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.

N-Acylethanolamines: formation and molecular composition of a new class of plant lipids

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120-126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [14C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [14C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133-142) raises the possibility that a similar mechanism may operate in plant cells.

N-Acylethanolamines in seeds. Quantification Of molecular species and their degradation upon imbibition

N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 +/- 89 ng g(-1) fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 +/- 309 ng g(-1) fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g(-1) fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35 degrees C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5, 5'-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K(m) of 65 &mgr;M and a V(max) of 83 nmol min(-1) mg(-1) protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.

Comparative toxicity of polychlorinated biphenyls to earthworms Eisenia foetida and Lumbricus terrestris.

Effects of polychlorinated biphenyls (PCB; Aroclor 1254) in the manure worm, Eisenia foetida, on survival (LC50/LD50), and ability of coelomic leukocytes (also called coelomocytes) to form secretory rosettes (SR) and erythrocyte rosettes (ER) with, and to phagocytose antigenic rabbit red blood cells were determined and compared with those published for the earthworm, Lumbricus terrestris. Using a 5-day filter paper contact exposure protocol, LC50 and LD50 were 30.4 microg cm(-2) and 4500 microg g(-1) dry mass, respectively. Nominal PCB exposure concentrations of 5.0 and 10.0 microg cm(-2) resulted in tissue levels of 1400 and 2900 microg g(-1) dry mass. These body burdens resulted in significant reduction in SR formation by 18 and 52%, respectively. ER formation and phagocytosis were reduced 52 and 61%, respectively, only at the higher tissue concentration. Compared to L. terrestris, E. foetida: (1) accumulated considerably more PCB at each exposure concentration; (2) showed lower LC50, but higher LD50; and (3) exhibited effects on coelmocytes only at tissue PCB concentrations that caused some mortality. In terms of lethality and immunomodulation of SR, ER and phagocytosis, E. foetida appears to be more resistant to PCB than L. terrestris.

Sperm count in earthworms (Lumbricus terrestris) as a biomarker for environmental toxicology: effects of cadmium and chlordane.

Earthworms, Lumbricus terrestris, exposed in artificial soil to sublethal concentrations of technical chlordane (6.25, 12.5, and 25 ppm) and cadmium nitrate (100, 200, and 300 ppm) exhibited significant reduction in spermatozoa from testes and seminal vesicles. The onset time of reduction varied with exposure concentration, but absolute depression in sperm count was independent of exposure concentration or exposure duration after reduction was first manifested, demonstrating a threshold effect. Earthworm sperm counts show potential as a rapid-measurement endpoint biomarker for measuring sublethal effects of chemical pollutants on reproduction.

Assessment of the estrogenicity and toxicity of a domestic wastewater effluent flowing through a constructed wetland system using biomarkers in male fathead minnows (Pimephales promelas rafinesque, 1820).

The toxicity and estrogenicity of a final treated municipal effluent was examined while flowing through a constructed wetland in north-central Texas, USA. Fish data were collected, and a baseline wetland characterization was performed to assess wetland treatment potential for these effluent properties. Vitellogenin (VTG), gonadosomatic index (GSI), hepatosomatic index (HSI), and secondary sexual characteristics were biomarkers used in fish models to assess aqueous estrogenicity. Biological indicators used to assess overall fish health included hematocrit and condition factor. Estrogenic nature of final sewage treatment works effluent was screened, concurrent with a three-week fish exposure, via gas chromatography/mass spectrometry for target estrogenic compounds, including 17beta-estradiol, ethynylestradiol, bisphenol A, nonylphenolic compounds, phthalates, and DDT. The VTG in Pimephales promelas was measured after exposure at four sites in a treatment wetland and was significantly elevated (p < 0.0001) in fish exposed at the inflow site. The GSIs were significantly less (alpha = 0.001) at the inflow site. At wetland sites closest to the inflow, secondary sexual characteristics, tubercle numbers, and fatpad thickness were less (alpha = 0.0001) than in laboratory controls. The HSIs and density of male breeding stripes were not significantly different from those of laboratory controls. However, elevated HSIs were found at the inflow site. Hematocrit and condition factors were both less (alpha = 0.001) in effluent-exposed fish at wetland sites closer to the inflow than in control fish or fish further downstream.

A non-invasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies.

We have compared two methods for collecting earthworm leukocytes (coelomocytes) with respect to cell yield, viability and behaviour in immunoassays. Non-invasive extrusion was more efficient than puncturing the coelomic cavity. Extrusion does not produce trauma to earthworms maintained under long term laboratory conditions. Neither technique modified immune assays, as determined by erythrocyte and secretory rosette formation, and phagocytosis, since all 3 were functionally equivalent. After an initial extrusion, sequential leukocyte collections by extrusion are possible at intervals of 6 weeks without affecting total and differential cell counts and rosette formation.

Use of watershed factors to predict consumer surfactant risk, water quality, and habitat quality in the upper Trinity River, Texas.

Surfactants are high production volume chemicals that are used in a wide assortment of "down-the-drain" consumer products. Wastewater treatment plants (WWTPs) generally remove 85 to more than 99% of all surfactants from influents, but residual concentrations are discharged into receiving waters via wastewater treatment plant effluents. The Trinity River that flows through the Dallas-Fort Worth metropolitan area, Texas, is an ideal study site for surfactants due to the high ratio of wastewater treatment plant effluent to river flow (>95%) during late summer months, providing an interesting scenario for surfactant loading into the environment. The objective of this project was to determine whether surfactant concentrations, expressed as toxic units, in-stream water quality, and aquatic habitat in the upper Trinity River could be predicted based on easily accessible watershed characteristics. Surface water and pore water samples were collected in late summer 2005 at 11 sites on the Trinity River in and around the Dallas-Fort Worth metropolitan area. Effluents of 4 major waste water treatment plants that discharge effluents into the Trinity River were also sampled. General chemistries and individual surfactant concentrations were determined, and total surfactant toxic units were calculated. GIS models of geospatial, anthropogenic factors (e.g., population density) and natural factors (e.g., soil organic matter) were collected and analyzed according to subwatersheds. Multiple regression analyses using the stepwise maximum R(2) improvement method were performed to develop prediction models of surfactant risk, water quality, and aquatic habitat (dependent variables) using the geospatial parameters (independent variables) that characterized the upper Trinity River watershed. We show that GIS modeling has the potential to be a reliable and inexpensive method of predicting water and habitat quality in the upper Trinity River watershed and perhaps other highly urbanized watersheds in semi-arid regions.

Incorporation of Molecular Oxygen and Water during Enzymatic Oxidation of Cyanide by Pseudomonas fluorescens NCIMB 11764.

Cell extracts (high-speed [150,000 x g] supernatants) from Pseudomonas fluorescens NCIMB 11764 catalyzed the oxidation of cyanide to CO(inf2) (and NH(inf3)). Conversion was both oxygen and NADH dependent, with 1 mol of each being consumed per mol of cyanide degraded. Analysis of (sup13)CO(inf2) by mass spectrometry indicated that one atom each of isotopically labelled oxygen 18 from molecular oxygen and water were incorporated during enzymatic conversion. The results confirm earlier reports of oxygenase-mediated cyanide conversion in this organism. A reaction pathway for cyanide oxidation involving initial monooxygenation followed by hydrolysis of a hypothetical oxygenated intermediate to CO(inf2) (and NH(inf3)) is proposed.

Analysis of lipids by one-dimensional thin-layer chromatography.

A high-efficiency silica gel, (type HLF) thin-layer chromatography plate (HETLC), linear high-performance thin-layer chromatography plate (HPTLC) and densitometry method has been devised in order to resolve the major lipid classes obtained from rat brain tissues. This methodology, which has largely overcome prior problems, enhances the opportunity for assessing the glycerophospholipid and glycolipid compositions of tissues. DEAE-Sephadex column chromatography was used to separate the crude lipids extract into neutral and acidic lipid fractions. The lipid fractions were then spotted on separate HPTLC and HETLC plates and chromatographed in one dimension using one solvent system. Quantitation was by in situ densitometry with the absolute quantity of the lipid classes determined from co-chromatographed standards. Sensitivity was increased by using cupric sulfate reagent, which was found to be more sensitive than the conventional cupric acetate reagent. This method is applicable to a broader separation of lipid classes and has improved sensitivity.

The impact of depressed pH and elevated aluminum concentrations on specific dynamic action in Somatochlora cingulata (de Selys).

Differences in oxygen consumption attributable to apparent specific dynamic action (SDA) were measured in relation to feeding level in the dragonfly naiad Somatochlora cingulata exposed to low pH and sublethal aluminum concentration plus low pH. The average increases in respiration among the treatments following feeding were 50-60% that of controls. The average treatment peak height ratios (post/prefeeding respiration rates) were proportionately reduced and not significantly different from the average controls indicating that the resting metabolism, rather than SDA, was reduced.