David G. Poirier

1H 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.

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.

Characterization and Biological Potency of Mono- to Tetra-Halogenated Carbazoles

This paper deals with the characterization and aryl hydrocarbon receptor (AhR) agonist activities of a series of chlorinated, brominated, and mixed bromo/chlorocarbazoles, some of which have been identified in various environmental samples. Attention is directed here to the possibility that halogenated carbazoles may currently be emitted into the environment as a result of the production of carbazole-containing polymers present in a wide variety of electronic devices. We have found that any carbazole that is not substituted in the 1,3,6,8 positions may be lost during cleanup of environmental extracts if a multilayer column is utilized, as is common practice for polychlorinated dibenzo-p-dioxin (dioxin) and related compounds. In the present study, (1)H NMR spectral shift data for 11 relevant halogenated carbazoles are reported, along with their gas chromatographic separation and analysis by mass spectrometry. These characterization data allow for confident structural assignments and the derivation of possible correlations between structure and toxicity based on the halogenation patterns of the isomers investigated. Some halogenated carbazoles exhibit characteristics of persistent organic pollutants and their potential dioxin-like activity was further investigated. The structure-dependent induction of CYP1A1 and CYP1B1 gene expression in Ah-responsive MDA-MB-468 breast cancer cells by these carbazoles was similar to that observed for other dioxin-like compounds, and the magnitude of the fold induction responses for the most active halogenated carbazoles was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 2,3,6,7-Tetrachlorocarbazole was one of the most active halogenated carbazoles and, like TCDD, contains 4 lateral substituents; however, the estimated relative effect potency for this compound (compared to TCDD) was 0.0001 and 0.0032, based on induction of CYP1A1 and CYP1B1 mRNA, respectively.

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.

Bioaccumulation of perfluorinated carboxylates and sulfonates and polychlorinated biphenyls in laboratory-cultured Hexagenia spp., Lumbriculus variegatus and Pimephales promelas from field-collected sediments

Polychlorinated biphenyls (PCBs) and perfluorinated carboxylates and sulfonates (PFASs) are persistent pollutants in sediment that can potentially bioaccumulate in aquatic organisms. The current study investigates variation in the accumulation of PCBs and PFASs in laboratory-cultured Hexagenia spp., Lumbriculus variegatus and Pimephales promelas from contaminated field-collected sediment using 28-day tests. BSAF(lipid) (lipid-normalized biota-sediment accumulation factor) values for total concentration of PCBs were greater in Hexagenia spp. relative to L. variegatus and P. promelas. The distribution of congeners contributing to the total concentration of PCBs in tissue varied among the three species. Trichlorobiphenyl congeners composed the greatest proportion of the total concentration of PCBs in L. variegatus while tetra- and pentabiphenyl congeners dominated in Hexagenia spp. and P. promelas. Perfluorooctane sulfonate (PFOS) was present in all three species at concentrations greater than all other PFASs analyzed. Hexagenia spp. also produced the greatest BSAF(lipid) and BSAF(ww) (non-lipid-normalized biota-sediment accumulation factor) values for PFOS relative to the other two species. However, this was not the case for all PFASs. The trend of BSAF values and number of carbon atoms in the perfluoroalkyl chain of perfluorinated carboxylates varied among the three species but was similar for perfluorinated sulfonates. Differences in the dominant pathways of exposure (e.g., water, sediment ingestion) likely explain a large proportion of the variation in accumulation observed across the three species.

Aquatic hazard assessment of MON 0818, a commercial mixture of alkylamine ethoxylates commonly used in glyphosate‐containing herbicide formulations. Part 2: Roles of sediment, temperature, and capacity for recovery following a pulsed exposure

A series of toxicity tests with MON 0818, a commercial surfactant mixture of polyoxyethylene tallow amines, were performed: 1) in the presence of sediment for benthic invertebrates and fish: 2) to examine the recovery capacity of Daphnia magna and 4 primary producers after a pulsed (24-h) exposure; and 3) to examine the potential effect of increased water temperature on toxicity of MON 0818 to 2 cold-water fishes. In the presence of sediment, no acute (24-h) mortality was observed for 3 of the 5 species up to 10 mg L-1 . The median effective concentrations for the other 2 species were significantly greater than for water only tests. The EC50 at 15 °C for Salvelinus alpinus was statistically lower than that at 10 °C. Latent effects of a 24-h exposure (1 mg L-1 ) were observed for Rhabdocelis subcapitata and Chlorella vulgaris, as indicated by delayed growth during recovery phase; however, both cultures were able to recover, as indicated by a lack of changes in maximum absolute growth rates. No significant effects of a 24-h exposure to MON 0818 were observed for Oophila sp. (1.5 mg L-1 ) or Lemna minor (100 mg L-1 ). Latent mortality after a 24-h exposure to 5 mg L-1 was observed during the recovery phase for D. magna; however, reproduction endpoints on surviving individuals were not altered. The results indicate that quick dissipation of MON 0818 in the presence of sediment can reduce the effects on exposed organisms, and that full recovery from 24-h exposures to concentrations of MON 0818 equal to, or greater than, those expected in the environment is possible. Environ Toxicol Chem 2017;36:512-521.

Aquatic hazard assessment of MON 0818, a commercial mixture of alkylamine ethoxylates commonly used in glyphosate‐containing herbicide formulations. Part 1: Species sensitivity distribution from laboratory acute exposures

The sensitivity of 15 aquatic species, including primary producers, benthic invertebrates, cladocerans, mollusks, and fish, to MON 0818, a commercial surfactant mixture of polyoxyethylene tallow amines, was evaluated in standard acute (48-96-h) laboratory tests. In addition, the potential for chronic toxicity (8 d) was evaluated with Ceriodaphnia dubia. Exposure concentrations were confirmed. No significant effects on any endpoint were observed in the chronic test. A tier-1 hazard assessment was conducted by comparing species sensitivity distributions based on the generated data, as well as literature data, with 4 exposure scenarios. This assessment showed moderate levels of hazard (43.1% of the species exposed at or above median effective concentration levels), for a chosen worst-case scenario-unintentional direct over-spray of a 15-cm-deep body of water with the maximum label application rate for the studied formulations (Roundup Original, Vision Forestry Herbicide; 12 L formulation ha-1 , equivalent to 4.27 kg acid equivalent [a.e.] ha-1 ). The hazard decreased to impairment of 20.9% of species under the maximum application rate for more typical uses (6 L formulation ha-1 , 2.14 kg a.e. ha-1 ), and down to 6.9% for a more frequently employed application rate (2.5 L formulation ha-1 , 0.89 kg a.e. ha-1 ). Finally, the percentage (3.8%) was less than the hazardous concentration for 5% of the species based on concentrations of MON 0818 calculated from maximum measured concentrations of glyphosate in the environment. Environ Toxicol Chem 2017;36:501-511.

Aquatic hazard assessment of MON 0818, a commercial mixture of alkylamine ethoxylates commonly used in glyphosate-containing herbicide formulations. Part 2: Roles of sediment, temperature, and capacity for recovery

A series of toxicity tests with MON 0818, a commercial surfactant mixture of polyoxyethylene tallow amines, were performed: 1) in the presence of sediment for benthic invertebrates and fish: 2) to examine the recovery capacity of Daphnia magna and 4 primary producers after a pulsed (24-h) exposure; and 3) to examine the potential effect of increased water temperature on toxicity of MON 0818 to 2 cold-water fishes. In the presence of sediment, no acute (24-h) mortality was observed for 3 of the 5 species up to 10 mg L-1 . The median effective concentrations for the other 2 species were significantly greater than for water only tests. The EC50 at 15 °C for Salvelinus alpinus was statistically lower than that at 10 °C. Latent effects of a 24-h exposure (1 mg L-1 ) were observed for Rhabdocelis subcapitata and Chlorella vulgaris, as indicated by delayed growth during recovery phase; however, both cultures were able to recover, as indicated by a lack of changes in maximum absolute growth rates. No significant effects of a 24-h exposure to MON 0818 were observed for Oophila sp. (1.5 mg L-1 ) or Lemna minor (100 mg L-1 ). Latent mortality after a 24-h exposure to 5 mg L-1 was observed during the recovery phase for D. magna; however, reproduction endpoints on surviving individuals were not altered. The results indicate that quick dissipation of MON 0818 in the presence of sediment can reduce the effects on exposed organisms, and that full recovery from 24-h exposures to concentrations of MON 0818 equal to, or greater than, those expected in the environment is possible. Environ Toxicol Chem 2017;36:512-521.

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.

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.