species-specific EC10s from the present study (for all remaining neonicotinoids). The resulting hazard quotients (HQs) indicated little to no hazard (HQ <1) in terms of chronic toxicity for acetamiprid, dinotefuran, thiacloprid, or thiamethoxam. A moderate hazard (HQ >1) was found for emergence of N. triangulifer for clothianidin, and a high hazard (HQ = 74) was found for imidacloprid. Environ Toxicol Chem 2018;37:2727-2739. © 2018 SETAC.

Chronic effects of an environmentally-relevant, short-term neonicotinoid insecticide pulse on four aquatic invertebrates.

Neonicotinoid insecticides used in agriculture can enter freshwater environments in pulses; that is, a short-term period of a higher concentration, followed by a period of a comparatively lower concentration. Non-target aquatic arthropods are exposed to these fluctuating concentrations of neonicotinoids. The present study investigated the potential latent effects of a single environmentally-relevant 24-h pulse of imidacloprid and thiamethoxam, in separate experiments, on the early life-stages of four aquatic arthropods (Hyalella azteca, Chironomus dilutus, Hexagenia spp., and Neocloeon triangulifer). At least three nominal pulse concentrations were tested for each neonicotinoid-species combination: 2.5, 5, and 10 µg L-1, which were based on environmental monitoring in Ontario, Canada. After exposure to the pulse, organisms were assessed for survival and immobilization. Surviving organisms were then moved into clean water for a chronic post-treatment period, where endpoints including survival, growth, reproduction or emergence, depending on the species, were evaluated. Immediately after the 24-h pulse, immobilization was seen in C. dilutus and N. triangulifer in the highest imidacloprid concentrations tested (8.8 and 8.9 µg L-1, respectively). After transfer to clean water, immobilized organisms recovered, and no latent toxicity was seen for any of the evaluated endpoints. H. azteca and Hexagenia spp. showed no effects immediately after the imidacloprid pulse, or after the chronic post-treatment period. No effects were seen in any species after the thiamethoxam pulse, or the post-treatment period. The present study shows that toxic effects due to short-term pulse exposures of ~9 µg L-1 imidacloprid can occur in sensitive insect species. However, organisms can recover when the stressor ceases, with no long-term effects on test organisms.

Acute toxicity of 6 neonicotinoid insecticides to freshwater invertebrates.

Neonicotinoids are a group of insecticides commonly used in agriculture. Due to their high water solubility, neonicotinoids can be transported to surface waters and have the potential to be toxic to aquatic life. The present study assessed and compared the acute (48- or 96-h) toxicity of 6 neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam) to 21 laboratory-cultured and field-collected aquatic invertebrates spanning 10 aquatic arthropod orders. Test conditions mimicked species' habitat, with lentic taxa exposed under static conditions, and lotic taxa exposed under recirculating systems. Median lethal concentrations (LC50s) and median effect concentrations (EC50s; immobility) were calculated and used to construct separate lethal- and immobilization-derived species sensitivity distributions for each neonicotinoid, from which 5th percentile hazard concentrations (HC5s) were calculated. The results showed that the most sensitive invertebrates were insects from the orders Ephemeroptera (Neocloeon triangulifer) and Diptera (Chironomus dilutus), whereas cladocerans (Daphnia magna, Ceriodaphnia dubia) were the least sensitive. The HC5s were compared with neonicotinoid environmental concentrations from Ontario (Canada) monitoring studies. For all neonicotinoids except imidacloprid, the resulting hazard quotients indicated little to no hazard in terms of acute toxicity to aquatic communities in Ontario freshwater streams. For the neonicotinoid imidacloprid, a moderate hazard was found when only invertebrate immobilization, and not lethality, data were considered. Environ Toxicol Chem 2018;37:1430-1445. © 2018 SETAC.

Effects of the herbicide surfactant MON 0818 on oviposition and viability of eggs of the ramshorn snail (Planorbella pilsbryi).

The surfactant mixture MON 0818 is an adjuvant in various commercial formulations of the herbicide glyphosate. Initial studies have shown that MON 0818 is more toxic to aquatic animals than the active ingredient. However, few studies have examined the effect of exposure to MON 0818 on species of mollusks, and no studies have examined the effect on gastropods. The present study investigated the effect of acute exposure (96 h) of MON 0818 to the eggs, juveniles, and adults of the file ramshorn snail (Planorbella pilsbryi). Concentrations of MON 0818 up to 9.9 mg/L did not have a significant effect on the viability of eggs (p > 0.05). Juvenile snails (50% lethal concentration [LC50] = 4.0 mg/L) were more sensitive than adult snails (LC50 = 4.9-9.1 mg/L). Oviposition was inhibited by exposure to MON 0818 (median effective concentration [EC50] = 0.4-2.0 mg/L). However, oviposition resumed when snails were removed to clean water, even after 96-h exposure to up to 4.9 mg/L of MON 0818. Exposure to a concentration ≥2.7 mg/L caused visible damage to the tentacles of adult snails, which could potentially impact chemoreception. A deterministic hazard assessment indicated that environmentally relevant concentrations of MON 0818 could pose a hazard to the deposition of eggs. However, because of the relatively short half-life of MON 0818 in aquatic systems and the ability of snails to resume oviposition following the dissipation of MON 0818, environmentally relevant concentrations of MON 0818 likely pose a de minimis risk to populations of ramshorn snails. Environ Toxicol Chem 2017;36:522-531. © 2016 SETAC.

The evaluation of 3 diets for rearing Hyalella azteca and the influence of diet on acute ammonia toxicity.

Three Hyalella azteca cultures were reared on different diets since birth, reflecting the recommended diets of various investigators. The 3 diets consisted of fish flakes (FF), a mixture of fish flakes supplemented with the diatom Thalassiosira weissflogii (FF-D), and a mixture of fish flakes supplemented with yeast, cereal grass media, and trout chow (FF-YCT). The 3 diets were evaluated by comparing 20 wk of culturing data, along with the organism's response to standard 96-h toxicity testing with ammonium chloride over a range of pH and temperature. Hyalella azteca fed the FF-D diet had the highest overall survival rate (96.6%, standard deviation [SD] 4.3%) compared to those fed the FF diet (92.0%, SD 12.7%), or the FF-YCT diet (91.1%, SD 14.8%), although difference in survival was not statistically significant. Organisms fed the FF-D diet produced a higher number of young per week per adult (6.1, SD 2.8) than the FF diet (5.1, SD 2.2), or the FF-YCT diet (4.0, SD 1.2), although differences were not statistically significant. Of the diets evaluated, H. azteca reared on the FF-D diet were often significantly more resistant to total and un-ionized ammonia toxicity in acute 96-h testing than those reared on the other 2 diets across the 2 temperatures and 5 pHs tested, suggesting this may be the most optimal diet for this species. Environ Toxicol Chem 2016;35:2416-2424. © 2016 SETAC.

(1)H NMR-based metabolomics investigation of Daphnia magna responses to sub-lethal exposure to arsenic, copper and lithium.

Metal and metalloid contamination constitutes a major concern in aquatic ecosystems. Thus it is important to find rapid and reliable indicators of metal stress to aquatic organisms. In this study, we tested the use of (1)H nuclear magnetic resonance (NMR) - based metabolomics to examine the response of Daphnia magna neonates after a 48h exposure to sub-lethal concentrations of arsenic (49µgL(-1)), copper (12.4µgL(-1)) or lithium (1150µgL(-1)). Metabolomic responses for all conditions were compared to a control using principal component analysis (PCA) and metabolites that contributed to the variation between the exposures and the control condition were identified and quantified. The PCA showed that copper and lithium exposures result in statistically significant metabolite variations from the control. Contributing to this variation was a number of amino acids such as: phenylalanine, leucine, lysine, glutamine, glycine, alanine, methionine and glutamine as well as the nucleobase uracil and osmolyte glycerophosphocholine. The similarities in metabolome changes suggest that lithium has an analogous mode of toxicity to that of copper, and may be impairing energy production and ionoregulation. The PCA also showed that arsenic exposure resulted in a metabolic shift in comparison to the control population but this change was not statistically significant. However, significant changes in specific metabolites such as alanine and lysine were observed, suggesting that energy metabolism is indeed disrupted. This research demonstrates that (1)H NMR-based metabolomics is a viable platform for discerning metabolomic changes and mode of toxicity of D. magna in response to metal stressors in the environment.

Accumulation and depuration of polychlorinated biphenyls from field-collected sediment in three freshwater organisms.

As laboratory-based bioaccumulation methods are standardized and expanded to include other test species, kinetic studies assessing the major classes of contaminants with these species are needed to adequately select the standard duration for bioaccumulation tests. In the present study we measured the uptake (28-d exposure) of polychlorinated biphenyls (PCBs; total and selected congeners) from field-contaminated sediment in the oligochaete Lumbriculus variegatus, mayfly nymph Hexagenia spp., and fathead minnow Pimephales promelas. Depuration (25 d) of PCBs was measured in organisms that had been transferred to clean sediment after the 28-d exposure. Uptake and elimination of PCBs was rapid in L. variegatus and Hexagenia spp. Tissue residues reached steady-state concentrations within 28 d; elimination rates and biota-sediment accumulation factors (BSAFs) of the PCB congeners were not correlated with K(OW). Uptake and elimination rates of PCBs were slower in P. promelas, and it is not clear whether steady-state was reached in fish tissues. Elimination rates of the PCB congeners significantly decreased with increasing K(OW) in fish. The appropriateness of a 28-d exposure for measuring steady-state concentrations in tissue of the invertebrates was confirmed, but further study is required for fish.

Validation of Ontario's new laboratory-based bioaccumulation methods with in situ field data.

To validate the standardization of a laboratory protocol for measuring bioaccumulation, laboratory-derived tissue residues and biota-sediment accumulation factors (BSAFs) were compared with historical field data from nine sites in Ontario, Canada. As a result of temporal discontinuity between the field and the laboratory studies, a priori considerations, such as changes in site conditions or concentrations of contaminants in sediment, were necessary to assess whether to compare absolute or relative measures of bioaccumulation. For the majority of sites, BSAFs for field-collected and laboratory-exposed fish were within a factor of 2. Biota-sediment accumulation factors for laboratory-exposed oligochaetes were typically greater than those for mussels caged in the field, by a factor of 2 to 9. Overall, the laboratory methods for all species generally overestimated the relative bioavailability of contaminants compared with field conditions by a factor of 1.1 to 9.2. Other than the great disparity observed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) between field and laboratory studies, on average the laboratory-derived BSAFs with Pimephales promelas and Lumbriculus variegatus overestimated those from field-collected fish and field-exposed mussels by factors of 1.6 and 3.6, respectively. The laboratory method reflects a potentially worst-case exposure scenario and provides an appropriately conservative estimate of bioaccumulation. Laboratory-based estimates can be comparable to bioaccumulation data from the field but may be confounded by species-specific differences in routes of exposure and bioaccumulation of certain compounds or other environmental and biological factors that should be considered in these comparisons.

A comparison of the bioaccumulation potential of three freshwater organisms exposed to sediment-associated contaminants under laboratory conditions.

In the field of sediment quality assessment, increased support has been expressed for using multiple species that represent different taxa, trophic levels, and potential routes of exposure. However, few studies have compared the bioaccumulation potential of various test species over a range of sediment contaminants (hydrophobic organics and metals). As part of the development and standardization of a laboratory bioaccumulation method for the Ontario Ministry of the Environment, the oligochaete Lumbriculus variegatus, mayfly nymph Hexagenia spp., and juvenile fathead minnow Pimephales promelas were exposed to a variety of field-contaminated sediments (n = 10) to evaluate their relative effectiveness for accumulating different contaminants (e.g., dichlorodiphenyltrichloroethane [DDT] and metabolites, polychlorinated biphenyls [PCBs), polycyclic aromatic hydrocarbons [PAHs), polychlorinated dibenzo-p-dioxins and dibenzofurans [PCDD/Fs), and heavy metals). Bioaccumulation was usually highest in L. variegatus but also most variable within and (relative measures) between sediments. Bioaccumulation was similar between L. variegatus and Hexagenia spp. in most of the sediments tested. Significant differences in bioaccumulation between species were observed for DDT, dichlorodiphenyldichloroethane (DDD), PAHs, and PCDD/Fs. The present study indicates that species-specific differences in bioaccumulation may, but do not always, exist and can vary with contaminant and sediment type. The choice of test species or combination to use in a standard test method may depend on the objectives of the sediment quality assessment and data requirements of an ecological risk assessment. The results of the present study provide insight for selection of test species and validation of laboratory methods for assessing bioaccumulation with these species, as well as valuable information for interpreting results of bioaccumulation tests.

Measuring bioaccumulation of contaminants from field-collected sediment in freshwater organisms: a critical review of laboratory methods.

To be effective, decision-making frameworks require data from robust and reliable test methods. Using standard methods allows for more effective comparison between studies and application of data, and it reduces unnecessary duplication of efforts. Laboratory methods to assess the toxicity of sediment have been standardized and extensively used; however, procedures for measuring the bioaccumulation of contaminants from sediment into aquatic organisms need further standardization. Bioaccumulation methods using freshwater invertebrates and fish exposed to field-contaminated sediment were reviewed to identify important similarities and differences in method protocols, test conditions that need to be controlled, and data gaps. Although guidance documents are available, great variation still exists in exposure techniques used in tests, which may potentially affect the estimation of bioaccumulation. The techniques most consistent across studies include the use of Lumbriculus variegatus as a test species, test temperatures between 20 and 25°C, and a 28-d exposure with no addition of food, followed by purging of organisms. Issues that were inconsistent between studies or remained unspecified, which should be addressed, include the bioaccumulation potential of other test species, loading density of organisms, and sediment-to-water ratio. In addition to proper evaluation of the various exposure techniques and conditions, a need exists for more consistent inclusion of quality control procedures during testing.