The Effects of Sertraline on Fathead Minnow (Pimephales promelas) Growth and Steroidogenesis.

The purpose of this study was to evaluate the steroidogenic effects of sertraline, a popular selective serotonin reuptake inhibitor, on larval fathead minnow (FHM; Pimephales promelas) and adult FHM. Larvae were exposed to 0.1, 1, and 10 µg/L sertraline for 28 days and analyzed for differential mRNA expression of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), 20ß-hydroxysteroid dehydrogenase (20ß-HSD), aromatase (CYP19a), nuclear thyroid receptor alpha (TRα), and normalized to RP-L8. Adult FHM were exposed to 3 or 10 µg/L sertraline for 7 days and analyzed for differential expression of the same genes with the addition of thyroid receptor beta (TRß). Larval FHM exposed to 0.1 µg/L had a significant upregulation of both 20ß-HSD and TRα while adult FHM exposed to 10 µg/L had a significant upregulation of 11ß-HSD expression in brain tissue. The significance of these findings with respect to survival, growth and reproduction are currently unknown, but represent areas for future research.

Investigating Research Gaps of Pharmaceutical take back Events: An Analysis of take back Program Participants' Socioeconomic, Demographic, and Geographic Characteristics and the Public Health Benefits of take back Programs.

Research continues to show that pharmaceutical environmental contamination causes adverse effects to aquatic life. There are also public health risks associated with pharmaceuticals because in-home reserves of medications provide opportunities for accidental poisoning and intentional medication abuse. Pharmaceutical take back programs have been seen as a potential remedy for these issues; however, a thorough review of past programs indicates limited research has been conducted on take back programs. Furthermore, there are significant gaps in take back program research. To address these gaps and ultimately determine if take back programs could improve public health, research was conducted in conjunction with the take back program Denton drug disposal days held in Denton, Texas. Socioeconomic, demographic, and geographic characteristics of Denton drug disposal days participants were investigated using surveys and Geographic Information Systems. Potential impacts of the Denton drug disposal days program on public health were determined by comparing data from Denton drug disposal days events with data supplied by the North Texas Poison Center. Results suggest that Denton drug disposal days events may have prevented accidental poisonings or intentional abuse, however only qualitative comparisons support this statement and there was insufficient empirical evidence to support the conclusion that Denton drug disposal days events were exclusively responsible for public health improvements. An interesting finding was that there was a definitive travel threshold that influenced participation in Denton drug disposal days events. Overall, this study fills some geographic, socioeconomic, and demographic data gaps of take back programs and proposes methods to analyze and improve participation in future take back programs. These methods could also be applied to improve participation in other local environmentally-focused programs such as household hazardous collection events.

Bioconcentration and endocrine disruption effects of diazepam in channel catfish, Ictalurus punctatus.

Recently, the detection of pharmaceuticals in surface waters has increased worldwide. Pharmaceuticals are typically found in the environment at concentrations well below therapeutic levels in humans; however, their mechanisms of action may be largely unknown in non-target organisms, such as teleost species. Thus, chronic exposure to these types of compounds warrants further investigation. The goal of this study was to examine the potential for diazepam, a model benzodiazepine drug, to bioconcentrate in tissues of channel catfish and to examine its ability to interact with the endocrine system through modulation of steroid hormones and/or steroidogenic genes. To investigate the bioconcentration potential of diazepam, channel catfish (Ictalurus punctatus) were exposed to 1 ng/mL diazepam for seven days, followed by clean water for another seven days, using an abbreviated OECD 305 Fish Bioconcentration Test study design. This concentration of diazepam is well below environmentally relevant concentrations of diazepam (ng/L). To evaluate steroidogenic effects, fish were exposed to 1 ng/mL diazepam for seven days only. Steroid hormone concentrations were analyzed for various tissues, as well as expression of selected steroidogenic genes. Calculated bioconcentration factors for diazepam were well below regulatory threshold values in all tissues analyzed. No changes in steroid hormone concentration were detected in any tissue analyzed; however, the steroidogenic gene cytochrome P450 side chain cleavage (P450scc) was significantly down-regulated at day 5 and 3ß-hydroxy steroid dehydrogenase (3ß-HSD) was significantly down-regulated at day 7 in the gonad. These results indicate that although diazepam does not significantly bioconcentrate, low-level chronic exposure to diazepam may have the potential to interact with endocrine function by altering gene expression.

Wastewater Effluent Hydrocodone Concentrations as an Indicator of Drug Disposal Program Success.

Drug disposal programs have been seen as a remedy to the concern of environmental contamination resulting from pharmaceutical disposal down the toilet or sink; however a thorough review of peer-reviewed literature and publicly available information on these programs indicates limited research has been conducted to validate the effectiveness of these programs at reducing pharmaceuticals in the environment. The purpose of this research was to determine if drug disposal programs could actually reduce pharmaceutical residues in the environment. The concentration of hydrocodone in wastewater effluent released from a wastewater treatment plant in Denton, Texas was monitored before and after a take back program called Denton Drug Disposal Day (D4). Data collected and analyzed suggests D4 events were successful in contributing to a reduction of pharmaceutical loading to the environment; however there was insufficient evidence to demonstrate that D4 events were exclusively responsible for these improvements.

Temperature and species-specific effects on ß3-adrenergic receptor cardiac regulation in two freshwater teleosts: Channel catfish (Ictalurus punctatus) and common carp (Cyprinus carpio).

ß3-adrenergic receptors (AR) are important in teleost cardiovascular regulation. To date, it is unknown whether temperature acclimation changes ß3-AR functionality and consequently the involvement of this AR subtype in teleost cardiac regulation. Common carp (Cyprinus carpio) were acclimated at 12 °C or 23 °C (minimum 3 weeks) after which cardiovascular variables (cardiac output (Q), stroke volume (Sv) and heart rate (fH)) were measured upon injection of the ß3-AR agonist, BRL(37344), and antagonist, SR(59230A). In both 12 °C and 23 °C acclimated carp, BRL(37344) induced significant increases in fH and Q whereas Sv was significantly decreased. While temperature did not affect the change (increase vs. decrease) in cardiac variables, the magnitude and on-set of responses differed. For instance, fH, Sv and Q responded significantly faster to ß3-AR stimulation in 23 °C carp. In contrast, maximum responses of fH and Q were significantly higher in 23 °C carp whereas the maximum response of Sv was significantly greater in 12 °C carp. These findings suggest that temperature acclimation induced changes in ß3-AR receptor functionality (e.g. density and/or affinity). Stimulation of ß3-ARs in 23 °C acclimated channel catfish (Ictalurus punctatus) caused significant increases in fH, Sv and Q. The increase in Sv was opposite to the decrease observed in 23 °C acclimated common carp. SR(59230A) induced significant decreases in Sv and Q but had no effect in carp (23 °C). Results suggest species diversity in the density and affinity or structure of ß3-ARs which may explain the different cardiac responses to ß3-AR ligands.

Early life stage (ELS) toxicity of sucralose to fathead minnows, Pimephales promelas.

Sucralose, an intense artificial sweetener, has been detected in wastewater and surface waters at concentrations ranging from ng/L to low µg/L. Although over a hundred studies have been conducted to evaluate the safety of sucralose for human consumption, few studies have focused on the chronic ecotoxicological effects of this compound in fish. As a remedy to this data gap, an early-life stage toxicity test was conducted to assess the effects of sucralose on hatching, survival, and growth of fathead minnows (Pimephales promelas). Hatching, survival, and growth were unaffected by 98 mg/L of sucralose. The Lowest-Observed-Effect Concentration (LOEC) and the No-Observed-Effect Concentration (NOEC) for fathead minnows determined by this study are >98 and 98 mg/L, respectively. The results from this study suggest that the concentrations of sucralose detected in the environment are well below those required to cause adverse effects to developing aquatic organisms.

Environmental stressors and the epigenome.

Epigenetic modification and transgenerational transfer of phenotype at the individual or population level, particularly in response to environmental change, is at the forefront of biological investigation. The plasticity of this process allows an organism to respond to changes in environmental conditions, potentially conferring a survival advantage. In this review, we discuss epigenetic transgenerational phenomena in the specific context of environmental stressors including hypoxia and environmental toxicants.:

In silico predicted structural and functional robustness of piscine steroidogenesis.

Assessments of metabolic robustness or susceptibility are inherently dependent on quantitative descriptions of network structure and associated function. In this paper a stoichiometric model of piscine steroidogenesis was constructed and constrained with productions of selected steroid hormones. Structural and flux metrics of this in silico model were quantified by calculating extreme pathways and optimal flux distributions (using linear programming). Extreme pathway analysis showed progestin and corticosteroid synthesis reactions to be highly participant in extreme pathways. Furthermore, reaction participation in extreme pathways also fitted a power law distribution (degree exponent γ=2.3), which suggested that progestin and corticosteroid reactions act as 'hubs' capable of generating other functionally relevant pathways required to maintain steady-state functionality of the network. Analysis of cofactor usage (O2 and NADPH) showed progestin synthesis reactions to exhibit high robustness, whereas estrogen productions showed highest energetic demands with low associated robustness to maintain such demands. Linear programming calculated optimal flux distributions showed high heterogeneity of flux values with a near-random power law distribution (degree exponent γ≥2.7). Subsequently, network robustness was tested by assessing maintenance of metabolite flux-sum subject to targeted deletions of rank-ordered (low to high metric) extreme pathway participant and optimal flux reactions. Network robustness was susceptible to deletions of extreme pathway participant reactions, whereas minimal impact of high flux reaction deletion was observed. This analysis shows that the steroid network is susceptible to perturbation of structurally relevant (extreme pathway) reactions rather than those carrying high flux.

Early life-stage toxicity of eight pharmaceuticals to the fathead minnow, Pimephales promelas.

Human pharmaceuticals are routinely being detected in the environment, and there is growing concern about whether these drugs could elicit effects on aquatic organisms. Regulatory paradigms have shifted accordingly, with a greater emphasis on chronic toxicity data compared with acute data. The Organisation for Economic Co-operation and Development 210 Early Life Stage Test has been proposed as a good measure of the potential for pharmaceuticals to elicit chronic toxicity. To begin building a data set regarding the early life-stage toxicity of pharmaceuticals to fish, fathead minnows (FHM) were exposed to amiodarone, carbamazepine, clozapine, dexamethasone, fenofibrate, ibuprofen, norethindrone, or verapamil. Survival and growth were used to assess chronic toxicity in FHM at 28 days posthatch. Exposure of FHM to carbamazepine, fenofibrate, and ibuprofen resulted in no significant adverse effects at the concentrations tested. FHM survival was not impacted by verapamil exposure; however, growth was significantly decreased at 600 µg/L. Dexamethasone-exposed FHM showed a significant decrease in survival at a concentration of 577 µg/L; however, growth was not impacted at the concentration tested. Norethindrone exposure resulted in a significant decrease in survival and dry weight at 14.8 and 0.74 µg/L, respectively. Exposure to amiodarone and clozapine resulted in a significant decrease in survival and a significant increase in growth at concentrations of 1020 and 30.8 µg/L, respectively. Although the effect levels derived in this study are greater then concentrations observed in the environment, these data suggest that synthetic progestins may require additional research.

Effects of the artificial sweetener sucralose on Daphnia magna and Americamysis bahia survival, growth and reproduction.

The artificial sweetener sucralose has been detected in municipal wastewater effluent and surface waters at concentrations ranging from ng/L to low µg/L. Few chronic ecotoxicological data are available in the peer reviewed literature with respect to sucralose. To address this data gap, 21 d Daphnia magna and 28 d Americamysis bahia (mysid shrimp) studies were conducted to assess the effects of sucralose on the survival, growth and reproduction of these organisms. Concentrations ⩽1800mg/L resulted in no statistically significant reduction in D. magna survival or reproduction. Survival, growth and reproduction of mysid shrimp were unaffected by ⩽93mg/L sucralose. The no observable effect concentration (NOEC) and lowest observable effect concentration (LOEC) for the D. magna study were 1800 and >1800mg/L, respectively. The NOEC and LOEC for the mysid study were 93 and >93mg/L, respectively. Collectively, these data suggest that the concentrations of sucralose detected in the environment are well below those required to elicit chronic effects in freshwater or marine invertebrates.

The interactive effects of a gradual temperature decrease and long-term food deprivation on cardiac and hepatic blood flows in rainbow trout (Oncorhynchus mykiss).

The aim of the present study was to determine the extent to which the fish liver is perfused with blood. Transonic® flow probes were therefore implanted around the ventral aorta and hepatic vein(s) to record baseline blood flows in rainbow trout (Oncorhynchus mykiss) previously held under two different feeding regimes (food-deprived or fed to satiation, 8-12 weeks). Fish from both groups were exposed to a gradual temperature decrease (12°C to 5°C) and physical disturbance. Cardiac output (Q), stroke volume (Sv) and hepatic venous blood flow (HVBF) were significantly reduced in food-deprived trout at 12°C. Heart rate was not significantly affected by nutritional status, but was significantly reduced when temperature was decreased to 5°C. Physically disturbing each fish at 12°C and 5°C showed that the performance capacity of the heart was not affected by food deprivation as the capacity to increase Q and Sv was not reduced in the food-deprived group. Overall this study showed that food deprivation in rainbow trout reduced cardiac and hepatic blood flows. However, long-term food deprivation did not affect the capacity of the heart to acutely increase performance.

Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (Pimephales promelas) blood plasma using LC-ESI+/MS/MS.

Fathead minnows (Pimephales promelas) comprise a species-of-choice for the hazard assessments of various environmental contaminants, including compounds capable of disrupting endocrine function. Towards this end, the use of liquid chromatography coupled with mass spectrometry (LC-MS) and/or tandem mass spectrometry (MS/MS) is gaining common use for the quantification of steroid hormones as biomarkers of endocrine stress in small-fish toxicological studies. In this work, 2-hydrazinopyridine (2-HP) was used to derivatize and quantify the physiologically relevant steroid hormones of: 17α-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17α,20ß-dihydroxypregnenone, in the blood plasma of male and female fathead minnows. Liquid chromatographic separation was achieved using a Waters™ Sunfire C(18) column (2.1 mm×50 mm with a 3.5 µm particle size) and Milli-Q water:methanol (both with 0.1% formic acid) mobile phase over a gradient of 15 min. All mass analyses were conducted using electrospray ionization in the positive mode with tandem mass spectrometry (ESI+/MS/MS). This is the first such application of 2-HP derivatization for the quantifications of the structurally and functionally diverse C19 androgen of 11-ketotestosterone; C21 progestogens of 17α-hydroxypregnenolone, progesterone and17α,20ß-dihydroxypregnenone; and C21 corticosteroid of 11-deoxycortisol, in fathead minnow blood plasma. The limits of detection (LOD) were set to the lowest calibration standard that gave a signal-to-background response of ≥3, and were: 0.16 ng/ml for progesterone, 0.63 ng/ml for 17α-hydroxypregnenolone, 11-deoxycortisol and 17α,20ß-dihydroxypregnenone, and 1.25 ng/ml for 11-ketotestosterone. This study demonstrates the application of 2-HP derivatization for the analysis of a variety of steroid hormones representative of endocrine function in a species of fish commonly used in toxicological studies.

The influence of gill and liver metabolism on the predicted bioconcentration of three pharmaceuticals in fish.

The potential for xenobiotic compounds to bioconcentrate is typically expressed through the bioconcentration factor (BCF), which has gained increased regulatory significance over the past decade. Due to the expense of in vivo bioconcentration studies and the growing regulatory need to assess bioconcentration potential, BCF is often calculated via single-compartment models, using K(OW) as the primary input. Recent efforts to refine BCF models have focused on physiological factors, including the ability of the organism to eliminate the compound through metabolic transformation. This study looks at the ability of in vitro biotransformation assays using S9 fractions to provide an indication of metabolic potential. Given the importance of the fish gill and liver in metabolic transformation, the metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions. Metabolic transformation rates (k(M)) were calculated and integrated into a refined BCF model. A significant difference was noted between BCF solely based on K(OW) and BCF including k(M). These studies indicate that the inclusion of k(M) in BCF models can bring predicted bioconcentration estimates closer to in vivo values.

A comparison of the chronic effects of human pharmaceuticals on two cladocerans, Daphnia magna and Ceriodaphnia dubia.

Scientific researchers and regulators are focusing attention on trace quantities of pharmaceuticals in wastewater effluents and surface waters, resulting in an increased level of concern regarding the potential environmental impact of these compounds. The current European regulatory guideline requires evaluation of the chronic effects of active pharmaceutical ingredients on Daphniamagna. Based on the life cycle of D. magna, chronic studies to establish survival and reproductive endpoints require a 21 d exposure period. A similar organism, Ceriodaphniadubia, has a shorter life cycle and therefore survival and reproductive endpoints may be established following 7d of exposure. No observed effect concentrations and lowest observed effect concentrations for survival and reproduction were obtained for D. magna and C. dubia following exposure to six human pharmaceuticals and two metabolites (i.e. celecoxib, linezolid, varenicline, sunitinib, Compound A, ziprasidone and the M1 and M4 metabolites of torcetrapib). These data were evaluated to determine whether one organism may be considered more sensitive. Survival and reproduction data obtained from the C. dubia study provide similar outcomes to D. magna when determining the predicted environmental concentration/predicted no effect concentration (PEC/PNEC) ratios for surface water. Based on these data, C. dubia may be used as a cost-effective alternative and representative invertebrate species when assessing the potential risk of human pharmaceuticals.

Determination of beta-adrenergic receptor blocking pharmaceuticals in United States wastewater effluent.

Beta adrenergic receptor antagonists (beta-Blockers) are frequently prescribed medications in the United States and have been identified in European municipal wastewater effluent, however no studies to date have investigated these compounds in United States wastewater effluent. Municipal wastewater effluent was collected from treatment facilities in Mississippi, Texas, and New York to investigate the occurrence of metoprolol, nadolol, and propranolol. Propranolol was identified in all wastewater samples analyzed (n = 34) at concentrations < or = 1.9 microg/l. Metoprolol and nadolol were identified in > or = 71% of the samples with concentrations of metoprolol < or = 1.2 microg/l and nadolol < or = 0.36 microg/l. Time course studies at both Mississippi plants and the Texas plant indicate that concentrations of propranolol, metoprolol, and nadolol remain relatively constant at each sampling period. This study indicates that beta-Blockers are present in United States wastewater effluent in the ng/l to microg/l range.

Toxicity of select beta adrenergic receptor-blocking pharmaceuticals (B-blockers) on aquatic organisms.

One class of pharmaceutical compounds identified in U.S. and European waters are the B-adrenergic receptor blocking compounds (B-blockers). However, little information is available on the potential aquatic toxicity of these compounds. Therefore, Hyalella azteca, Daphnia magna, Ceriodaphnia dubia, and Oryias latipes (Japanese medaka) were exposed to metoprolol, nadolol, and propranolol to determine potential toxicity. Average 48-h LC(50) for propranolol to H. azteca was 29.8 mg/L. The no-observed-effects concentration (NOEC) and lowest-observed-effects concentration (LOEC) for propranolol affecting reproduction of H. azteca were 0.001 and 0.1 mg/L, respectively. The average propranolol and metoprolol 48-h LC(50)s for D. magna were 1.6 and 63.9 mg/L, respectively. C. dubia 48-h LC(50)s were 0.85 and 8.8 mg/L for propranolol and metoprolol, respectively. The NOEC and LOEC of propranolol affecting reproduction in C. dubia were 0.125 and 0.25 mg/L, respectively. In O. latipes, the propranolol 48-h LC(50) was 24.3 mg/L. Medaka growth was decreased at 0.5 mg/L propranolol. A 2-week medaka reproductive study indicated significant changes in plasma steroid levels; however, no changes in the average number of eggs produced or number of viable eggs which hatched was observed. In a 4-week follow-up propranolol exposure, the total number of eggs produced by medaka and the number of viable eggs that hatched were decreased at concentrations as low as 0.5 microg/L. Based on this study and the expected aqueous environmental exposure levels, adverse effects of propranolol to invertebrate populations is unlikely; however, further reproductive studies are need to elucidate the risk to teleosts.

Toxicity of fipronil and its degradation products to Procambarus sp.: field and laboratory studies.

Fipronil is a phenylpyrazole insecticide that is the active ingredient in the pesticide Icon 6.2 FS which is applied to rice seeds targeting the rice water weevil. An arthropod-selective insecticide, fipronil blocks the GABA-gated chloride channel and is unique in that several of its degradation products have been indicated to be equal or more potent than fipronil. After application of rice seeds (2-3 days postplant) to flooded rice fields, water is typically pumped from the rice fields and can be used for the culture of crayfish (Procambarus sp.). Because fipronil is selective for arthropods, is transported via organic sediment, and crayfish consume organic sediment, 96-h LC(50) experiments were conducted with fipronil and three of its environmental derivatives in crayfish under conditions without carrier solvents in water of similar pH, alkalinity, and hardness as observed in south Louisiana crayfish culture ponds. Measured LC(50)s for fipronil to red swamp (Procambarus clarkii) and white river (Procambarus zonangulus) crayfish were 14.3 (95% CI; 5.1-23.4) and 19.5 (95% CI; 11.1-27.9) microg/L, respectively. LC(50)s of fipronil sulfone (11.2; 9.2-13.2 microg/L), fipronil sulfide (15.5; 13-18 microg/L); and the photoproduct, desulfinyl fipronil (68.6; 46-95.2 microg/L) displayed very high toxicity in crayfish. In situ toxicity studies using caged crayfish in culturing ponds receiving effluent from drained rice fields indicated that effluent from rice fields planted with Icon-treated seed was significantly more toxic compared to untreated surface water (40% survival compared to 83% survival). Hazard quotient comparisons using measured water concentrations in the field and laboratory-based LC(50)s indicated that fipronil and its metabolites in water resulting from Icon-treated rice seed planting poses a significant risk to crayfish survival.

Toxicity of copper in an oxic stream sediment receiving aquaculture effluent.

Sediments were collected from a stream (upstream, outfall and downstream) receiving copper laden catfish pond effluent to assess toxicity to non-target biota. No significant reduction in Hyalella azteca survival or growth (10 d), or Typha latifolia germination and root and shoot growth (7 d) were observed after exposure to upstream and outfall sediments. A significant reduction in H. azteca survival was observed after exposure to the downstream sediment sample; however, no reduction in T. latifolia germination or seedling growth was detected. Bulk sediment copper concentrations in the upstream, outfall and downstream samples were 29, 31, and 25 mg Cu/kg dry weight, respectively. Interstitial water (IW) concentrations ranged from 0.053 to 0.14 mg Cu/l with 10 d IW toxicity units > or = 0.7. Outfall samples were amended with additional concentrations of copper sulfate so that bulk sediment measured concentrations in the amended samples were 172, 663, 1245, and 1515 mg Cu/kg dry weight. Survival was the most sensitive endpoint examined with respect to H. azteca with a no observed effects concentration (NOEC) and lowest observed effects concentration (LOEC) of 1245 and 1515 mg Cu/kg, respectively. NOEC and LOEC for T. latifolia root growth were 663 and 1245 mg Cu/kg, respectively. IW copper concentrations were > or = 0.86 mg Cu/l with H. azteca intersitial water toxicity unit (IWTU) concentrations > or = 1.2. Sequential extraction qualitatively revealed the carbonate and iron oxide fractions which accounted for a majority of the copper binding. In this instance, the copper which was applied to catfish ponds does not appear to be adversely impacting the receiving stream system.

Accumulation, distribution, and toxicity of copper in sediments of catfish ponds receiving periodic copper sulfate applications.

Copper sulfate (CuSO4) is applied periodically to commercial channel catfish (Ictalurus panctatus) ponds as an algicide or parasiticide. Current understanding of the chemistry of copper in soil-water systems suggests that copper may accumulate in pond sediments, although the forms and potential bioavailability of copper in catfish pond sediments are not known. This study investigated the accumulation and distribution of copper in the sediment of catfish ponds receiving periodic additions of CuSO4.5H2O. All ponds were constructed in Sharkey (very-fine, smectitic, thermic Chromic Epiaquert) soil. Nine 0.40-ha ponds received 59 applications of 2.27 kg CuSO4.5H2O per application per pond over 3 yr; no CuSO4.5H2O applications were made to nine additional ponds. Total Cu concentration in the sediments of CuSO4.5H2O-amended catfish ponds (172.5 mg kg(-1)) was four to five times higher than that in the sediments of nonamended ponds (36.1 mg kg(-1)). Copper accumulated in catfish pond sediments at a rate of 41 microg kg(-1) dry sediment for each 1 kg ha(-1) of CuSO4. 5H2O applied to ponds. Copper in the sediments of amended ponds was mainly in the organic matter-bound (30.7%), carbonate-bound (31.8%), and amorphous iron oxide-bound (22.1%) fractions with a considerable fraction (3.4%; 3 to 8 mg kg(-1)) in soluble and exchangeable fractions. This indicates that Cu accumulates differentially in various fractions, with proportionally greater initial accumulation in potentially bioavailable forms. However, toxicity bioassays with amphipods (Hyallela azteca) and common cattail (Typha latifolia L.) indicated that the effect of exposure to amended or nonamended pond sediments was not different.