Graham van Aggelen

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

Background In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics—Moving into Regulation and Monitoring, held 21–23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). Objectives The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. Discussion Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. Conclusions The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

TOXICITY OF α-, β-, (α+β)-ENDOSULFAN AND THEIR FORMULATED AND DEGRADATION PRODUCTS TO DAPHNIA MAGNA, HYALELLA AZTECA, ONCORHYNCHUS MYKISS, ONCORHYNCHUS KISUTCH, AND BIOLOGICAL IMPLICATIONS IN STREAMS

The static acute toxicities of α-endosulfan, β-endosulfan, (α + β)-endosulfan, endosulfan sulfate (their transformation product), and formulated materials were determined for a representative freshwater amphipod (Hyalella azteca), cladoceran (Daphnia magna), and salmonid fish (Oncorhynchus mykiss). Acute lethality tests also were conducted on these organisms and coho salmon (Oncorhynchus kisutch) by exposing them to simulated field water endosulfan concentrations, using either a single compound and/or in combinations. As well, growth/survival bioassays of H. azteca were carried out in simulated sediment endosulfan concentrations that were detected in farm ditches contiguous to fish streams in the Lower Fraser Valley (BC, Canada). All materials tested were very highly toxic to these nontarget aquatic indicator organisms. As the most potent compound, α-endosulfan is about 1.3, 6.6, and 58 times more toxic than the β-isomer to Daphnia, rainbow trout, and Hyalella, respectively. Some possible biological and toxicological implications of our findings are discussed in the context of endosulfan concentrations found in farm ditches flowing to fish streams in the Lower Fraser Valley.

Reverse Engineering Adverse Outcome Pathways

The toxicological effects of many stressors are mediated through unknown, or incompletely characterized, mechanisms of action. The application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, metabolic, signaling) can be used to overcome these limitations. This approach was used to characterize adverse outcome pathways (AOPs) for chemicals that disrupt the hypothalamus-pituitary-gonadal endocrine axis in fathead minnows (FHM, Pimephales promelas). Gene expression changes in FHM ovaries in response to seven different chemicals, over different times, doses, and in vivo versus in vitro conditions, were captured in a large data set of 868 arrays. Potential AOPs of the antiandrogen flutamide were examined using two mutual information-based methods to infer gene regulatory networks and potential AOPs. Representative networks from these studies were used to predict network paths from stressor to adverse outcome as candidate AOPs. The relationship of individual chemicals to an adverse outcome can be determined by following perturbations through the network in response to chemical treatment, thus leading to the nodes associated with the adverse outcome. Identification of candidate pathways allows for formation of testable hypotheses about key biological processes, biomarkers, or alternative endpoints that can be used to monitor an AOP. Finally, the unique challenges facing the application of this approach in ecotoxicology were identified and a road map for the utilization of these tools presented.

Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17β-estradiol exposed rainbow trout (Oncorhynchus mykiss)

While the endocrine system is known to modulate immune function in vertebrates, the role of 17β-estradiol (E2) in cellular immune function of teleosts is poorly understood. The cellular and molecular responses of juvenile rainbow trout (Oncorhynchus mykiss) to E2 treatment were evaluated by exposing fish to 0.47±0.02μg/L E2 (mean±SEM) for either 2 or 7d, with a subsequent 14d recovery period. After 2 and 7d of exposure to E2, hematocrit was significantly lower than in control fish. Lipopolysaccharide-induced lymphocyte proliferation was elevated on day 2 and concanavalin A-induced lymphocyte proliferation was reduced following 7d of E2 exposure. Four estrogen receptor (ER) transcripts were identified in purified trout head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL). While the mRNA abundance of ERβ1 and ERβ2 was unaffected by treatment, ERα1 was up-regulated in HKL and PBL following 7d of E2 exposure. ERα2 was up-regulated in HKL after 7d of E2 exposure, but down-regulated in PBL after 2 and 7d of treatment. All parameters that were altered during the E2 exposure period returned to baseline levels following the recovery period. This study reports the presence of the full repertoire of ERs in purified HKL for the first time, and demonstrates that ERα transcript abundance in leukocytes can be regulated by waterborne E2 exposure. It also demonstrated that physiologically-relevant concentrations of E2 can modulate several immune functions in salmonids, which may have widespread implications for xenoestrogen-associated immunotoxicity in feral fish populations inhabiting contaminated aquatic environments.

Triclosan Affects Thyroid Hormone–Dependent Metamorphosis in Anurans

Our original work on Rana catesbeiana (bullfrog) tadpoles exposed to triclosan (TCS) during induction of precocious metamorphosis that was presented in Veldhoen et al. (2006) suggested that frog tadpoles exposed to the antibacterial agent exhibit changes through prometamorphosis. In a recent study, Fort et al. (2010) exposed premetamorphic Xenopus laevis tadpoles to triclosan for 21 days into prometamorphosis. This work, published in Toxological Sciences (Fort et al., 2010), stated that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model. Herein, we describe the apparent discrepancy between these two studies, and on examination of the data presented in the study of Fort et al. (2010), we do not come to the same conclusion that triclosan had no effects on metamorphosis. Fort et al. (2010) stated that ‘‘larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters.’’ A major indicator of exposure effect is progression through the developmental stages as defined by Nieuwkoop and Faber (NF; Nieuwkoop and Faber, 1994). The relevant stages in the study are between NF stages 54 and 66; the range of stages attained by NF stage 51 tadpoles after 21 days of exposure. Even though these are numerical stages, they are categorical, defined by distinctive morphological characteristics. The number of tadpoles that were at these stages was reported in Table 3 of the study of Fort et al. (2010), and chi-square analysis was performed. The authors stated that ‘‘Overall, the frequency of each developmental stage in each of the treatment concentrations following 21 days of exposure to TCS was not significantly different from each other (chi-square, p 1⁄4 0.319).’’ It was not clear how the analysis was done, but using the same data as presented in Table 3 in the study of Fort et al. (2010) in a 9 3 5 contingency table with NF 54–56, 63–64, and 65–66 stages grouped and all other stage categories kept separate gave a chi-square 1⁄4 48.7, p 1⁄4 0.029, degrees of freedom 1⁄4 32. In addition, a pairwise 7 3 2 contingency table comparison of the control with each of the TCS treatments individually gave the results shown in Table 1. Therefore, the data presented by Fort et al. (2010) do indeed show that TCS at the three lower exposure concentrations had an effect on the postembryonic development of X. laevis tadpoles. This is consistent with our observations with the bullfrog (Veldhoen et al., 2006.). Moreover, the authors observed that tails from stagematched NF 60 tadpoles showed significantly elevated thyroid hormone receptor b (TRb) transcript levels in the two middle concentrations examined. The 1.5-fold increase observed relative to the control animals was interpreted as ‘‘likely not biologically significant,’’ and the authors point to a lack of concentration dependence of the TCS exposure on altered transcript abundance. Putting this information in context, TRb transcript levels increase in the tadpole tail by approximately sevenfold between NF stages 54 and 60 (Wagner and Helbing,

Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA)

The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 μg/L thyroxine (T(4)), 0.3, 3 and 30 μg/L (nominal) TCS, or combined T(4)/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T(4) treatment alone accelerated development concomitant with altered levels of TH receptors α and β, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 μg/L) was protective against tadpole mortality, this protection was lost in the presence of T(4). The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.

Trace analysis of total naphthenic acids in aqueous environmental matrices by liquid chromatography/mass spectrometry-quadrupole time of flight mass spectrometry direct injection

A rapid and sensitive liquid chromatography quadrupole time of flight method has been established for the determination of total naphthenic acid concentrations in aqueous samples. This is the first methodology that has been adopted for routine, high resolution, high throughput analysis of total naphthenic acids at trace levels in unprocessed samples. A calibration range from 0.02 to 1.0μgmL(-1) total Merichem naphthenic acids was validated and demonstrated excellent accuracy (97-111% recovery) and precision (1.9% RSD at 0.02μgmL(-1)). Quantitative validation was also demonstrated in a non-commercial oil sands process water (OSPW) acid extractable organics (AEOs) fraction containing a higher percentage of polycarboxylic acid isomers than the Merichem technical mix. The chromatographic method showed good calibration linearity of ≥0.999 RSQ to 0.005μgmL(-1) total naphthenic acids with a precision <3.1% RSD and a calculated detection limit of 0.0004μgmL(-1) employing Merichem technical mix reference material. The method is well suited to monitoring naturally occurring and industrially derived naphthenic acids (and other AEOs) present in surface and ground waters in the vicinity of mining developments. The advantage of the current method is its direct application to unprocessed environmental samples and to examine natural naphthenic acid isomer profiles. It is noted that where the isomer profile of samples differs from that of the reference material, results should be considered semi-quantitative due to the lack of matching isomer content. The fingerprint profile of naphthenic acids is known to be transitory during aging and the present method has the ability to adapt to monitoring of these changes in naphthenic acid content. The methods total ion scan approach allows for data previously collected to be examined retrospectively for specific analyte mass ions of interest. A list of potential naphthenic acid isomers that decrease in response with aging is proposed and a quantitative assay of an adamantane carboxylic acid is reported.

Evaluating the treatment of a synthetic wastewater containing a pharmaceutical and personal care product chemical cocktail: Compound removal efficiency and effects on juvenile rainbow trout

Pharmaceutical and personal care products (PPCPs) can evade degradation in sewage treatment plants (STPs) and can be chronically discharged into the environment, causing concern for aquatic organisms, wildlife, and humans that may be exposed to these bioactive chemicals. The ability of a common STP process, conventional activated sludge (CAS), to remove PPCPs (caffeine, di(2-ethylhexyl)phthalate, estrone, 17α-ethinylestradiol, ibuprofen, naproxen, 4-nonylphenol, tonalide, triclocarban and triclosan) from a synthetic wastewater was evaluated in the present study. The removal of individual PPCPs by the laboratory-scale CAS treatment plant ranged from 40 to 99.6%. While the efficiency of removal for some compounds was high, remaining quantities have the potential to affect aquatic organisms even at low concentrations. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to influent recreated model wastewater with methanol (IM, solvent control) or with PPCP cocktail (IC), or CAS-treated effluent wastewater with methanol (EM, treated control) or with PPCP cocktail (EC). Alterations in hepatic gene expression (evaluated using a quantitative nuclease protection plex assay) and plasma vitellogenin (VTG) protein concentrations occurred in exposed fish. Although there was partial PPCP removal by CAS treatment, the 20% lower VTG transcript levels and 83% lower plasma VTG protein concentration found in EC-exposed fish compared to IC-exposed fish were not statistically significant. Thus, estrogenic activity found in the influent was retained in the effluent even though typical percent removal levels were achieved raising the issue that greater reduction in contaminant load is required to address hormone active agents.

Effects of acute exposure to the non-steroidal anti-inflammatory drug ibuprofen on the developing North American Bullfrog (Rana catesbeiana) tadpole.

A variety of pharmaceutical chemicals can represent constituents of municipal effluent outflows that are dispersed into aquatic receiving environments worldwide. Increasingly, there is concern as to the potential of such bioactive substances to interact with wildlife species at sensitive life stages and affect their biology. Using a combination of DNA microarray, quantitative real-time polymerase chain reaction, and quantitative nuclease protection assays, we assessed the ability of sub-lethal and environmentally relevant concentrations of ibuprofen (IBF), a non-steroidal anti-inflammatory agent and prevalent environmental contaminant, to function as a disruptor of endocrine-mediated post-embryonic development of the frog. While the LC50 of IBF for pre-metamorphic Rana catesbeiana tadpoles is 41.5 mg/L (95% confidence interval: 32.3-53.5 mg/L), exposure to concentrations in the ppb range elicited molecular responses both in vivo and in organ culture. A nominal concentration of 15 μg/L IBF (actual = 13.7 μg/L) altered the abundance of 26 mRNA transcripts within the liver of exposed pre-metamorphic R. catesbeiana tadpoles within 6 d. IBF-treated animals demonstrated subsequent disruption of thyroid hormone-mediated reprogramming in the liver transcriptome affecting constituents of several metabolic, developmental, and signaling pathways. Cultured tadpole tail fin treated with IBF for 48 h also demonstrated altered mRNA levels at drug concentrations as low as 1.5 μg/L. These observations raise the possibility that IBF may alter the post-embryonic development of anuran species in freshwater environs, where IBF is a persistent or seasonal pollutant.

Hatching success and pesticide exposures in amphibians living in agricultural habitats of the South Okanagan Valley, British Columbia, Canada (2004-2006).

In 2004 to 2006, in the Okanagan Valley, British Columbia, Canada, we measured pesticides, water chemistry, and hatching success of Great Basin spadefoot (Spea intermontana), Pacific treefrog (Pseudacris regilla), Western toad (Bufo boreas), and Columbia spotted frog (Rana luteiventris). Predator-proof cages containing Gosner Stage 4 eggs were placed in ponds in nonagricultural reference sites in conventionally sprayed and organic orchards. Seventeen pesticides were detected in ponds in sprayed orchards but occurred at low concentrations (ng/L) except for diazinon (1,410 ng/L). Chloride, sulfate, conductivity, nitrate, and phosphorus showed significant differences among sites. Spadefoot mean hatching success ranged from 0 to 92% among sprayed orchards, whereas the range was 48 to 98.6% among organic orchards and 51 to 95.5% among reference sites. Mean hatching success for Pacific treefrog was 22.1 to 76.1% among sprayed orchards, whereas the range was 83.4 to 97.1% among reference sites. Although sample sizes were small and replication was low, we found that trends in hatching success of eggs of Western toad and Columbia spotted frogs were consistent with the other species. Variables that correlated negatively with amphibian hatching success included 12 pesticides and seven water chemistry parameters. However, stepwise regression found that, in 2005, atrazine accounted for 79% of the variation in spadefoot hatching success and, in 2006, atrazine, total nitrate, and chlorpyrifos accounted for 80%. For Pacific treefrog there were no significant correlations with pesticide concentrations; rather, hatching success correlated with water chemistry parameters. The present study also emphasizes the variability in species sensitivity and importance of incorporating water chemistry into the interpretation of water quality for amphibians.