Daniel Schlenk

Target organ toxicity in marine and freshwater teleosts

1. General Mechanisms of Toxicity Daniel Schlenk 2. Toxic Responses of the Nervous System Russell L. Carr and Janice E. Chambers 3. Fish Immunotoxicology: Mechanisms of Action and New Directions Charles Rice 4. Neurobehavioural Toxicity in Fish Edward E. Little and Sandra K. Brewer 5. Toxic Responses of the Reproductive System Lisa D. Arcand-Hoy and William H. Benson L. Carr, Mississippi State University, USA, Janice E. Chambers, Mississippi State University, USA, Charles Rice, Clemson University, USA, Edward E. Little, Columbia Environmental Research Center, USA, Sandra K. Brewer, University of Missouri, USA, Lisa D. Arcand-Hoy, Blasland, Bouck & Lee, Inc., USA, William H. Benson, US Environmental Protection Agency.

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

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.

Evaluation of estrogenic activity from a municipal wastewater treatment plant with predominantly domestic input

The purpose of this study was to survey estrogenic releases from two primarily domestic wastewater treatment plants over three seasons (1996-1999). Mature male channel catfish were maintained at two sites within each WWTP and a reference site for 21 days. Estrogenic activity of effluent was assessed by the Yeast Estrogen Screen (YES) assay (in 1999) and the expression of the female egg yolk precursor protein, vitellogenin (Vtg) in caged male channel catfish (1996-1998). Serum Vtg of animals exposed at WWTP-A was induced 220% above reference values in the Fall of 1996 and 480% in Spring of 1997. In animals exposed to effluent of WWTP-B, serum Vtg was elevated 370% in Spring of 1997 and 480% in Fall of 1997 relative to fish held in a reference location. Serum 17-beta-estradiol (E2) levels were also significantly elevated 13 and 16-fold in the Fall 1997 and Summer 1998 in the fish exposed to WWTP-A effluent. A 13.5-fold increase in serum E2 was observed in fish exposed to WWTP-B during Fall 1997. Utilizing an E2 concentration-Vtg response curve generated in the laboratory, effluent from both plants (in 1997 and 1998) had estrogen equivalent values ranging from 23 to 123 ng/l E2 equivalents. These values were comparable with YES values obtained from 1999, which indicated the presence of 21 to 147 ng/l E2 equivalents. E2 was responsible for 3 (fall) to 100% (summer) of the YES activity. Glucuronides of E2 were also observed in the treated effluent. These studies indicate that variable estrogenic activity is present in municipal wastewater resulting from domestic activities and that this activity may be significantly altered by environmental factors.

Estrogenic activity and reproductive effects of the UV-filter oxybenzone (2-hydroxy-4-methoxyphenyl-methanone) in fish

Previous studies in extracts of sediments surrounding municipal outfalls off the coast of California, USA and effluents of New York City, NY, USA indicated the UV-filtering agent, oxybenzone (CAS# 131-57-7; benzophenone-3) as a potential estrogen. The effects of oxybenzone on estrogenic activity and reproduction were evaluated using a 14-day juvenile rainbow trout assay for plasma vitellogenin and a subsequent 21-day Japanese medaka reproduction assay. Significant induction of vitellogenin was observed in the rainbow trout at the 1000 microg/L nominal concentration (749 microg/L median measured value) of oxybenzone which was approximately 75 times greater than the concentrations observed in previous wastewater effluent. Vitellogenin induction was also observed in the 1000 microg/L nominal concentration (620 microg/L median measured) of oxybenzone in male Japanese medaka (Oryzias latipes) after 21 days of exposure. The number of eggs produced per female per day exposed to the same concentration (620 microg/L) were significantly lower after 7 days, but returned to control values after 21 days. Fertilized eggs were then monitored for 20 days to assess hatching success. The overall percentage of fertilized eggs collected during the 21-day exposure that hatched was significantly lower in the 620 microg/L oxybenzone concentration. There was also a temporal effect at this concentration as egg viability (percentage of fertilized eggs that hatched) was diminished 13-15 days after eggs were collected. All three oxybenzone concentrations (16, 132, and 620 microg/L) and the 50 ng/L estradiol positive control showed reduced hatching of eggs at day 15, and the 132 and 620 microg/L oxybenzone concentrations diminished the percentage of eggs that hatched on days 13-15. These data indicate that the UV-filter oxybenzone alters endocrine or reproduction endpoints in two fish species, but at concentrations significantly higher than those measured in the environment.

Effects of Environmental Estrogens and Antiandrogens on Endocrine Function, Gene Regulation, and Health in Fish

A number of studies have indicated widespread reproductive endocrine disruption in wild fish populations. A number of laboratory studies have been conducted to determine the sources and to elucidate potential mechanisms of the disruption. This review explores the varied mechanisms of estrogenic and antiandrogenic effects in fish including effects at the steroid receptor level, effects on steroid synthesis, distribution, and excretion, actions up the hypothalamus-pituitary-gonad axis, as well as indirect mechanisms including thyroid and growth hormone disruption. Consequences of reproductive endocrine disruption will be touched on including non-reproductive responses such as impaired gene regulation, effects of unscheduled protein synthesis and DNA damage, and reproductive responses such as intersex, sex reversal and reproductive failure.

IN VIVO BIOASSAY-GUIDED FRACTIONATION OF MARINE SEDIMENT EXTRACTS FROM THE SOUTHERN CALIFORNIA BIGHT, USA, FOR ESTROGENIC ACTIVITY

The exposure and uptake of environmental estrogenic compounds have been reported in previous studies of demersal flatfish species in the central Southern California Bight (SCB), USA. The objective of this study was to evaluate the estrogenic or feminizing activity of marine sediments from the SCB by using in vivo vitellogenin (VTG) assays in male or juvenile fish. In 2003, sediments were collected near wastewater outfalls serving the counties of Los Angeles (LACSD) and Orange (OCSD), and the city of San Diego (SD), California, USA. Cultured male California halibut (CH; Paralichthys californicus) were either directly exposed to sediments for 7 d or treated with two intraperitoneal injections of sediment extract over 7 d. The 17beta-estradiol (E2) equivalent values ranged from 1 to 90 microg/kg with LACSD > SD > OCSD. Measurable concentrations of E2 were observed in all sediment extracts and ranged from 0.16 to 0.45 ng/g. Estrone (El) was only observed in sediments near the LACSD outfall (0.6 ng/g). Alkylphenols and alkylphenol ethoxylates were observed in all sediment samples, but were highest near the OCSD outfall, where concentrations of nonylphenol were 3,200 ng/g. Fractionation studies of the LACSD sediment extract collected in 2004 failed to demonstrate relationships between VTG expression and 62 analytes, including E2, which was observed in the whole extract (2.9 ng/g). Oxybenzone (1.6 ng/g) was identified in bioactive fractions as well as unknown compounds of relatively high polarity. These results indicate that estrogen receptor-based assays may underestimate environmental estrogenic activity and estrogenic compounds other than classic natural and xenoestrogens may contribute to estrogenic activity of sediments from the SCB.

Biochemical and Clinical Aspects of the Human Flavin-Containing Monooxygenase Form 3 (FMO3) Related to Trimethylaminuria

Trimethylaminuria is a rare metabolic disorder that is associated with abnormal amounts of the dietary-derived trimethylamine. Excess unmetabolized trimethylamine in the urine, sweat and other body secretions confers a strong, foul body odor that can affect the individuals ability to work or engage in social activities. This review summarizes the biochemical aspects of the condition and the classification of the disorder into: 1) primary genetic form, 2) acquired form, 3) childhood forms, 4) transient form associated with menstruation, 5) precursor overload and 6) disease states. The genetic variability of the flavin-containing monooxygenase (form 3) that is responsible for detoxication and deodoration of trimethylamine is discussed and put in context with other variant forms of the flavin-containing monooxygenase (forms 1-5). The temporal-selective expression of flavin-containing monooxygenase forms 1 and 3 is discussed in terms of an explanation for childhood trimethylaminuria. Information as to whether variants of the flavin-containing monooxygenase form 3 contributes to hypertension and/or other diseases are presented. Discussion is provided outlining recent bioanalytical approaches to quantify urinary trimethylamine and trimethylamine N-oxide and plasma choline as well as data on self-reporting individuals tested for trimethylaminuria. Finally, trimethylaminuria treatment strategies and nutritional support are described including dietary sources of trimethylamine, vitamin supplementation and drug treatment and issues related to trimethylaminuria in pregnancy and lactation are discussed. The remarkable progress in the biochemical, genetic, clinical basis for understanding the trimethylaminuria condition is summarized and points to needs in the treatment of individuals suffering from trimethylaminuria.

Effect of arsenite, arsenate, and the herbicide monosodium methyl arsonate (MSMA) on hepatic metallothionein expression and lipid peroxidation in channel catfish

Arsenic exerts its toxicity by the generation of reactive oxygen intermediates which caused lipid peroxidation and cellular damage. Metallothioneins (MTs) have been shown to be induced by oxidative stress and act as scavengers of reactive oxygen intermediates. Thus, hepatic MT was examined in channel catfish treated with the herbicide monosodium methyl arsonate (MSMA) and compared to equal doses of trivalent and pentavalent arsenic. Fish were exposed to 0.01, 0.1, and 1.0 mg/L of each compound for 1 week by static renewal. Hepatic MT was measured by the cadmium/hemoglobin (Cd/Hb) saturation assay, ELISA using antibodies raised against the first 10 amino acids of piscine MT, and Northern blot analysis using a cDNA encoding winter flounder hepatic MT. Cd/Hb and ELISA measurements of low molecular weight fractions from the hepatic cytosolic component of fish exposed to MSMA revealed a dose dependent increase in MT. MTs and MT mRNA of fish receiving the 1.0 mg/L dose were significantly induced vs control. Responses to arsenate exposure were more variable, but showed a trend toward a dose-dependent induction of MT and MT mRNA. MT mRNA and protein also showed a dose-dependent increase with arsenite exposure with no significant differences with untreated animals. Hepatic lipid peroxidation (as determined by TBARS) and glutathione was unaltered by any of the arsenical treatments. Thus, the lack of correlation between oxidative stress and MT expression suggest MT may not be a reliable indicator of oxidative stress. In addition, the induction of hepatic MT by various forms of As does not appear to be mediated through an oxidative stress mechanism in the liver.

Efficacy of Copper Sulfate for the Treatment of Ichthyophthiriasis in Channel Catfish

Abstract Ichthyophthirius multifiliis is a protozoan that may infest and significantly damage cultured fish species. The purpose of this study was to measure the efficacy of copper sulfate in treating ichthyophthiriasis. Fingerling channel catfish Ictalurus punctatus exposed to at least 2,000 theronts of I. multifiliis per liter of water developed consistent infestations of I. multifiliis (20 or more trophonts on the dorsal surface of the head of the fish). Infestation was observed in untreated controls at day 5 after exposure and mortality occurred after day 10. Coexposure studies with theronts and different concentrations of copper sulfate revealed that all theronts were killed at concentrations greater than 0.05 mg/L. To determine the effect of copper sulfate in the treatment of ichthyophthiriasis, fish were exposed to the parasite until trophonts were observed (day 5), and they were subsequently treated with copper sulfate. The lowest effective concentration of copper sulfate for treatment of ichthyop...