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The accumulation and tissue distribution of toxicants in aquatic biota can be determinative of their toxic impact to both exposed organisms and their potential human consumers. In the present study, accumulation of the trace metal thallium (Tl) in gill, muscle, plasma, and otoliths of rainbow trout (Oncorhynchus mykiss) following acute (96-h) and subchronic (28-day) waterborne exposures was investigated. Owing to known interactions between Tl and potassium ions (K + ), plasma and muscle K + concentrations were also determined. Branchial Tl accumulated in a dose-dependent manner in both acute and subchronic exposures, while plasma Tl was rapidly mobilized to tissues and accumulated only at exposure concentrations of 141 µg L -1 or higher. For muscle tissue, Tl concentrations at 28 days were markedly lower than those at 96 h at comparable exposure concentrations (0.9 µg L -1 ), indicating the presence of mechanisms that act to reduce Tl accumulation over time. However, after acute exposure, muscle Tl reached concentrations that, if consumed, would exceed acceptable daily intake values for this element, indicating some risk to human health from the consumption of fish from waters heavily contaminated with Tl. Otoliths showed Tl concentrations that reflected exposure concentration and length, confirming their capacity to provide insight into fish exposure history. No changes in tissue K + concentrations were observed, suggesting that accumulation of Tl in rainbow trout plasma and muscle does not occur at the expense of K + homeostasis. In addition to highlighting the capacity of rainbow trout to accumulate Tl to levels that exceed recommended dietary doses to human consumers, the present study provides the first data of tissue-specific Tl accumulation in an important regulatory species. Environ Toxicol Chem 2023;42:1553-1563. © 2023 SETAC., (© 2023 SETAC.)

Diltiazem is ubiquitously prescribed and has been reported in many effluents and freshwater bodies. Being a calcium channel blocker, diltiazem could disrupt the function of the sensory and central nervous systems. In the present study, using electro-olfactography (EOG), we investigated the interaction of diltiazem with the olfactory sensory neurons (OSNs) of rainbow trout by looking into the detection threshold and effects of immediate (~5 min) and acute (24 h) exposure to diltiazem at 6.6, 66, and 660 µg/L. We also studied the accumulation of the drug in fish plasma and whole body. Brief exposure to diltiazem impaired the OSN response to a chemosensory stimulus in a concentration-dependent manner at 6.6 µg/L and higher, whereas OSNs exposed for 24 h only displayed an impairment at 660 µg/L. Chemical analysis showed that the accumulation of diltiazem in fish plasma and body correlated with the EOG response because it was 10 times higher in the group that displayed a significant impairment (660 µg/L) compared to the other 2 groups (6.6, 66 µg/L). This correlation suggests that the impact of diltiazem on OSNs might partially be through the accumulated molecules in the fish bloodstream. Fish did not detect diltiazem as a sensory stimulus even at concentrations as high as 660 µg/L; thus, fish could potentially swim toward or fail to escape harmful concentrations of diltiazem. Environ Toxicol Chem 2022;41:554-550. © 2020 SETAC., (© 2020 SETAC.)

Information on the effects of silver nanoparticles (AgNPs) in fish has mostly been generated from standard laboratory species and short-term toxicity tests. However, there is significant uncertainty regarding AgNP toxicity to native species of concern in North America, particularly in northern freshwater ecosystems. We assessed the chronic toxicity of AgNPs in early life stages of three North American fish species: rainbow trout (Oncorhynchus mykiss), lake trout (Salvelinus namaycush), and northern pike (Esox lucius). Newly fertilized embryos were exposed to nominal aqueous concentrations of 0.1, 0.3, 1.0, 3.0, 10.0, or 30.0 nM AgNPs for 126 (rainbow trout), 210 (lake trout), and 25 (northern pike) days. Endpoints included cumulative developmental time (°C × day or degree-days to 50% life-stage transition), mortality, fork length, embryonic malformations, cumulative survival, and histopathology of gill and liver in larvae/alevins. The results showed life stage-specific differences in responses, with endpoints during the embryonic stage occurring more often and at lower concentrations compared to larval/alevin and juvenile stages. Sensitivities among species were highly dependent on the endpoints measured, although developmental time appeared to be the most consistent endpoint across species. At embryonic and larval/alevin stages, northern pike was the most sensitive species (lowest observable effect concentration of 0.1 nM using developmental time). Rainbow trout displayed similar responses to lake trout across multiple endpoints and therefore seems to be an adequate surrogate for trout species in ecotoxicology studies. Moreover, while mortality during individual life stages was not generally affected, the cumulative mortality across life stages was significantly affected, which highlights the importance of chronic, multi-life-stage studies. Environ Toxicol Chem 2021;40:3337-3350. © 2021 SETAC., (© 2021 SETAC.)

Multiple linear regression (MLR) modeling has been successfully used to predict how water chemistry variables influence the toxicity of cationic metals to aquatic organisms, but no MLR model exists for vanadium (V). Recent research has indicated that an increase in pH (from 6 to 9), or high concentrations of sodium (473 mg Na + /L), increase V toxicity to Daphnia pulex. In contrast, increases in alkalinity (>100 mg as CaCO 3 ) and sulfate (>100 mg SO 4 2- /L) reduce V toxicity. How these variables influence V toxicity to Oncorhynchus mykiss (rainbow trout) was still unknown. Our results show that increasing pH from 6.2 to 8.9 tended to decrease the 96-h median lethal concentration (LC50) for V toxicity to O. mykiss by 9.6 mg V/L. An alkalinity increase from 71 to 330 mg/L as CaCO 3 tended to increase the 96-h LC50 by 3.3 mg V/L, whereas when SO 4 2- rose from 150 to 250 mg/L, the LC50 significantly increased by 0.3 mg V/L followed by a significant decrease of 1 mg V/L when SO 4 2- was >250 mg/L. Sodium (between 100 and 336 mg/L) showed no effect on V toxicity to O. mykiss. The toxicity patterns for O. mykiss were similar to those observed for D. pulex, except for that of SO 4 2- , potentially indicating different mechanisms of V uptake or regulation in the 2 species. The LC50s and associated water chemistry were combined to develop an MLR model for O. mykiss and D. pulex. Alkalinity and pH modified V toxicity to both species, whereas SO 4 2- influenced V toxicity to D. pulex. Overall, MLR models should be considered for creating new local benchmarks or water quality guidelines for V. Environ Toxicol Chem 2020;39:1737-1745. © 2020 SETAC., (© 2020 SETAC.)

The present study assessed the chronic toxicity of 2 chemically enhanced water accommodated fractions (CEWAFs) of diluted bitumens (dilbits), Access Western Blend (AWB) and Cold Lake Blend (CLB), to rainbow trout alevins. Chemical dispersion was used to overcome the resistance to dispersion of dilbits and to generate test solutions that contained more and smaller oil droplets for increased partitioning of petroleum hydrocarbons into water. Test solutions were characterized by fluorescence spectroscopy, a rapid and inexpensive analytical tool to compare toxicity endpoints measured by fluorescence (total petroleum hydrocarbons measured by fluorescence [TPH-F]). Cumulative mortality and the prevalence and severity of malformations increased following exposure of alevins to dispersed dilbits. Toxicity curves overlapped for AWB and CLB when expressed as TPH-F and 22- to 24-d median lethal and effect concentrations ranged from 0.36 to 1.5 mg/L. Gene expression in alevins was also altered following exposure to dispersed dilbit, with relative cytochrome P450-1A mRNA levels increasing up to 170-fold for AWB and up to 240-fold for CLB. Access Western Blend and CLB caused similar toxicity to rainbow trout alevins as light to medium conventional crude oils, and rainbow trout alevins were more sensitive than yellow perch, Japanese medaka, and fathead minnow embryos exposed to dispersed AWB and CLB. The present study is the first to assess the embryotoxicity of dilbits to a Canadian freshwater salmonid species. Environ Toxicol Chem 2020;39:1620-1633. © 2020 SETAC., (© 2020 SETAC.)

3-Trifluoromethyl-4-nitrophenol (TFM) has been used for more than 60 yr to control the invasive parasitic sea lamprey (Petromyzon marinus) in the Great Lakes Basin (USA/Canada). In the early 1990s, researchers reported that TFM induced vitellogenin in fish and that TFM was an agonist for the rainbow trout estrogen receptor. To support continued registration of TFM for sea lamprey control, regulatory agencies required further testing to evaluate potential endocrine disruption effects. Fathead minnow (Pimephales promelas) were exposed to TFM at measured concentrations of 0.0659, 0.181, 0.594, 1.79, and 5.11 mg active ingredient (a.i.)/L for 21 d. No-observable- and lowest-observable-effect concentrations (NOEC and LOEC, respectively) were determined to be 1.79 mg/L or greater for each endpoint. Male survival in the highest treatment group was reduced relative to the controls. Percentage of egg fertility was reduced in the highest treatment group, resulting in an estimated NOEC of 1.79 mg/L. Whereas no effect on the gonadosomatic index (GSI) was observed for males, female GSI was increased in the 5.11-mg/L treatment. Vitellogenin production was not altered relative to the controls for all TFM treatment groups. However, female testosterone was elevated in the 5.11-mg/L treatment. The results suggest that prolonged exposure to TFM at concentrations exceeding 1.79 mg/L has the potential to disrupt endocrine function. Biologically relevant effects were found at the highest exposure concentration following a 21-d exposure. However, the duration of exposure in our study is not consistent with typical treatment durations (12 h) for sea lamprey control. Environ Toxicol Chem 2020;39:1599-1607. © 2020 SETAC., (© 2020 SETAC.)

Elevated nitrate (NO 3 ) and sulfate (SO 4 ) in surface water are of global concern, and studies are needed to generate toxicity data to develop environmental guideline values for NO 3 and SO 4 . The present study was designed to fill existing gaps in toxicity databases by determining the acute and/or chronic toxicity of NO 3 (tested as NaNO 3 ) to a unionid mussel (Lampsilis siliquoidea), a midge (Chironomus dilutus), a fish (rainbow trout, Oncorhynchus mykiss), and 2 amphibians (Hyla versicolor and Lithobates sylvaticus), and to determine the acute and/or chronic toxicity of SO 4 (tested as Na 2 SO 4 ) to 2 unionid mussels (L. siliquoidea and Villosa iris), an amphipod (Hyalella azteca), and 2 fish species (fathead minnow, Pimephales promelas and O. mykiss). Among the different test species, acute NO 3 median effect concentrations (EC50s) ranged from 189 to >883 mg NO 3 -N/L, and chronic NO 3 20% effect concentrations (EC20s) based on the most sensitive endpoint ranged from 9.6 to 47 mg NO 3 -N/L. The midge was the most sensitive species, and the trout was the least sensitive species in both acute and chronic NO 3 exposures. Acute SO 4 EC50s for the 2 mussel species (2071 and 2064 mg SO 4 /L) were similar to the EC50 for the amphipod (2689 mg SO 4 /L), whereas chronic EC20s for the 2 mussels (438 and 384 mg SO 4 /L) were >2-fold lower than the EC20 of the amphipod (1111 mg SO 4 /L), indicating the high sensitivity of mussels in chronic SO 4 exposures. However, the fathead minnow, with an EC20 of 374 mg SO 4 /L, was the most sensitive species in chronic SO 4 exposures whereas the rainbow trout was the least sensitive species (EC20 > 3240 mg SO 4 /L). The high sensitivity of fathead minnow was consistent with the finding in a previous chronic Na 2 SO 4 study. However, the EC20 values from the present study conducted in test water containing a higher potassium concentration (3 mg K/L) were >2-fold greater than those in the previous study at a lower potassium concentration (1 mg K/L), which confirmed the influence of potassium on chronic Na 2 SO 4 toxicity to the minnow. Environ Toxicol Chem 2020;39:1071-1085. © 2020 SETAC., (© 2020 SETAC.)

Physiologically based toxicokinetic (PBTK) modeling enables researchers to predict internal tissue concentrations for various species exposed to exogenous compounds through different routes at varying concentrations without having to run in vivo experiments for each scenario. Parameters for the models may be gathered from in vivo or in vitro measurements, cross-species or cross-chemical extrapolations, literature reviews, or other models. The PBTK models, described using ordinary differential equations (ODEs), are then simulated using these parameters for a given compound/exposure/species scenario. Although they are potentially useful for regulatory toxicology, the complexity of ODE programming and simulation remains a barrier for many would-be researchers. Petri nets, a graphical modeling framework, offers a more intuitive approach to PBTK modeling. To demonstrate their utility and ease of use, we present a model of waterborne fluoranthene exposure to rainbow trout (Oncorhynchus mykiss) written and simulated in Snoopy, a graphical Petri net development and simulation software package. We converted an existing ODE PBTK model and evaluated the Petri net model against the ODE model results. The simulated tissue concentrations of the Petri net model closely mirrored the simulated concentrations of the ODE model. To convert the ODE model to a Petri net model, we introduced a new parameter, blood volume (V BLOOD ). Sensitivity analysis found V BLOOD to be very robust when varied over an order of magnitude. The resulting Petri net PBTK model has a number of advantages over ODE models, while maintaining equivalent predictive functionality. Environ Toxicol Chem 2019;00:1-10. © 2019 SETAC., (© 2019 SETAC.)

The world-class Alaskan Bristol Bay salmon fishery and vast deposits of copper (Cu) and other metals in the watershed warrant further investigation into the potential toxicity of Cu to salmonids under the low water-hardness conditions that occur in the watershed. Therefore we investigated the acute toxicity of Cu to rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimephales promelas) in low-hardness water (∼ 30 mg/L as CaCO 3 ) formulated in the laboratory and collected from the Bristol Bay watershed. The median lethal concentration (LC50) for rainbow trout exposed to Cu in low-hardness laboratory water was 16 μg Cu/L (95% confidence intervals [CIs]: 12, 21; dissolved Cu, filtered to 0.45 μm). The LC50 values for fathead minnows exposed to Cu in low-hardness laboratory water or site water were 29 and 79 μg Cu/L (95% CIs: 23, 35 and 58, 125; dissolved Cu), respectively. The biotic ligand model (BLM) LC50 estimates for these bioassays were 1.3 to 2.3 times higher than the actual LC50 values. We also calculated and analyzed acute Cu water quality criteria, also known as criterion maximum concentration (CMC), using hardness-based methods and the BLM for water samples collected throughout the Bristol Bay watershed in 2007. Biotic ligand model CMCs ranged from 0.05 to 17.5 μg Cu/L and hardness-based CMCs ranged from 2.3 to 6.1 μg Cu/L for the 65 samples analyzed. Our results show the need for site-specific research and subsequent water quality guidelines in low-hardness aquatic habitats. Environ Toxicol Chem 2019;38:190-197. © 2018 SETAC., (© 2018 SETAC.)

We investigated the olfactory toxicity of copper (Cu) to rainbow trout in low-hardness (27 mg/L as CaCO 3 ) water formulated in the laboratory over a 120-h period using a flow-through design. The fish's response to an alarm cue (e.g., reduction in activity) was recorded to determine the exposure concentrations and durations that inhibited olfactory detection of the cue after 3, 24, 48, and 96 h of Cu exposure and after 24 h of clean water recovery following the 96-h exposure period. Exposures were conducted with a range of Cu concentrations from 0.13 (control) to 7.14 μg Cu/L (dissolved Cu). We observed a dose-dependent response in olfactory inhibition with a 20% reduction in the probability of responding to the alarm cue, relative to controls, at 2.7 and 2.4 μg Cu/L after 24 or 96 h of exposure, respectively. Olfactory inhibition manifested between 3 and 24 h of exposure. Our 24- and 96-h 20% olfactory inhibition estimates fell between the criteria derived using the biotic ligand model (BLM; criterion maximum concentration [CMC] and criterion continuous concentration [CCC] values were 0.63 and 0.39 μg Cu/L, respectively) and water hardness-based criteria (CMC and CCC values were 3.9 and 2.9 μg Cu/L, respectively). Therefore, the hardness-based criteria do not appear to be protective and the BLM-derived criteria do appear to be protective against Cu-induced olfactory inhibition given our test water chemistry. Neither the hardness-based criteria nor the BLM-derived criteria appear to be protective against our estimated Cu behavioral avoidance response concentrations at 24- and 96-h exposures (0.54 and 0.50 μg Cu/L, respectively). Environ Toxicol Chem 2019;38:198-209. © 2018 SETAC., (© 2018 SETAC.)

The present study investigated oil dispersant toxicity to fish species typical of the cooler regions of Canada, together with less well-documented issues pertaining to oil dispersant monitoring. The oil dispersant toxicity of Corexit EC9500A was assessed for the freshwater fish species rainbow trout and the seawater species coho, chinook, and chum, with a final median lethal concentration (LC50) acute lethality range between 35.3 and 59.8 mg/L. The LC50 range was calculated using confirmed 0-h dispersant concentrations that were justified by fish mortality within the first 24 h of exposure and by variability of the dispersant indicator dioctyl sulfosuccinate (DOSS) used to monitor concentrations at later time points. To investigate DOSS as an oil dispersant indicator in the environment, microcosm systems were prepared containing Corexit EC9500A, Finasol OSR52, Slickgone NS, and Slickgone EW dispersants together with diluted bitumen. The DOSS indicator recovery was found to be stable for up to 13 d at 5 °C, 8 d at 10 °C, but significantly less than 8 d at ≥15 °C. After 3 d at temperatures ≥15 °C, the DOSS indicator recovery became less accurate and was dependent on multiple environmental factors including temperature, microbial activity, and aeration, with potential for loss of solvents and stabilizers. A final assessment determined DOSS to be a discrepant indicator for long-term monitoring of oil dispersant in seawater. Environ Toxicol Chem 2018;37:1309-1319. © 2018 SETAC., (© 2018 SETAC.)

Warming of freshwaters as a result of climate change is expected to have complex interactions with the toxicity of contaminants to aquatic organisms. The present study evaluated the effects of temperature on the acute toxicity of endosulfan, chlorpyrifos, and phenol to 3 warm water species of fish-silver perch, rainbowfish, and western carp gudgeon-and 1 cold water species, rainbow trout. Endosulfan was more toxic to silver perch at 30 °C and 35 °C than at 15 °C, 20 °C and 25 °C during short exposures of 24 h, but at 96 h, temperature had no effect on toxicity. Toxicity to rainbow trout increased with increasing temperature, whereas warm water species exhibited maximum toxicity at around 30 °C, decreasing again toward 35 °C. Chlorpyrifos became more toxic to all species with increasing temperature. Phenol toxicity to all species decreased at low to intermediate temperatures; but as temperatures increased further toward the upper thermal limit, phenol became more toxic. Increasing toxicity in the upper thermal range of cold water species may contribute to upstream range contraction in rivers with high toxicant loads. In contrast, warm water species may not exhibit a range shift within rivers as a result of interactions between temperature and toxicity. Catchment management to offset global warming at local scales may present opportunities to mitigate increased toxicity of contaminants to fish., (© 2015 SETAC.)

The authors tested the applicability of a previously developed biotic ligand model (BLM) to predict acute toxicity of single metals and metal mixtures (cadmium, lead, and zinc) to rainbow trout fry (Oncorhynchus mykiss) from a single available dataset. The BLM used in the present study hypothesizes that metals inhibit an essential cation (calcium) and organisms die as a result of its deficiency, leading to an assumption that the proportion of metal-binding ligand (f) is responsible for the toxic effects of metals on the survival of rainbow trout. The f value is a function of free-ion concentrations of metals computed by a chemical speciation model, and the function has affinity constants as model parameters. First, the survival effects of single metals were statistically modeled separately (i.e., f-survival relationship) by using the generalized linear mixed model with binomial distribution. The modeled responses of survival rates to f overlapped reasonably irrespective of metals tested, supporting the theoretical prediction from the BLM that f-survival relationships are comparable regardless of metal species. The authors thus developed the generalized linear mixed model based on all data pooled across the single-metal tests. The best-fitted model well predicted the survival responses observed in mixture tests (r = 0.97), providing support for the applicability of the BLM to predict effects of metal mixtures., (© 2014 SETAC.)

Traditionally, aquatic toxicity studies examine the toxicity of a single chemical to an organism. Organisms in nature, however, may be exposed to multiple toxicants. Given this is a more realistic exposure scenario in situ, the authors sought to understand the interactive toxicity of multiple metals to aquatic organisms. The authors performed a series of studies using equitoxic mixtures of cadmium, copper, and zinc to 2 aquatic organisms, rainbow trout (Oncorhynchus mykiss) and the waterflea, Ceriodaphnia dubia. Single metal toxicity tests were conducted to determine the acute median lethal concentration (LC50) values for O. mykiss and short-term, chronic median effective concentration (EC50) values for C. dubia. All 3 metals were then combined in equitoxic concentrations for subsequent mixture studies using a toxic unit (TU) approach (i.e., 1 TU = EC50 or LC50). For C. dubia, the mixture study showed greater-than-additive effects in hard water (TU-based EC50 = 0.74 TU), but less-than-additive effects in soft water (TU-based EC50 = 1.93 TU). The mixture effects for O. mykiss showed less-than-additive effects in both hard and soft waters, with TU-based LC50 values of 2.33 total TU and 2.22 total TU, respectively. These data are useful in helping understand metal mixture toxicity in aquatic systems and indicate that although in most situations the assumption of additivity of metal mixture toxicity is valid, under certain conditions it may not be sufficiently protective., (© 2014 SETAC.)

As a result of the stringent data requirements of the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation, a vast amount of ecotoxicological data has become available through the dissemination portal of the European Chemicals Agency (ECHA). As of April 2014, the database contained 12,439 unique substances from 47,909 dossiers. This vast database could be used to refine existing, or to create new, non-testing methods, such as quantitative structure-activity relationships (QSARs). Acute fish toxicity data were mined from the ECHA database using the eChemPortal; after filtering for single organic substances, 1159 experimental data points remained, representing 564 compounds. To evaluate the quality and accessibility of this data, the authors used the data to refine and improve an existing QSAR. The reliability of the data submitted to the ECHA database, as well as the effectiveness of the Klimisch scoring system, were assessed by comparing the refined QSAR with established QSAR benchmarks. The model developed meets all Organisation for Economic Co-operation and Development principles, has strong internal (leave-one-out internally cross-validated correlation coefficient [Q(2)(LOO)] = 0.91) and external (external coefficient of determination (predicted vs experimental [test set])) validation statistics, and can provide reliable fish median lethal concentration (LC50) predictions for non-polar narcotics. Although some issues with dossier misinformation were discovered, it was found that the ECHA dissemination portal is a valuable and reliable data source. When queried using the eChemPortal, chemical dossiers containing reliable data could be found quickly. The ECHA dissemination portal holds great potential for future QSAR development and improvement, such as updating QSARs within the Ecological Structure-Activity Relationships (ECOSAR) program., (© 2014 SETAC.)

Chronic toxicity of cadmium, copper, lead, or zinc to white sturgeon (Acipenser transmontanus) and rainbow trout (Oncorhynchus mykiss) was evaluated in water-only exposures started with newly hatched larvae or approximately 1-mo-old juveniles. The 20% effect concentration (EC20) for cadmium from the sturgeon tests was higher than the EC20 from the trout tests, whereas the EC20 for copper, lead, or zinc for the sturgeon were lower than those EC20s for the trout. When the EC20s from the present study were included in compiled toxicity databases for all freshwater species, species mean chronic value for white sturgeon was in a relatively low percentile of the species sensitivity distribution for copper (9th percentile) and in the middle percentile for cadmium (55th percentile), zinc (40th percentile), or lead (50th percentile). However, the species mean chronic value for rainbow trout was in a high percentile for copper, lead, and zinc (∼68th-82nd percentile), but in a low percentile for cadmium (23rd percentile). The trout EC20s for each of the 4 metals and the sturgeon EC20s for cadmium or lead were above US Environmental Protection Agency chronic ambient water quality criteria (AWQC) or Washington State chronic water quality standards (WQS), whereas the sturgeon EC20s for copper or zinc were approximately equal to or below the chronic AWQC and WQS. In addition, acute 50% effect concentrations (EC50s) for copper obtained in the first 4 d of the chronic sturgeon test were below the final acute value used to derive acute AWQC and below acute WQS for copper., (Published 2014 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

The present study sought to determine whether environmentally realistic mixtures of agriculturally important pesticides are stressful to fish. Juvenile rainbow trout were exposed for 96 h to concentrations of a pesticide mixture found in a waterway that is the focus of salmon restoration efforts (Nicomekl River, BC, Canada). This mixture contained organochlorine, organophosphorus, phenylurea, and triazine classes of pesticides. Fish given a realistic mixture exposure (total concentration, 1.01 µg/L) had increased plasma cortisol concentration, packed red cell volume, hematocrit (Hct), as well as decreased white cell volume, leukocrit (Lct). Similar changes in Hct and Lct were apparent after exposure to a lower concentration (0.186 µg/L). Interestingly, no changes in plasma cortisol concentration, Hct, or Lct were noted after exposure to a higher concentration (13.9 µg/L). This suggests that the exposure likely impaired the mechanisms enabling the stress response. Across all exposures, plasma glucose concentration was related to plasma cortisol concentration, not to pesticide mixture concentration. This suggests that a secondary stress response may be more related to variability in individual primary stress response than to differences in pesticide exposure concentrations. In summary, the present study indicates that salmon living in agrichemical-contaminated waterways may be experiencing stress, and this may pose a threat to their survival., (Copyright © 2011 SETAC.)

Normal transcriptomic patterns along the brain-pituitary-gonad-liver (BPGL) axis should be better characterized if endocrine-disrupting compound-induced changes in gene expression are to be understood. Female rainbow trout were studied over a complete year-long reproductive cycle. Tissue samples from pituitary, ovary, and liver were collected for microarray analysis using the 16K Genomic Research on Atlantic Salmon Project (GRASP) microarray and for quantitative polymerase chain reaction measures of estrogen receptor (ER) isoform messenger RNA (mRNA) levels. Plasma was collected to determine levels of circulating estradiol-17β (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). As an a priori hypothesis, changes in gene expression were correlated to either circulating levels of E2, FSH, and LH, or ER mRNAs quantified by quantitative polymerase chain reaction. In the liver, most transcriptomic patterns correlated to levels of either E2, LH, or ERs. Fewer ovarian transcripts could be correlated to levels of E2, ERα, or FSH. No significant associations were obvious in the pituitary. As a post hoc hypothesis, changes in transcript abundance were compared with microarray features with known roles in gonadal maturation. Many altered transcripts in the ovary correlated to transcript levels of estradiol 17-beta-dehydrogenase 8 or 17 B HSD12, or to glycoprotein alpha chain 1 or 2. In the pituitary, genes involved with the growth axis (e.g., growth hormone, insulin-related growth factor binding protein) correlated with the most transcripts. These results suggest that transcriptional networks along the BPGL axis may be regulated by factors other than circulating steroid hormones., (© 2010 SETAC.)

The sense of smell in fish is thought to be important for behaviors associated with alarm scent recognition and predator avoidance. Olfactory function of juvenile chinook salmon was evaluated in two separate experiments following 2-h immersion exposures to 0, 1, 10, or 100 microg diazinon/L. In the first test system, groups of 10 salmon were transferred to the prechoice zone of a Y-maze fluviarium with parallel streams of water. A dilute alarm scent composed of either chinook skin extract or L-serine was pumped into the water supply of one arm. Fish were allowed to choose either upstream arm by raising a containment screen for 10 min, then their positions were photographed and the screen lowered. In the second test system the relationship of diazinon exposure to predation susceptibility was directly examined by transferring groups of 20 salmon (10 diazinon-exposed, 10 control) to troughs containing a protective cover. These salmon were given two olfactory warnings by delivering conspecific skin extract to both their transfer container and the predation tank they entered. After a 2-min prey acclimation period, larger predator rainbow trout were allowed access to the downstream section of the tank containing the test salmon. The combined fish were observed until approximately 50% of the prey fish had been consumed. Statistical comparisons indicated that although the salmon did avoid the water streams containing alarm scents, none of the diazinon exposure levels resulted in significantly different outcomes from the controls (alpha=0.05) in either the two-choice maze study or the predation study. Possible environmental and population implications are discussed in light of related olfaction and diazinon studies., (Copyright (c) 2010 SETAC.)

The chronic (early life stage) toxicity of silver to rainbow trout (Oncorhynchus mykiss) was determined in flow-through exposures. Rainbow trout embryos were exposed to silver (as AgNO3) from 48 h or less postfertilization to 30 d postswimup in soft water in the presence and absence of 49 mg/L of NaCl (30 mg/L of Cl). The studies determined effect levels for rainbow trout exposed throughout an extended development period and assessed possible protective effects of sodium chloride. Lowest-observed-effect concentrations were greater than 1.25 microg/L of dissolved silver for survival, mean day to hatch, mean day to swimup, and whole-body sodium content in both studies. Whole-body silver concentrations increased significantly at 0.13 microg/L of dissolved silver in unmodified water and at 1.09 microg/L of dissolved silver in amended water. The maximum-acceptable toxicant concentration for growth was greater than 1.25 microg/L of dissolved silver in unmodified water and 0.32 microg/L of dissolved silver in amended water. Whole-body silver concentrations were more sensitive than survival and growth end points in unmodified water. Interpretation of sodium chloride effects on chronic silver toxicity to rainbow trout was complicated by differences in measured effect levels that were potentially the result of strain differences between test organisms in the two studies.

Rainbow trout (Oncorhynchus mykiss) were exposed to 1.5 and 15% v/v secondary treated sewage effluent for 32 weeks in flow-through mesocosms. The exposure encompassed the full period of reproductive development for rainbow trout. Trout did not show any evidence of a dose-dependent change in growth. Fish exposed to 15% effluent were the only group to show mortality (5%) over the duration of the experiment. Trout at the highest effluent concentration had significantly higher liver size than reference water fish. Both male and female trout in the 15% exposure group also exhibited significantly higher gonad weight than the reference group. In female trout, this gonad size increase could be explained by higher egg numbers. Female and male trout both displayed a significant increase in plasma 17 beta-estradiol levels after exposure to 15% effluent, while neither sex had dose-dependent differences in plasma testosterone. Male trout displayed elevated vitellogenin levels and reduced plasma 11-ketotestosterone concentration after exposure to 15% effluent. Chemical examination of steroidal compounds, including both estrogens and androgens, in the wastewater revealed that only estrone was detectable at a mean concentration of 4.5 ng/L. It is assumed that the effects observed in trout exposed to 15% effluent were consistent with stimulation of reproductive development due to very low levels of estrogens. Overall, long-term exposure to treated sewage effluent containing low levels of estrogen did not have significant negative implications for reproductive development in rainbow trout.

Caging experiments were conducted using hatchery-reared, immature, female rainbow trout (Oncorhynchus mykiss) in three previously defined areas of the Biobio River (south central Chile) representing a pollution gradient from the pulp and paper mill discharges area: a pre-impact area (upstream area, reference location), an impact area (area directly influenced), and a postimpact area (downstream area, less influenced). No significant changes were observed in the physiological index as represented by condition factor (K) and liver somatic index during different sampling times (after 11, 21, and 30 d of exposure). Ethoxyresorufin-O-deethylase activities were significantly higher in trout caged at the impact and postimpact discharges areas (two- to fourfold) compared with the reference (pre-impact) area, and a strong inhibition of acetylcholinesterase activity, reaching 50%, was observed mainly in fish caged at the impact area. A significant endocrine-disrupting effect (reproductive level) was evidenced by significant increments in gonad somatic index and plasma vitellogenin levels combined with an induction of gonad maturation (presence of vitellogenic oocytes) in trout caged at the impact and postimpact areas. These results, generated by an in situ approach, confirmed our group's findings for trout exposed to sediment in the laboratory: discharges of pulp mill effluent in the Biobio River are associated with the effects evaluated at different biological levels.

Four pulp and paper mills discharge their effluents in the same section of the Biobio River in central southern Chile. Pulp mill effluents are a very complex mixture with characteristics that depend on the type of raw material, the process technology, and the effluent treatment. To investigate the effect of pulp mill effluent discharges, immature Oncorhynchus mykiss were exposed to river sediments in the laboratory for 29 d. Three sampling areas were defined in a spatial gradient in the river: Preimpact, impact, and postimpact zones relative to the pulp and paper mill discharge areas. Ethoxyresorufin-O-deethylase activities were significantly higher in fish exposed to impact and postimpact sediments when compared to those exposed to preimpact sediments, and higher levels of vitellogenin were observed in the plasma of female fish exposed to impact and postimpact sediments. Histological analysis of the gonadal tissue showed an induction of gonadal maturation in fish exposed to sediment coming from the impact and postimpact zones (oocytes in a vitellogenic state). No site differences were observed in erythrocytes, although differences were noted in the leukocytes in the exposure areas. Finally, the biomarker approach showed evidence that the sediment associated with pulp mill effluent discharges produces some effects in fish under laboratory conditions.

Of the fish species tested in chronic Ni exposures, rainbow trout (Oncorhynchus mykiss) is the most sensitive. To develop additional Ni toxicity data and to investigate the toxic mode of action for Ni, we conducted acute (96-h) and chronic (85-d early life-stage) flow-through studies using rainbow trout. In addition to standard toxicological endpoints, we investigated the effects of Ni on ionoregulatory physiology (Na, Ca, and Mg). The acute median lethal concentration for Ni was 20.8 mg/L, and the 24-h gill median lethal accumulation was 666 nmol/g wet weight. No effects on plasma Ca, Mg, or Na were observed during acute exposure. In the chronic study, no significant effects on embryo survival, swim-up, hatching, or fingerling survival or growth were observed at dissolved Ni concentrations up to 466 microg/L, the highest concentration tested. This concentration is considerably higher than the only other reported chronic no-observed-effect concentration (<33 microg/L) for rainbow trout. Accumulation of Ni in trout eggs indicates the chorion is only a partial barrier with 36%, 63%, and 1% of total accumulated Ni associated with the chorion, yolk, and embryo, respectively. Whole-egg ion concentrations were reduced by Ni exposure. However, most of this reduction occurred in the chorion rather than in the embryos, and no effects on hatching success or larval survival were observed as a result. Plasma ion concentrations measured in swim-up fingerlings at the end of the chronic-exposure period were not significantly reduced by exposure to Ni. Nickel accumulated on the gill in an exponential manner but plateaued in trout plasma at waterborne Ni concentrations of 118 microg/L or greater. Consistent with previous studies, Ni did not appear to disrupt ionoregulation in acute exposures of rainbow trout. Our results also suggest that Ni is not an ionoregulatory toxicant in long-term exposures, but the lack of effects in the highest Ni treatment precludes a definitive conclusion.

The effect of cytochrome P4501A (CYP1A) induction on cell-specific benzo[a]pyrene (BaP) adduct formation was studied in rainbow trout (Oncorhynchus mykiss) gills. Fish preexposed to beta-naphthoflavone (betaNF) or caged in a polluted river were exposed to waterborne 3H-benzo[a]pyrene (3H-BaP). The 3H-benzo[a]pyrene adducts in the gill filaments were localized by autoradiography and CYP1A protein by immunohistochemistry. Ethoxyresorufin O-deethylase (EROD) activity was measured using a gill filament-based ex vivo assay. Branchial 3H-BaP binding and EROD activity were enhanced by exposure to betaNF or to the river water, and completely blocked by the CYP1A inhibitor ellipticine. The predominant sites of adduct formation were in epithelium of the secondary lamellae and in epithelium of the efferent edge of the gill filament. In betaNF-exposed fish, the strongest CYP1A immunoreactivity was observed in differentiating cells and in pillar cells. In fish caged in the polluted river, strong CYP1A immunoreactivity was found in most cells in the secondary lamellae, whereas the primary lamellae were almost devoid of immunoreactivity. Our results reveal a discrepancy between the localization of CYP1A protein and BaP adducts in the gill. Consequently, other factors, such as bioavailability of waterborne polycyclic aromatic hydrocarbons (PAHs) to the target cells, are important for the localization of PAH adducts in the gill.

Fire-retardant chemicals often are applied in relatively pristine and environmentally sensitive areas that are potentially inhabited by endangered or threatened aquatic species. Avoidance of contaminants is an adaptive behavior that may reduce exposure to harmful conditions. We evaluated the avoidance responses of rainbow trout (Oncorhynchus mykiss) to concentrations of fire-retardant chemicals and alternate constituent formulations ranging from 0.65 to 26 mg/L. Countercurrent avoidance chambers were used in a flow-through design with receiving water at each end and a drain at the center to create a distinct boundary between treatment water and reference water. Rainbow trout consistently avoided water treated with retardants at all concentrations tested. The magnitude of the avoidance response did not appear to follow a concentration-response relationship, but rather was an all-or-none response.

Rubber tire material contains toxic compounds including oils rich in polycyclic aromatic hydrocarbons (PAH), so-called highly aromatic (HA) oils, as well as other reactive additives used as antioxidants, antiozonants, and vulcanization accelerators. The toxicity of rubber tire leachates to aquatic organisms has been demonstrated before. However, previous studies have focused on lethal rather than sublethal effects. We kept rainbow trout (Oncorhynchus mykiss) in tanks with two types of tires: a tire containing HA oils in the tread or a tire free of HA oils in the tread. After 1 d of exposure, an induction of cytochrome P4501A1 (CYP1A1) was evident in both exposed groups, measured as elevated ethoxyresorufin-O-deethylase (EROD) activity and increased CYP1A1 mRNA levels. After two weeks of exposure, EROD activity and CYP1A1 mRNA were still high in fish exposed to leachate from HA oil-containing tire, whereas the effect was somewhat lower in fish exposed to leachate from HA oil-free tread tire. Compounds in the tire leachates also affected antioxidant parameters. Total glutathione concentration in liver as well as hepatic glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase activities were markedly elevated after two weeks of exposure in both groups. The responses were greater in the group exposed to leachate from HA oil-free tread tire. Vitellogenin measurements did not indicate leakage of estrogenic compounds from the tires. Chemical analyses of bile from exposed fish revealed the presence of hydroxylated PAH as well as aromatic nitrogen compounds indicating uptake of these compounds by the fish.

We have investigated the response dynamics of the estrogen-dependent genes vitellogenin (VTG) and the vitelline envelope proteins (VEPs) as well as circulating VTG in immature female rainbow trout (Oncorhynchus mykiss) exposed to 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) for periods of 7 and 14 d. Gene responses were quantified by measurement of messenger RNA (mRNA) in liver extracts using a chemiluminescent hybridization protection assay. Circulating VTG was measured by a homologous enzyme-linked immunosorbent assay. Exposure to both E2 and EE2 induced concentration-dependent increases in all biomarkers. The data presented indicate that VEP genes may be more sensitive to estrogens than the VTG gene. The biomarker lowest-observed-effect concentrations (biomarkerLOEC) in the 14-d study with E2 were 14 ng/L (VTG protein, VTG mRNA, VEPbeta, and VEPgamma) or 4.8 ng/L (VEPalpha). The EE2 was 5- to 66-fold more potent depending on the biomarker studied. In the 7-d study, all biomarkers were elevated after 48-h exposure to E2, with biomarkerLOECs of 30 ng/L (VTG protein, VTG mRNA, and VEPgamma) or 9.7 ng/L (VEPalpha and VEPbeta). Vitellogenin mRNA was induced up to 1,000-fold above baseline, and this translated into an increase of approximately 50,000-fold in circulating VTG. In conclusion, all biomarkers responded to estrogen exposure at environmentally relevant concentrations.

Wild female and male white sucker (Catostomus commersoni) inhabiting an area receiving pulp mill effluent had reduced hepatic levels of retinol, didehydroretinol, retinyl esters, and didehydroretinyl esters, while vitamin E levels were unaffected. This disruption of the retinoid system led us to test methanol and dichloromethane extracts from the effluent of 11 pulp mills from across Canada for their ability to bind to rainbow trout (Oncorhynchus mykiss) retinoic acid receptors (RARs) from the gill and retinoid X receptors (RXRs) from the liver. Concentrated extracts of the final effluent from 6 of the 11 pulp mills were able to displace greater than 25% of the receptor-bound [3H]all-trans retinoic acid (RA) or [3H]9-cis RA from trout RARs and RXRs, respectively. The ability of the extracts to displace retinoic acid did not appear to be linked to the pulping or treatment processes. Moreover, extracts with the greatest activity came from thermomechanical mills, suggesting the compounds may originate from the wood furnish. In addition, extracts prepared from wood furnish (wood chips: white spruce [50%], lodgepole pine [47%], and balsam fir [3%]) from one mill were able to displace [3H]RA from the RARs and RXRs. The 4-hydroxy RA, a metabolite of RA that has been shown to be generated in greater quantities in fish exposed to P450-inducing xenobiotics, was able to displace [3H]all-trans RA from trout RARs as effectively as unlabeled all-trans RA. These results suggest that pulp mill effluent may impact the retinoid system of fish at multiple sites, either by decreasing hepatic retinoid stores or through contributing additional ligands (from the wood furnish) that can bind to RA receptors.

The main objective of the study was to use a species comparison approach in order to understand sensitivity and tolerance differences to copper. We hypothesized that species differences in toxicity would be reflected by differences in copper binding to high-affinity sites on the gill. Specifically, the strength of copper binding (affinity, logK) and maximum number of binding sites (saturation, Bmax) for copper at the gill surface would vary among different species of fish. Two species that are different in their copper sensitivity are the rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens). We explicitly compared acute toxicity (median lethal concentrations via 96-h LC50s) and whole-body Na+ loss in both organisms in two distinct water chemistries (i.e., hard and soft water). For both species, the copper binding sites at the gill surface were characterized for their affinity and saturability. The binding properties of the gill were quite similar between the two species in each water chemistry. Based on estimations of the free cupric ion concentration, the affinity, or logK, was 8.4 for both species in soft water, whereas in hard water, the affinity was higher (approximately 9.7). The Bmax value in soft water was 1.88 nmol/g for rainbow trout and yellow perch, while in hard water, saturation occurred at 3.63 nmol/g for rainbow trout and 9.01 nmol/g for yellow perch. More importantly, the amount of copper bound to the gills at 50% mortality (i.e., lethal accumulation; the LA50) was different between the two species (yellow perch LA50s were nine times higher than those of rainbow trout in soft water and hard water), indicating that the copper binding to the yellow perch gill must not have been 'biologically reactive.' According to 96-h LC50s, yellow perch were less sensitive to copper than were rainbow trout; however, the difference between the two species was similar in hard water (1.05 vs 4.16 microM) and soft water (approximately 0.10 vs 0.44 microM). Perch were more tolerant because they lost less sodium upon exposure to copper; yet this mechanism of tolerance was not reflected by the amount of copper at the gill surface. The influence of water chemistry on the binding properties of the gill demonstrates the dynamic nature of the gill in maintaining ionoregulatory homeostasis, a key issue in the future development of the chronic biotic ligand model.

Perfluorinated acids (PFAs) recently have emerged as persistent global contaminants after their detection in wildlife and humans from various geographic locations. The highest concentrations of perfluorooctane sulfonate are characteristically observed in high trophic level organisms, indicating that PFAs may have a significant bioaccumulation potential. To examine this phenomenon quantitatively, we exposed juvenile rainbow trout (Oncorhynchus mykiss) simultaneously to a homologous series of perfluoroalkyl carboxylates and sulfonates for 34 d in the diet, followed by a 41-d depuration period. Carcass and liver concentrations were determined by using liquid chromatography-tandem mass spectrometry, and kinetic rates were calculated to determine compound-specific bioaccumulation parameters. Depuration rate constants ranged from 0.02 to 0.23/d, and decreased as the length of the fluorinated chain increased. Assimilation efficiency was greater than 50% for all test compounds, indicating efficient absorption from food. Bioaccumulation factors (BAFs) ranged from 0.038 to 1.0 and increased with length of the perfluorinated chain; however, BAFs were not statistically greater than 1 for any PFA. Sulfonates bioaccumulated to a greater extent than carboxylates of equivalent perfluoroalkyl chain length, indicating that hydrophobicity is not the sole determinant of PFA accumulation potential and that the acid function must be considered. Dietary exposure will not result in biomagnification of PFAs in juvenile trout, but extrapolation of these bioaccumulation parameters to larger fish and homeothermic organisms should not be performed.

Rainbow trout (Oncorhynchus mykiss) were exposed simultaneously to a homologous series of perfluoroalkyl carboxylates and sulfonates in a flow-through system to determine compound-specific tissue distribution and bioconcentration parameters for perfluorinated acids (PFAs). In general, PFAs accumulated to the greatest extent in blood > kidney > liver > gall bladder. Carboxylates and sulfonates with perfluoroalkyl chain lengths shorter than seven and six carbons, respectively, could not be detected in most tissues and were considered to have insignificant bioconcentration factors (BCFs). For detectable PFAs, carcass BCFs increased with increasing length of the perfluoroalkyl chain, ranging from 4.0 to 23,000, based on wet weight concentrations. Carboxylate carcass BCFs increased by a factor of eight for each additional carbon in the perfluoroalkyl chain between 8 and 12 carbons, but this relationship deviated from linearity for the longest PFA tested, possibly because of decreased gill permeability. In general, half-lives (3.9-28 d) and uptake rates (0.053-1.700 L/kg/d) also increased with increasing length of the perfluoroalkyl chain in all tissues. Sulfonates had greater BCFs, half-lives, and rates of uptake than the corresponding carboxylate of equal perfluoroalkyl chain length, indicating that hydrophobicity, as predicted by the critical micelle concentration, is not the only determinant of PFA bioaccumulation potential and that the acid function must be considered.

On the basis of a new fractionation method combined with in vitro ethoxyresorufin-O-deethylase (EROD) induction in a rainbow trout liver cell line (RTL-W1) and chemical analysis, halogenated aromatic hydrocarbons with dioxin-like activity were identified in a sediment extract from Bitterfeld, Germany. The fractionation method allowed a separation of different nonplanar and coplanar polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) with different degrees of chlorination. The dioxin-like activity at the investigated site could be quantitatively assigned to PCDD/Fs. Both PCBs and PCNs could be excluded as the cause of the measured effects on the basis of the fractionation procedure and bioanalytical results. Thus, the method allowed the chemical analysis to focus on PCDD/Fs, with significant reduction of the analytical expense. The EROD-induction potency of sediment-extract fractions was quantified, and toxicants were confirmed by the application of induction equivalent quantities on the basis of fixed-effect-level concentrations that exhibit 15% of the maximum induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. This approach was designed to minimize methodological limitations due to superimposing inhibitory effects.

The polycyclic aromatic hydrocarbons (PAHs) phenanthrene and retene (7-isopropyl-1-methyl phenanthrene) are lethal to rainbow trout (Oncorhynchus mykiss) larvae during chronic exposures. Phenanthrene is a low-toxicity, non-cytochrome P4501A (CYP1A)-inducing compound that accumulates in fish tissues during exposure to lethal concentrations in water. Retene is a higher toxicity CYP1A-inducing compound that is not detectable in tissue at lethal exposure concentrations. The metabolism, excretion, and toxicity of retene and phenanthrene were examined in juvenile and larval rainbow trout during coexposure to the model CYP1A inducer beta-naphthoflavone (betaNF), or to the inducer-inhibitor piperonyl butoxide to determine if modulating CYP1A activity affected PAH metabolism and toxicity. Phenanthrene metabolism, excretion rate, and toxicity increased with coexposure to betaNE Piperonyl butoxide inhibited phenanthrene metabolism and reduced the excretion of all phenanthrene metabolites. As a consequence, embryo mortality rates increased but rates of sublethal effects did not. Coexposure of trout to retene and betaNF caused no change in retene metabolism and excretion, but retene toxicity increased, perhaps due to additivity. Piperonyl butoxide inhibited retene metabolism, decreased the excretion of some retene metabolites while increasing the excretion of others, and increased the toxicity of retene. These results support the role of CYP1A activity in PAH metabolism and excretion, and the role ofthe CYP1A-generatedmetabolites of PAHs in chronic toxicity to larval fish.

Extensive season-by-season sampling was used to establish the normal range of whole-body Na+ and Cl- and Na+ uptake in healthy populations of two fish species, rainbow trout, Oncorhynchus mykiss, and fathead minnow, Pimephales promelas, of known differences in sensitivity to ionoregulatory toxicants (low pH, trace metals). These data together with responses of both species to six different ionoregulatory challenge tests of increasing severity (mild handling, exposure to low Ca2+ water, epinephrine injection, net-confinement stress, exposure to copper, and osmotic shock) were evaluated for their potential as biomarkers of sensitivity and of effect of ionoregulatory toxicants. There were no obvious biomarkers of sensitivity in the ion measures themselves, but four of the six challenges (exposure to low Ca2+ water, epinephrine injection, exposure to copper, and osmotic shock) produced a significantly greater effect in the more sensitive of the two species, fathead minnow. Based on the responses of both species, this article makes a number of recommendations for the application of ion measures alone and in combination with challenge tests to the assessment of chronic effects in populations experiencing sublethal ionoregulatory stress.

The organochlorine o,p'- dichlorodiphenyldichloroethane (o,p'-DDD) is a metabolite of dichlorodiphenyltrichloroethane (DDT), known for its adrenolytic actions in birds and mammals. The effects of o,p'-DDD on the cortisol stress response were investigated in rainbow trout, Oncorhynchus mykiss, in a dose-response study in vivo. A dose-dependent decrease in plasma cortisol levels was observed on days 7 and 14 after a single i.p. injection of o,p'-DDD. Treatment with o,p'-DDD had no effect on weight gain, hematocrit, and gonado- or hepatosomatic index but decreased liver glycogen reserves. The results indicate that o,p'-DDD is an adrenotoxic compound in rainbow trout and that its effects can be detected even 14 d postinjection.

The interactions of sublethal waterborne copper exposure and social dominance behavior were examined in juvenile rainbow trout. Dominance hierarchies were determined between pairs of fish by behavioral observations and among groups of 10 fish by the use of passive integrated transponder (PIT) tagging equipment. The present study is one of the first to utilize this novel PIT tag method for behavioral assessment. Feeding behavior was quantified by placing a PIT tag recorder at the entrance to the feeding area. Linear dominance hierarchies were documented based on these observations of feeding behavior. Dominance hierarchies established in control water were not altered by exposure to 30 microg/L of copper; however, physiological responses of fish to sublethal concentrations of copper were related to social rank. Subordinate fish exhibited a higher accumulation of copper in both gill and liver tissue. Subordinates of paired fish were also shown to have a higher uptake of sodium than dominant fish, and the uptake of sodium was correlated with uptake of copper from the water. Therefore, within a population of fish, it cannot be assumed that individuals of different social status will exhibit the same physiological responses to the presence of copper.

An often overlooked problem in the use of radiotracers is the possibility of isotopic contamination. Commercially available silver 110mAg was used to study silver uptake and depuration in rainbow trout and European eel. Quality control by means of comparative gamma and beta counting brought our attention to a contamination of the 110mAg stock with 109Cd, which could be seen only because the 109Cd was markedly bioconcentrated by trout gills. The contamination could not be detected in eel gills or in other tissues of both species. The difference between trout and eel gill structure and function is the probable explanation for the marked difference in 109Cd accumulation. This contamination was identified as 109Cd by gamma spectroscopy and its origin by transmutation of natural silver as a result of neutron activation is described. Failure to recognize this contamination problem would have resulted in serious misinterpretation of the data set. Guidance for avoiding this problem is given.

As part of an effort to validate the use of heat shock proteins (HSPs) as biomarkers of exposure to and effects of contaminants, we evaluated the effect of two handling regimens on the induction of HSP 60 and 70 in rainbow trout (Oncorhynchus mykiss). Fish were acclimated to laboratory conditions for several weeks before the beginning of the experiment. Fish were then captured by net, placed in a cooler for 1 h while being transported in a truck, returned to their original tanks, then sacrificed 6 to 8 h later. Tricaine methane sulfonate (MS-222) was used during different phases of handling to reduce handling stress. Heat-stressed fish were included in the experiment as a positive control. Muscle, liver, gills, and heart were analyzed for HSP 60 and 70 by immunoblotting. We found no effect of any handling regimen on the induction of HSPs. These findings suggest that the capture and transport of fish for environmental monitoring purposes should not interfere with the use of stress proteins as biomarkers.

Field and laboratory experiments were carried out to assess the influence of Cd and Zn on the contamination levels of 110Ag, 57Co, and 134Cs in rainbow trout. During a four-week prior exposure phase, two fish groups were held in tanks in the Lot River (France) at a reference (<0.05 microg Cd/L and 68 microg Zn/L) and at a polluted site (1.5 microg Cd/L and 152 microg Zn/L). During a subsequent phase, organisms were brought back to the laboratory, where the radionuclide accumulation and depuration were studied for 14 and 7 d, respectively. During this second phase, the water used in the experiments was brought back from the two sites on the Lot River in order to work under the same chemical conditions. The potential effect of chronic exposure to stable metals on several biomarkers has been explored: Plasma analysis indicated the disruption of certain variables linked to the energetic metabolism and to the maintenance of the ionic balance. In contrast, no significant disruption of the measured enzyme activities was observed. With regard to the bioaccumulation of radionuclides, concentrations in fish exposed to metals are much lower than those in fish from the control group. Various hypotheses are proposed to link fish metabolic profiles due to metal exposure to the radiocontamination of organisms.

From the large data set available on the toxicity of boron to aquatic organisms, the toxicity of boron to the early life stages of rainbow trout (Oncorhyncus mykiss) is the seminal issue relative to setting water quality criteria and effluent standards. Issues associated with the early life stage studies are the flat concentration-response curve, the low threshold of toxicity, and teratogenic effects observed. Recent laboratory and field studies offer new experimental data that make a weight-of-the-evidence assessment timely. In a re-examination of the effect of boron on the embryo-larval stage in rainbow trout and zebrafish, adverse effects due to boron deficiency are observed which decrease with increasing dose. It was found that low concentrations of boron stimulate embryonic growth in rainbow trout and increase the viability and survival of embryonic zebrafish. As boron concentration is further increased, the dose-response curve becomes flat as homeostatic processes are active; this is followed at higher doses by a new adverse response that increases with increasing dose. As a result, the dose-response relationship is U shaped, consistent with the characteristic shape of an essential micronutrient. Thus, effects originally reported to be toxicity at low exposures rather may be due to boron deficiency. Water analyses in trout hatcheries and field studies in wild trout streams add additional information on the toxicity of boron to trout. Of particular note is a controlled field study carried out in the Firehole River in Yellowstone Park (WY, USA), where trout populations survive and reproduction successfully occurs in natural water containing boron concentrations up to and in some cases greater than 1.0 mg B/L. Teratogenic effects due to boron exposure were not observed in any of these more recent studies.