Edward Wirth

Antifouling biocides in water and sediments from California marinas.

Irgarol 1051 is a common antifouling biocide and is highly toxic to non-target plant species at low ng/L concentrations. We measured up to 254 ng/L Irgarol in water and up to 9 ng/g dry weight Irgarol in sediments from Southern California recreational marinas. Irgarols metabolite, M1, concentrations were up to 62 ng/L in water and 5 ng/g dry weight in sediments. Another antifouling biocide, diuron, reached up to 68 ng/L in water and 4 ng/g dry weight in sediments. The maximum Irgarol concentrations in water were greater than the Irgarol concentration recommended as the plant toxicity benchmark (136 ng/L), suggesting that Irgarol concentrations may be high enough to cause changes in phytoplankton communities in the sampled marinas. Irgarol concentrations measured in sediments were greater than calculated Environmental Risk Limits (ERLs) for Irgarol in sediments (1.4 ng/g). Antifouling pesticide accumulation in sediments may present a potential undetermined risk for benthic organisms.

Effects of fluoxetine exposure on serotonin-related activity in the sheepshead minnow (Cyprinodon variegatus) using LC/MS/MS detection and quantitation.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor and is among the top 100 drugs prescribed yearly in the United States and the United Kingdom. Tissue and water extraction methods were developed to detect and quantify FLX, norfluoxetine and the associated biological compounds serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophan (TRP) and melatonin (MEL) using LC/MS/MS. Acute mortality and sublethal physiological effects of FLX were assessed using standard static renewal toxicity tests in which juvenile sheepshead minnows (Cyprinodon variegatus) were exposed to FLX. Fluoxetine did not cause significant mortality at levels near currently reported environmental concentrations. Significant changes in neurotransmitter levels were observed within the serotonergic system in juvenile sheepshead minnows exposed at concentrations approximately one order of magnitude above those currently reported in the environment. Transformation activity ratios of a product to a precursor compound (5-HT/TRP, 5-HIAA/5-HT and MEL/5-HT) also exhibited significant changes with FLX treatment. Fluoxetine exposure did not only affect 5-HT but had additional effects both upstream and downstream of 5-HT within its synthesis and metabolic pathways. These sublethal changes within the serotonergic pathway may result in behavioral changes which could, in turn, have implications for the ecological response of populations to additional environmental stressors.

Development of Species-Specific Ah Receptor-Responsive Third Generation CALUX Cell Lines with Enhanced Responsiveness and Improved Detection Limits.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

Analysis of selected natural and synthetic hormones by LC-MS-MS using the US EPA method 1694

The US EPA Method 1694 (which is specific to the analysis of pharmaceuticals and personal care products) was evaluated for analysis of selected hormones: 17α- and 17β-estradiol, estriol, estrone, 17α-ethynylestradiol, diethylstilbestrol, equilin, equilenin, testosterone, progesterone and norgestrel in filtered sewage influent/effluent and seawater. The extraction of hormones from fortified water matrices with HLB Oasis solid phase extraction cartridges resulted in good recovery efficiencies: 86–113% for seawater and 80–114% for sewage samples with a relative standard deviation of 6–16% (seawater) and 7–14% (wastewater). Natural and synthetic estrogens (17α- and 17β-estradiol, estriol, estrone, 17α-ethynylestradiol, diethylstilbestrol, equilin, equilenin) were separated with a Gemini-NX C18 (Phenomenex, Inc.) column and detected by Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS) in negative ElectroSpray Ionization (ESI) multiple reaction monitoring (MRM) mode. Three other hormones (testosterone, progesterone and norgestrel) were separated using an Xterra MS C18 (Waters Corp.) column along with the Group I compounds of the EPA Method 1694 and detected by MS–MS in ESI positive mode. Most compounds were quantified by isotope dilution with matched labeled internal standards. The method was applied to the analysis of effluent and influent from wastewater treatment facilities in South Carolina, USA, and surface seawater samples from Charleston Harbor, South Carolina. Only estrone was detected in surface seawater at a concentration of 0.5 ng L−1, which was below the method reporting limit of 1 ng L−1. Estriol, estrone and testosterone were found in influent samples at 155–179 ng L−1, 27–28 ng L−1 and 41–49 ng L−1, respectively. The estrone concentration was 24 ng L−1 in an effluent sample from one of the treatment plants. This study demonstrates that the US EPA Method 1694 can be successfully used for the analysis of natural and synthetic hormones along with the pharmaceuticals and personal care products (PPCPs) currently listed in the method.

Fate and transport of Irgarol 1051 in a modular estuarine mesocosm

Fate and transport of Irgarol 1051 were assessed using a modular estuarine mesocosm containing natural seawater, saltmarsh sediments, marsh grass, shrimp, clams, snails, and naturally derived planktonic and benthic microorganisms. The mesocosms were enclosed in a greenhouse under near ambient conditions, and included a saltwater sump and a simulated tidal flux. The exposure was comprised of four replicate treatments of 0, 100, 1,000 and 10,000 ng/L Irgarol. Solid-phase extraction (SPE) was used to extract Irgarol and its major metabolite M1 (aka GS26575) from water samples. Sediment samples were extracted using Accelerated Solvent Extraction (ASE) with 100% dichloromethane. Irgarol and M1 were separated and quantified utilizing Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS) with ElectroSpray Ionization in Multiple Reaction Monitoring mode. Aqueous concentrations of Irgarol declined rapidly (average 93% loss) over the course of the 35 day experiment while Irgarol was accumulated in the sediments (average mass balance of 75 +/- 5%). Loss of aqueous Irgarol occurred in two distinct phases, a relatively rapid phase up to 96 hours post-dose, and a slower phase following 96 hours. The rate constants of the initial rapid degradation phase (k(1)) for treatments 100 and 1,000 ng/L Irgarol were 2-3 times higher than the rate constants of the subsequent slow degradation phase (k(2)) for these treatments. The average half-life of Irgarol in water was 7 +/- 3 days. The aqueous concentration of Irgarols metabolite, M1 increased over the course of the experiment. By 35 days post-dose, M1 concentrations in water averaged about 3% of the parent compound. M1 also accumulated in mesocosm sediments (average 16.7 +/- 2.5% of total Irgarol dose). Mass balance calculations showed that by 35 days, Irgarol and M1 amounts in mesocosm water and sediments were close to 100% of the initial Irgarol dose. Average partition coefficient normalized for organic carbon (log K(oc)) calculated for Irgarol was 3.2 +/- 0.1 for 35 days post-dose. Accumulation of Irgarol and M1 in mesocosm sediments may warrant further study to assess toxicity for benthic communities.

Using procedural blanks to generate analyte-specific limits of detection for persistent organic pollutants based on GC-MS analysis.

Several methods are used to generate a limit of detection for organic pollutants measured by gas chromatography-mass spectroscopy (GC-MS); all have theoretical and practical drawbacks. The current project investigated two common existing methods (statistical and empirical) for applicability to chromatographic properties from real samples, comparing these with a new proposed method using procedural blanks to estimate a minimum detectable peak area. Weaknesses of all three methods are discussed. The proposed method was superior to other examined methods in that it provided analyte-specific limits of detection linked to the recovery of mass-labeled internal standards for every analyte within every sample. Other identified quality assurance benefits included the following: enhanced protection against false positives; providing a sensitivity performance metric across batch, analyst, and instrument; enabling chemists with discretionary decisions specific to every analyte regarding detectability and interferences; and some strengths of both statistical and empirical techniques without major drawbacks of either. In marine sediment samples, the proposed method of calculating the limit of detection increased reporting of trace level (low- to subppb) GC-MS data for polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs), and polycyclic aromatic hydrocarbons (PAHs) by up to 400% compared with the statistical method.

Baseline monitoring of organic sunscreen compounds along South Carolina's coastal marine environment

Organic ultraviolet filters (UV-F) are increasingly being used in personal care products to protect skin and other products from the damaging effects of UV radiation. In this study, marine water was collected monthly for approximately one year from six coastal South Carolina, USA sites and analyzed for the occurrence of seven organic chemicals used as UV filters (avobenzone, dioxybenzone, octocrylene, octinoxate, oxybenzone, padimate-o and sulisobenzone). The results were used to examine the relationship between beach use and the distribution of UV-F compounds along coastal South Carolina, USA. Five of the seven target analytes were detected in seawater along coastal South Carolina during this study. Dioxybenzone and sulisobenzone were not detected. The highest concentrations measured were >3700 ng octocrylene/L and ~2200 ng oxybenzone/L and beach use was greatest at this site; a local beach front park. Patterns in concentrations were assessed based on season and a measure of beach use.

Endocrine-mediated effects of UV-A irradiation on grass shrimp (Palaemonetes pugio) reproduction

Although much is known regarding photoperiodic effects on crustacean egg production, the effects of ultraviolet (UV) light on reproduction has not been investigated. Likewise, little is known concerning the interaction between UV and xenobiotic exposure on crustacean reproductive cycles. In this study, male and female grass shrimp, Palaemonetes pugio, were exposed to sublethal concentrations of endosulfan (200 ng/l and 400 ng/l ES) under both white fluorescent (WF) and UV-A (315-400 nm) light conditions for 50 days in laboratory bioassays. Female endocrine (vitellogenin, ecdysteroids, and cholesterol), reproductive (percent gravid, clutch size), and embryo (days to hatch, hatching success, and hatching survival) responses were assessed. UV-exposure alone caused a significant (>4-fold) increase in total Palaemonetes pugio female egg production over the course of 50 days. Exposure to ES and UV significantly lowered the percentage of gravid females relative to UV controls, whereas ES-exposed shrimp under WF lighting did not exhibit these trends. Although higher vitellogenin concentrations and lower ecdysteroid titers were correlated with increased female egg production, cholesterol titers only exhibited a dose-dependent change when exposed to ES. Embryos from females exposed to UV had significantly lower ecdysteroid titers and shorter hatching times but there were no differences in embryo vitellogenin concentrations, hatching success, or hatching survival. These results indicate that UV-A exposure has a pronounced effect on grass shrimp (Palaemonetes pugio) reproduction and is likely mediated through 5-hydroxytrptamine (5-HT)-related neuroendocrine pathways. The implications for decapod aquaculture and evaluating chronic contaminant effects are discussed.

In situ monitoring of dredged material spoil sites using the oyster Crassostrea virginica

In situ and laboratory bioassays using the eastern oyster, Crassostrea virginica, were undertaken in the Wright River Estuary, South Carolina, to determine the toxic potential of effluent and sediment from recently dredged sediments. Current standards (ASTM, USEPA, and USACE) rely solely on laboratory-based bioassays to assess toxicity of dredge spoils prior to disposal. These bioassays do not necessarily replicate the natural physicochemical estuarine processes, limiting the environmental realism of this approach. In this study, oysters were collected from a site on Leadenwah Creek (SC) and deployed in plastic cages anchored above the sediment and within the intertidal zone for 90 days at four dredge spoil disposal areas (18 sites total, one bushel/site). Oysters were also deployed at a reference site (New River Estuary, SC) and the original collection site. Trace metals and polycyclic aromatic hydrocarbons (PAHs) in tissue, sediment, and effluent samples as well as the assessment of oyster health in adults (% mortality and % reduction in potential yield) and larvae (larval development) were measured. Results indicated high arsenic concentrations in surface water samples (<10 to 147 μg/L), some of which exceeded the USEPA chronic marine water quality criteria and sediment concentrations (<1.0–82.2 mg/kg), which also exceeded the ERM (70 mg/kg) and the ERL (8.2 mg/kg) for arsenic, and which may have contributed to the toxic response seen in deployed oysters. A positive relationship was also seen between the in situ percent reduction in potential yield and laboratory-derived data from larval oyster development bioassays. The advantage of the combined in situ/laboratory approach used in this study is the ability to resolve probable factors influencing the toxicity of these effluents to oysters.

POLYCYCLIC MUSK FRAGRANCES IN SEDIMENTS AND SHRIMP TISSUES

Polycyclic musk fragrances are widely used as ingredients in personal care products, shampoos, lotions, and household cleaning agents. These chemicals have relatively high octanol-water partition coefficients, and therefore tend to accumulate in sediments, sludge, and biological tissues. We analyzed shrimp and sediment samples for the presence of synthetic musks. Samples were extracted using accelerated solvent extraction. Gel permeation chromatography and solid phase extraction with silica were used to clean the extracts. The extracts were analyzed with Gas Chromatography Mass Spectrometry with Electron Impact ionization (GC-MS-EI) in selected ion monitoring (SIM) mode. Sediment samples were collected from three tidal tributaries of the Chesapeake Bay. HHCB was detected in concentrations up to 9.2 (average 1.1 ± 2.2) ng/g dry weight. Shrimp samples were collected as part of a seafood market survey of wild and farmed shrimp from the USA and other countries (Mexico, India, Equador, Thailand, China and others). Detected HHCB concentrations ranged from 48 to 683 (average 198 ± 156) ng/g lipid in farmed shrimp, and from 66 to 762 (average 334 ± 236) ng/g lipid in wild shrimp. Estimated concentrations of AHTN were up to 185 ng/g lipid in farmed shrimp, and up to 384 ng/g lipid weight in wild shrimp. HHCB was detected in all tissue samples analyzed, thus indicating the widespread distribution of this synthetic fragrance in shrimp.