John D. Henderson

Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill.

Hydrocarbon-inducible cytochrome P4501A (CYP1A) expression was measured, as ethoxyresorufin-O-deethylase (EROD) activity, in livers of wintering harlequin ducks (Histrionicus histrionicus) captured in areas of Prince William Sound, Alaska, USA, oiled by the 1989 Exxon Valdez spill and in birds from nearby unoiled areas, during 2005 to 2009 (up to 20 years following the spill). The present work repeated studies conducted in 1998 that demonstrated that in harlequin ducks using areas that received Exxon Valdez oil, EROD activity was elevated nearly a decade after the spill. The present findings strongly supported the conclusion that average levels of hepatic EROD activity were higher in ducks from oiled areas than those from unoiled areas during 2005 to 2009. This result was consistent across four sampling periods; furthermore, results generated from two independent laboratories using paired liver samples from one of the sampling periods were similar. The EROD activity did not vary in relation to age, sex, or body mass of individuals, nor did it vary strongly by season in birds collected early and late in the winter of 2006 to 2007, indicating that these factors did not confound inferences about observed differences between oiled and unoiled areas. We interpret these results to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 20 years after the original spill. This adds to a growing body of literature suggesting that oil spills have the potential to affect wildlife for much longer time frames than previously assumed.

Factors in standardizing automated cholinesterase assays.

A scientific panel assembled by the U.S. Environmental Protection Agency (EPA) determined that variability in cholinesterase (ChE) activities in the agencys pesticide/animal study database likely was due to a lack of accepted guidelines for ChE methodology. A series of trials was held in which participating laboratories measured ChE activity in blood and brain samples from untreated and pesticide-treated rats using a colorimetric assay method. The degree of inhibition of ChE activity in plasma and brain samples compared to controls was consistent among most of the laboratories. The ChE activity in erythrocyte samples differed more between laboratories due to a high blank, low erythrocyte AChE activity and hemoglobin absorption at the wavelength of the assay. Strategies are suggested for minimizing the variability of ChE activity in hemoglobin-rich samples.

Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks.

To examine the phenomenon of apparent age resistance of young chicks to organophosphate-induced delayed neuropathy (OPIDN), groups of either 2- or 10-week-old chicks were exposed subcutaneously daily for 4 days to the neuropathic organophosphate (OP), di-isopropylfluorophosphate (DFP, 1 mg/kg), the non-neuropathic OP, paraoxon (PO, 0.25 mg/kg) or atropine (20 mg/kg). Subsequently, all birds were examined at post-exposure intervals (calculated from the last day of exposure) for up to 56 days for neurological deficits and morphological lesions in the central and peripheral nervous systems (CNS, PNS). Clinically, none of the birds in the 2-week-old groups, or in the 10-week-old PO or atropine exposed groups had neurological deficits. However, all birds in the 10-week-old DFP exposed group developed ataxia by 7 days post-exposure (DPE) and then progressive paralysis. Therefore, all birds in the 10-week-old groups were killed at 14 DPE. Pathologically, the 2-week-old DFP exposed chicks had increasingly severe lesions of Wallerian-like degeneration predominantly in the spinal cord from 7 DPE and subsequently. In the 10-week-old DFP exposed chicks, the degenerative lesions of OPIDN were first detected in the CNS at 3 DPE and then with equally increasing severity in the CNS and PNS up to 14 DPE. A higher incidence of neuronal necrosis and chromatolysis in ventral motor horn neurons of spinal cord grey matter and in dorsal root ganglia occurred in both the DFP exposed age groups compared with those lesions in other groups. These results demonstrate that after neuropathic DFP exposure, 2-week-old chicks develop pathological lesions in the spinal cord without neurological deficits. In both age groups, onset of degenerative lesions in the spinal cord preceeded those in the PNS. The claim of apparent age resistance of chicks to OPIDN needs to be re-evaluated.

Toxicity of storm‐water runoff after dormant spray application in a French prune orchard, Glenn County, California, USA: Temporal patterns and the effect of ground covers

Organophosphorous (OP) insecticides, especially diazinon, have been detected routinely in surface waters of the Sacramento and San Joaquin River watersheds, coincident with rainfall events following their application to dormant orchards during the winter months. Preventive best management practices (BMP) aim at reducing off-site movement of pesticides into surface waters. Two proposed BMPs are: The use of more hydrophobic pyrethroid insecticides believed to adsorb strongly to organic matter and soil and the use of various types of ground cover vegetation to increase the soils capacity for water infiltration. To measure the effectiveness of these BMPs, storm water runoff was collected in a California prune orchard (Glenn County, CA, USA) during several rainstorms in the winter of 2001, after the organophosphate diazinon and the pyrethroid esfenvalerate were applied to different orchard sections. We tested and compared acute toxicity of orchard runoff from diazinon- and esfenvalerate-sprayed sections to two species of fish (Pimephales promelas, Onchorhynchus mykiss) and three aquatic invertebrates (Ceriodaphnia dubia, Simocephalus vetelus, Chironomus riparius), and determined the mitigating effect of three ground cover crops on toxicity and insecticide loading in diazinon-sprayed orchard rows. Runoff from the esfenvalerate-sprayed orchard section was less toxic to waterflea than runoff from the diazinon-sprayed section. However, runoff from the orchard section sprayed with esfenvalerate was highly toxic to fish larvae. Samples collected from both sections one month later were not toxic to fish, but remained highly toxic to invertebrates. The ground cover crops reduced total pesticide loading in runoff by approximately 50%. No differences were found between the types of vegetation used as ground covers.

Standardization of Clinical Cholinesterase Measurements

Previous studies showed that commonly used kits for measuring cholinesterases were not optimal for determining acetyl-cholinesterase (ACh E) activity. Clinical use of different kits and methodologies resulted in ACh E levels being reported in different units and activities that were not reproducible among laboratories. Findings such as these led to a revision in California regulations (covering ACh E measurements for pesticide worker safety) calling for clinical laboratories to standardize their findings. The laboratories were contacted and invited to participate in a split-sample study of human blood ACh E and nonspecific cholinesterase (BCh E) assays. Participating laboratories measured erythrocyte (RBC) ACh E and/or plasma BCh E from undiluted and 50% diluted blood, according to their practices. Aliquots of blood samples were shipped to University of California Davis for measurement, using an optimized semiautomated plate reader version of the method of Ellman. Nine of 25 laboratories sent samples for comparison. Two others performed their own comparisons and submitted data to the state. Best correlations were obtained with BCh E activity. Correlations (r 2) were.88 or above for four of five laboratories for BCh E, and above.9 for two of seven laboratories for ACh E. Reasons for poor correlations may include difficulties in pipetting RBCs, storage, and processing. A bovine ACh E RBC ghost “standard” was devised and tested. Activity of the preparation was maintained at-70°C for approximately 11 months. A test with an East coast laboratory resulted in a high correlation, demonstrating the reliability of the RBC ghost standard and that one laboratory can replicate the ACh E findings of another. The overall poor correlation of interlaboratory cholinesterase results points to the need to further standardize sample handling and assay methods.

Toxicity of repeated doses of organophosphorus esters in the chicken

Hens were repeatedly exposed to paraoxon (PO, phosphonothioic acid, diethyl paranitrophenyl ester), the chemical warfare agent VX/phosphorofluoridic acid, methyl-S-(2-[bis(1-methylethyl)amino/ethyl)O-ethyl ester], or the neuropathic DFP [phosphorofluoridic acid, bis(1-methylethyl)ester] as evidence was sought for nerve or other tissue damage following long-term treatments at high dose levels. Thirty-day and 90-d trials were performed in which each bird was injected 3 or 5 times per week with atropine as protection, weighed, their eggs collected, and their blood enzymes (cholinesterases creatine kinase, and lactic dehydrogenase) and locomotion periodically examined. Muscle and brain enzymes were assayed at the end of the experiments. Doses of PO and VX were at or above LD50 levels. DFP doses were lowered with each run to estimate a no-observable-effect level for organophosphate-induced delayed-neuropathy (OPIDN). No abnormalities attributable to repeated exposures to either PO or VX were found, even though acute, short-term symptoms of toxicity appeared after each injection. No evidence for OPIDN was obtained with repeated exposures to PO and VX under conditions where OPIDN was caused by DFP. Histological signs of OPIDN appeared in the spinal cord without gross symptoms of ataxia following repeated treatments of 25 mg/kg of DFP. The results of one experiment suggested that exposure to protective injections of atropine delays the appearance of the locomotor symptoms of the DFP-induced neuropathy.

Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow's goldeneyes to residual Exxon Valdez oil.

We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrows goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/1997, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity (≥ 2 times unoiled average) declined from 41% in winter 1996/1997 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrows goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill.

Clinical Effects of Low-Level Exposures to Chemical Warfare Agents in Mice and Chickens

Concerns that chemical warfare (CW) agents themselves or in combination with other chemicals may cause long-term damage to nerve and muscle are reviewed and discussed. Experiments on mice and hens underway with agent GA and pyridostigmine bromide (PB) and their effects (either separately or together) are presented.

Toxicity of an acute dose of agent VX and other organophosphorus esters in the chicken

The neurotoxicities of single doses of a chemical warfare agent VX [phosphonothioic acid, methyl-S-(2-[bis(1-methylethyl)amino/ethyl) O-ethyl ester], a metabolite of the agricultural chemical parathion, paraoxon, PO (phosphonothioic acid, diethyl paranitrophenyl ester), and the known neuropathic agents DFP] phosphorofluoridic acid, bis(1-methylethyl) ester] and TOCP (phosphoric acid, tri-o-tolyl ester) were compared in the chicken. Single injections (subcutaneous, sc) of VX as high as 150 micrograms/kg (5 times the LD50, intramuscular, im) were tolerated by laying tens if atropine and 2-pralidoxime were used as antidotes before and immediately after injection. The 150 of VX for inhibition of chicken brain acetylcholinesterase was approximately 5 X 10(-10). Plasma acetylcholinesterase, but not butyrylcholinesterase, was depressed 2 h after injections of 2-20 micrograms VX/kg im without antidotes. Levels of plasma enzymes such as creatine kinase, indicative of tissue damage, were increased after exposure to both VX and PO. Injections of up to 150 micrograms/kg of VX with antidotes did not cause locomotor or histological signs of organophosphorus-induced delayed neuropathy, but single injections of 400 mg TOCP/kg did.

ORGANOCHLORINE CONCENTRATIONS AND EGGSHELL THICKNESS IN FAILED EGGS OF THE CALIFORNIA CLAPPER RAIL FROM SOUTH SAN FRANCISCO BAY

Abstract In 1992 we collected 22 failed California Clapper Rail (Rallus longirostris obsoletus) eggs from four tidal marshes of south San Francisco Bay for organochlorine analysis and determination of eggshell thickness. Mean eggshell thickness of these eggs (262 microns) was not statistically distinguishable from that of pre-1932 museum eggs (271 microns). Total PCB concentrations in eggs ranged from 0.65 to 5.01 μg g−1 on an adjusted fresh wet weight basis, with a geometric mean concentration of 1.30 μg g−1. DDE concentrations were extremely low at a geometric mean of 0.11 μg g−1. Geometric mean concentrations of all other organochlorines detected were below 0.10 μg g−1. The concentrations of all organochlorines except PCBs appear to have declined in California Clapper Rails since the mid 1980s. PCBs may still be high enough in some rail eggs to produce embryotoxic effects but additional work to quantify the more toxic PCB congeners in rail eggs is needed. Concentraciones de Organocloros y Espesor de los Cascarones en Huevos Fallados de Rallus longirostris obsoletus de la Bahía de San Francisco del Sur Resumen. En 1992 recolectamos 22 huevos de Rallus longirostris obsoletus que fallaron en salir del cascarón en cuatro ciénagas de marea del sur de la Bahía de San Francisco para su análisis de contenido de organocloros y la determinación del espesor de los cascarones de dichos huevos. La media proporcional del espesor de los cascarones de huevo recolectados en el campo en 1992 (262 micras) no era menor que la de aquellos huevos (en museo) anteriores a 1932 (256 micras). Las concentraciones totales de PCB en los huevos fluctuaban desde 0.65 hasta 5.01 μg g−1 en base a un ajuste del peso húmedo fresco, con una concentración media geométrica de 1.30 μg g−1. Las concentraciones de DDE eran extremadamente bajas a una media geométrica de 0.11 μg g−1. La concentración media geométrica de todos los otros organocloros descubiertos estuvo por debajo de 0.10 μg g−1. La concentración de todos los organocloros, excepto los PCBs parece haber disminuido en R. l. obsoletus desde mediados de los años ochenta. Los PCBs todavía pueden ser suficientemente altos en algunos huevos de gallinetas de California como para producir efectos tóxicos en embriones, pero es necesario realizar investigaciones adicionales para cuantificar mayores congeners de los PCBs.