Dietary Uptake of Highly Hydrophobic Chemicals by Rainbow Trout (Oncorhynchus Mykiss).

Rainbow trout (Oncorhynchus mykiss) was exposed through the diet to a mixture of non-ionic organic chemicals for 28 d, followed by a depuration phase, in accordance with OECD method 305. The mixture included hexachlorobenzene (HCB), 2,2',5,5'-tetrachlorobiphenyl (PCB-52), 2,2',5,5'-hexachlorobiphenyl (PCB-153), decachlorobiphenyl (PCB-209), decabromodiphenyl ether (BDE209), decabromodiphenyl ethane (DBDPE), bis-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH), perchloro-p-terphenyl (p-TCP), perchloro-m-terphenyl (m-TCP), and perchloro-p-quaterphenyl (p-QTCP), the latter six of which are considered highly hydrophobic based on n-octanol/water partition coefficients (KOW) greater than 108. All chemicals had first-order uptake and elimination kinetics except p-QTCP, whose kinetics could not be verified due to limitations of analytical detection in the elimination phase. For HCB and PCBs, the growth-corrected elimination rates (k2g), assimilation efficiencies (α), and biomagnification factors (BMFL) corrected for lipid content compared well with literature values. For the highly hydrophobic chemicals, elimination rates were faster than the rates for HCB and PCBs, and α's and BMFLs were much lower than those of HCB and PCBs, i.e., ranging from 0.019 to 2.8%, and from 0.000051 to 0.023 (g-lipid/g-lipid), respectively. As a result, the highly hydrophobic organic chemicals were found be much less bioavailable and bioaccumulative than HCB and PCBs. Based on the current laboratory dietary exposures, none of the highly hydrophobic substances would be expected to biomagnify, but Trophic Magnification Factors (TMFs) > 1 have been reported from field studies for TBPH and DBDPE. Additional research is needed to understand and reconcile the apparent inconsistencies in these two lines of evidence for bioaccumulation assessment.

Bioaccumulation of Bis-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate and Mono-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate by Lumbriculus variegatus.

The brominated flame retardant bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH) is used widely in consumer items including polyurethane foam used in furniture. Information on its bioaccumulation in aquatic species is limited. In the current study, sediment bioaccumulation tests with the oligochaete Lumbriculus variegatus were performed on a spiked natural sediment equilibrated for 14.5 months. Analysis showed the TBPH used to spike the sediment contained a small amount (0.046% by mass) of mono-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBMEHP), a potential biotransformation product of the parent chemical. Steady-state biota-sediment accumulation factors (BSAFs) of 0.254 and 1.50 (kg organic carbon/kg lipid) were derived for TBPH and TBMEHP, respectively. TBPH had biphasic elimination behavior where 94% of the body burden was depleted within the first 12 h of elimination (i.e., half-life of 1.2 h or less) and the remaining 6% eliminated very slowly thereafter (half-life of 15 days). There was little evidence for biotransformation of either chemical by L. variegatus. This investigation confirms the extremely hydrophobic behavior of TBPH and its impact on its bioavailability.

Metabolism of cyclic phenones in rainbow trout in vitro assays.

1. Cyclic phenones are chemicals of interest to the USEPA due to their potential for endocrine disruption to aquatic and terrestrial species.2. Prior to this report, there was very limited information addressing metabolism of cyclic phenones by fish species and the potential for estrogen receptor (ER) binding and vitellogenin (Vtg) gene activation by their metabolites.3. The main objectives of the current research were to characterize rainbow trout (rt) liver slice-mediated in vitro metabolism of model parent cyclic phenones exhibiting disparity between ER binding and ER-mediated Vtg gene induction, and to assess the metabolic competency of fish liver in vitro tests to help determine the chemical form (parent and/or metabolite) associated with the observed biological response.4. GC-MS, HPLC and LC-MS/MS technologies were applied to investigate the in vitro biotransformation of cyclobutyl phenyl ketone (CBP), benzophenone (DPK), cyclohexyl phenyl ketone (CPK) mostly in the absence of standards for metabolite characterization.5. It was concluded that estrogenic effects of the studied cyclic phenones are mediated by the parent chemical structure for DPK, but by active metabolites for CPK. A definitive interpretation was not possible for CBP and CBPOH (alcohol), although a contribution of both structures to gene induction is suspected.

Characterization and analysis of estrogenic cyclic phenone metabolites produced in vitro by rainbow trout liver slices using GC-MS, LC-MS and LC-TOF-MS.

Cyclic phenones are chemicals of interest to the USEPA and international organizations due to their potential for endocrine disruption to aquatic and terrestrial species. The metabolic conversion of cyclic phenones by liver hepatocytes and the structure of main metabolites yielded have not been assessed in fish species. As part of a larger project, in this study we investigated the structure of metabolites produced in vitro by rainbow trout (rt) liver slices after exposure to the model cyclic phenones benzophenone (DPK), cyclobutyl phenyl ketone (CBP) and cyclohexyl phenyl ketone (CPK). While only one distinct metabolite was detected for DPK and CBP (benzhydrol and CBPOH, respectively), CPK yielded nine positional isomers (M1-M9) as products. In absence of standards, improved inference of CPK metabolites tentative structures was achieved by combining GC-MS with and without derivatization, LC with tandem MS, LC with high resolution time of flight (TOF) MS and LC fractionation data with CPK phase II conjugative metabolism information. Data supported that CPK is metabolized by phase I oxidation of the cyclohexyl ring and not the phenyl group as predicted by metabolism simulators. CPK metabolites M1 and M2 (MW 186), were proposed to be cyclohexenyl-derivatives. Also, M6-M9 were proposed to be hydroxylated metabolites (MW 204), with the potential for undergoing phase II conjugative metabolism to glucuronides and sulfates. Finally, M3, M4 and M5 were proposed as cyclohexanone-derivatives of CPK (MW 202), resulting from the limited redox-interconversion of their hydroxylated pairs M8, M6 and M7, respectively. Assessment of metabolite role in biological responses associated with endocrine disruption will advance the development of methods for species extrapolation and the understanding of differential sensitivity of species to chemical exposure.

The relative importance of waterborne and dietborne As exposure on survival and growth of juvenile fathead minnows.

The survival and growth of juvenile fathead minnows were investigated at various combinations of waterborne exposure to arsenate and of dietborne exposure to oligochaete worms which had been exposed to inorganic arsenic. Previous work with rainbow trout established that dietborne arsenic can reduce fish growth at environmentally relevant concentrations and could be more important than waterborne exposures. This was found to be less true for fathead minnows, which were less sensitive to dietborne exposures than rainbow trout, while being as or more sensitive to waterborne exposures. When assessed on the basis of accumulation of total As by the fish, further differences between fathead minnows and rainbow trout were evident. Fathead minnows accumulated relatively more arsenic from water versus diet than trout, and the accumulations at which growth effects occurred in minnows were different for dietborne and waterborne exposure, whereas they were the same for trout. These results suggest complex relationships involving arsenic speciation, toxicokinetics, and toxicodynamics, and underscore a need for care in relating effects information to real-world exposures. The present study also demonstrated the challenges in testing and interpreting growth effects in long-term exposures to fish, because the expression of toxicity can be confounded by the relationship of fish growth to size, the feeding regime, and wet weight versus dry weight relationships.

The effects of arsenic speciation on accumulation and toxicity of dietborne arsenic exposures to rainbow trout.

The effects on juvenile rainbow trout survival, growth, food consumption, and food conversion efficiency from dietborne exposures to inorganic arsenic (arsenite, arsenate) and to the organoarsenicals monomethylarsonate (MMA), dimethylarsinate (DMA), and arsenobetaine (AsB) were investigated in two experiments: (1) a 28-d exposure using live diets of oligochaete worms separately exposed via water to these five arsenic compounds and (2) a 56-d exposure using pellet diets prepared from commercial fish food to which arsenite, MMA, or DMA were added. In the live diet experiment, the degree to which worms could be contaminated with the organoarsenicals was limited by toxicity to the worms and other experimental constraints, so that their toxicity relative to inorganic arsenic could not be fully established, but AsB was concluded to have low toxicity, consistent with published results for mammals. For the pellet diet experiment, MMA and DMA were found to be at least an order of magnitude less toxic than inorganic As on the basis of concentration in the diet, as well as much less toxic on the basis of accumulation in the fish. The need to consider speciation in aquatic risk assessments for arsenic was further demonstrated by tissue analyses of three macroinvertebrate species from a mining-impacted stream, which showed large variations in both total arsenic and the relative amounts of inorganic and organic arsenic. Additionally, although effects of arsenic on trout appear to be well correlated with inorganic arsenic, worms were found to be more sensitive to waterborne DMA than to inorganic arsenic, showing that low toxicity of organoarsenicals cannot be assumed for all aquatic organisms. Various difficulties in evaluating and applying studies on dietborne exposures and fish growth are also discussed.

Characterization of the Fundulus heteroclitus embryo transcriptional response and development of a gene expression-based fingerprint of exposure for the alternative flame retardant, TBPH (bis (2-ethylhexyl)-tetrabromophthalate).

Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (n = 16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.

Estimating n-octanol-water partition coefficients for neutral highly hydrophobic chemicals using measured n-butanol-water partition coefficients.

Direct measurement of the n-octanol partition coefficients (KOW) for highly hydrophobic organic chemicals is extremely difficult because of the extremely low concentrations present in the water phase. n-Butanol/water partition coefficients (KBW) are generally much lower than KOW due to the increased solubility of solute in the alcohol saturated aqueous phase, and therefore become easier to measure. We measured the KBW for 25 neutral organic chemicals having measured log KOWs ranging from 2 to 9 and 4 additional highly hydrophobic chemicals, with unmeasured KOWs, having estimated log KOWs ranging from 6 to 18. The measured log KBW and log KOW values were linearly related, r2 = 0.978, and using the regression developed from the data, KOWs were predicted for the 4 highly hydrophobic chemicals with unmeasured KOWs. The resulting predictions were orders of magnitude lower than those predicted by a variety of computational models and suggests the estimates of KOW in the literature for highly hydrophobic chemicals (i.e., log KOW greater than 10) are likely incorrect by several orders of magnitude.

Bioaccumulation of Highly Hydrophobic Chemicals by Lumbriculus variegatus.

Bioaccumulation of highly hydrophobic chemicals (log KOW > 8) from contaminated sediments by Lumbriculus variegatus has been studied for relatively few chemicals, and the measured and model predicted biota-sediment accumulation factors (BSAFs) can differ by orders of magnitude. In the current study, sediment bioaccumulation tests with L. variegatus were performed on sediments dosed with chemicals having a wide range of predicted n-octanol/water partition coefficients (KOW; 106-1018), including some higher than most highly hydrophobic chemicals studied to date. The highly hydrophobic chemicals had biphasic elimination kinetics with compartments A and B having fast and slow elimination kinetics, respectively, and for compartment B, elimination followed first-order kinetics. For compartment A with fast elimination kinetics, the mechanism and its kinetic-order could not be determined. Steady-state BSAFs (kg organic carbon/kg lipid) of 0.015, 0.024, and 0.022 were derived for tetradecachloro-p-terphenyl, tetradecachloro-m-terphenyl, and octadecachloro-p-quaterphenyl, respectively. The high uncertainty in predicted KOWs for highly hydrophobic chemicals limited the comparison and evaluation of predicted BSAFs from the Arnot-Gobas food web model and BSAFs measured in this study. The results of this study point to the need to perform dietary assimilation efficiency studies with highly hydrophobic compounds to resolve uncertainties surrounding the estimation of their KOW and the need to understand mechanism and models for the biphasic elimination kinetics.

Evaluation of micro-colorimetric lipid determination method with samples prepared using sonication and accelerated solvent extraction methods.

Two common laboratory extraction techniques were evaluated for routine use with the micro-colorimetric lipid determination method developed by Van Handel (1985) [2] and recently validated for small samples by Inouye and Lotufo (2006) [1]. With the accelerated solvent extraction method using chloroform:methanol solvent and the colorimetric lipid determination method, 28 of 30 samples had significant proportional bias (α=1%, determined using standard additions) and 1 of 30 samples had significant constant bias (α=1%, determined using Youden Blank measurements). With sonic extraction, 0 of 6 samples had significant proportional bias (α=1%) and 1 of 6 samples had significant constant bias (α=1%). These demonstrate that the accelerated solvent extraction method with chloroform:methanol solvent system creates an interference with the colorimetric assay method, and without accounting for the bias in the analysis, inaccurate measurements would be obtained.