Doris E. Vidal-Dorsch

Contaminants of emerging concern in municipal wastewater effluents and marine receiving water

The occurrence and concentrations of contaminants of emerging concern (CECs) were investigated in municipal effluents and in marine receiving water. Final effluent from four large publicly owned treatment works (POTWs) and seawater collected near the respective POTW outfall discharges and a reference station were collected quarterly over one year and analyzed for 56 CECs. Several CECs were detected in effluents; naproxen, gemfibrozil, atenolol, and tris(1-chloro-2-propyl)phosphate were the compounds most frequently found and with the highest concentrations (>1 µg/L). Gemfibrozil and naproxen had the highest seawater concentrations (0.0009 and 0.0007 µg/L) and also were among the most frequently detected compounds. Effluent dilution factors ranged from >400 to approximately 1,000. Fewer CECs were detected and at lower concentrations in seawater collected from the reference station than at the outfall sites. Effluent concentrations for some CECs (e.g., pharmaceuticals) were inversely related to the degree of wastewater treatment. This trend was not found in seawater samples. Few temporal differences were observed in effluent or seawater samples. Effluent CEC concentrations were lower than those currently known for chronic toxicity thresholds. Nevertheless, the evaluation of potential chronic effects for CECs is uncertain because aquatic life toxicity thresholds have been developed for only a few CECs, and the effluent and seawater samples had compounds, such as nonylphenol, known to bioaccumulate in local fish. Additional data are needed to better understand the significance of CEC presence and concentrations in marine environments.

Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray

Background Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. Objectives We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. Methods We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. Results Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. Conclusions The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Organic contaminants of emerging concern in sediments and flatfish collected near outfalls discharging treated wastewater effluent to the Southern California Bight

To investigate the occurrence and bioaccumulation of organic contaminants of emerging concern (CECs) near four major wastewater ocean outfalls in the Southern California Bight, more than 75 pharmaceutical and personal care products, current-use pesticides, and industrial/commercial chemicals were analyzed in sediment and liver tissues of hornyhead turbot (Pleuronichthys verticalis) using gas and liquid chromatography-mass spectrometry. Although most CECs targeted were infrequently detected or not detectable, triclosan, 4-nonylphenol (4-NP) and bis(2-ethylhexylphthalate) were detected in all sediments at median (maximum) concentrations of 5.1 (8.6), 30 (380), and 121 (470) µg/kg, respectively. In the liver, 4-NP and polybrominated diphenyl ether (PBDE) congeners 47 and 99 were detected in >90% of samples at median (maximum) concentrations of 85 (290) and 210 (480) µg/kg, respectively. The sedative diazepam was detected in all liver samples, but was infrequently detected in sediments. Sediment and liver concentrations across outfall locations ranged over several orders of magnitude and were elevated relative to a reference site. Relative to sediment, accumulation in liver of PBDEs 47 and 99 was comparable to that for legacy organochlorines, confirming their high bioaccumulation potential and suggesting their inclusion in future tissue monitoring studies. Mean tissue PBDE and diazepam concentrations were higher in livers from male versus female P. verticalis, suggesting that gender differences also be considered in designing such studies.

Comparison of national and regional sediment quality guidelines for classifying sediment toxicity in California

A number of sediment quality guidelines (SQGs) have been developed for relating chemical concentrations in sediment to their potential for effects on benthic macroinvertebrates, but there have been few studies evaluating the relative effectiveness of different SQG approaches. Here we apply 6 empirical SQG approaches to assess how well they predict toxicity in California sediments. Four of the SQG approaches were nationally derived indices that were established in previous studies: effects range median (ERM), logistic regression model (LRM), sediment quality guideline quotient 1 (SQGQ1), and Consensus. Two approaches were variations of nationally derived approaches that were recalibrated to California-specific data (CA LRM and CA ERM). Each SQG approach was applied to a standardized set of matched chemistry and toxicity data for California and an index of the aggregate magnitude of contamination (e.g., mean SQG quotient or maximum probability of toxicity) was calculated. A set of 3 thresholds for classification of the results into 4 categories of predicted toxicity was established for each SQG approach using a statistical optimization procedure. The performance of each SQG approach was evaluated in terms of correlation and categorical classification accuracy. Each SQG index had a significant, but low, correlation with toxicity and was able to correctly classify the level of toxicity for up to 40% of samples. The CA LRM had the best overall performance, but the magnitude of differences in classification accuracy among the SQG approaches was relatively small. Recalibration of the indices using California data improved performance of the LRM, but not the ERM. The LRM approach is more amenable to revision than other national SQGs, which is a desirable attribute for use in programs where the ability to incorporate new information or chemicals of concern is important. The use of a consistent threshold development approach appeared to be a more important factor than type of SQG approach in determining SQG performance. The relatively small change in classification accuracy obtained with regional calibration of these SQG approaches suggests that further calibration and normalization efforts are likely to have limited success in improving classification accuracy associated with biological effects. Fundamental changes to both SQG components and conceptual approach are needed to obtain substantial improvements in performance. These changes include updating the guideline values to include current use pesticides, as well as developing improved approaches that account for changes in contaminant bioavailability.

Molecular Analysis of Endocrine Disruption in Hornyhead Turbot at Wastewater Outfalls in Southern California Using a Second Generation Multi-Species Microarray

Sentinel fish hornyhead turbot ( Pleuronichthys verticalis ) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences.

Development and evaluation of sediment quality guidelines based on benthic macrofauna responses.

Toxicity-based sediment quality guidelines (SQGs) are often used to assess the potential of sediment contamination to adversely affect benthic macrofauna, yet the correspondence of these guidelines to benthic community condition is poorly documented. This study compares the performance of 5 toxicity-based SQG approaches to a new benthos-based SQG approach relative to changes in benthic community condition. Four of the toxicity-based SQG approaches--effects range median, logistic regression modeling (LRM), sediment quality guideline quotient 1 (SQGQ1), and consensus--were derived in previous national studies in the United States, and one was developed as a regional variation of LRM calibrated to California data. The new benthos-based SQG approach, chemical score index, was derived from Southern California benthic community data. The chemical-specific guidelines for each approach were applied to matched chemical concentration, amphipod mortality, and benthic macrofauna abundance data for Southern California. Respective results for each SQG approach were then combined into a summary metric describing the overall contamination magnitude (e.g., mean quotient) and assessed in accordance with a set of thresholds in order to classify stations into 4 categories of expected biological effect. Results for each SQG approach were significantly correlated with changes in sediment toxicity and benthic community condition. Cumulative frequency plots and effect category thresholds for toxicity and benthic community condition were similar, indicating that both types of effect measures had similar sensitivity and specificity of response to contamination level. In terms of discriminating among multiple levels of benthic community condition, the toxicity-based SQG indices illustrated moderate capabilities, similar to those for multiple levels of toxicity. The National LRM, California LRM, and the chemical score index had the highest overall agreement with benthic categories. However, only the benthos-based chemical score index was consistently among the highest performing SQG indices for all measures of association (correlation, percent agreement, and weighted kappa) for both toxicity and benthos.

Genomic and phenotypic response of hornyhead turbot exposed to municipal wastewater effluents

Laboratory tests with marine flatfish were conducted to investigate associations among gene expression, higher biological responses and wastewater effluent exposure. In the present study, male hornyhead turbot (Pleuronichthys verticalis) were exposed to environmentally realistic (0.5%) and higher (5%) concentrations of chemically enhanced advanced-primary (PL) and full-secondary treated (HTP) effluents from two southern California wastewater treatment plants (WWTP). Hepatic gene expression was examined using a custom low-density microarray. Alterations in gene expression (vs. controls) were observed in fish exposed to both effluent types. Fish exposed to 0.5% PL effluent showed changes in genes involved in the metabolism of xenobiotics, steroids, and lipids, among other processes. Fish exposed to 5% PL effluent showed expression changes in genes involved in carbohydrate metabolism, stress responses, xenobiotic metabolism, and steroid synthesis, among others. Exposure to 5% HTP effluent changed the expression of genes involved in lipid, glutathione and xenobiotic metabolism, as well as immune responses. Although no concentration-dependent patterns of response to effluent exposure were found, significant Spearman correlations were observed between the expression of 22 genes and molecular and/or higher biological responses. These results indicate that microarray gene expression data correspond to higher biological responses and should be incorporated in studies assessing fish health after exposure to complex environmental mixtures.

Integrated coastal effects study: synthesis of findings.

Municipal wastewater discharges constitute a major source of contaminants of emerging concern (CECs) to coastal waters, yet uncertainty exists regarding their linkage to adverse biological effects such as endocrine disruption. Limited information is available concerning the types and fate of CECs discharged to the Southern California Bight (SCB) from municipal wastewater and their potential for ecological impacts. The present study investigated the impacts of CECs from ocean wastewater discharges on SCB fish. Concentrations of CECs were measured in effluents from four major municipal wastewater dischargers. Seawater, sediment, and hornyhead turbot (Pleuronichthys verticalis) from the discharge sites and a reference area were collected and analyzed for chemical and biological indicators. Low concentrations of pharmaceuticals, personal care products, and industrial and commercial compounds were measured in effluent. Some CECs were also detected in sediment, seawater, and fish livers near the outfalls, confirming exposure to CECs. Fish plasma hormone analyses suggested the presence of physiological effects, including a reduced stress response, altered estrogen synthesis or estrogenic exposure, and reduced thyroxine. Most fish responses were found at all sites and could not be directly associated with effluent discharges. However, concentrations of thyroxine were lower at all discharge sites relative to the reference, and estradiol concentrations were lower at three of the four outfall sites. The physiological responses found were not associated with adverse impacts on fish reproduction or populations. Interpretation of molecular and physiological measurements in field organisms such as those used in the present study is challenging because of a lack of information on baseline conditions and uncertain linkages to apical endpoints such as survival and reproduction.

Using Gene Expression to Assess the Status of Fish from Anthropogenically Influenced Estuarine Wetlands

The diverse mixture of contaminants frequently present in estuaries complicates their assessment by routine chemical or biological analyses. We investigated the use of gene expression to assess contaminant exposure and the condition of southern California estuarine fish. Liver gene expression, plasma estradiol concentrations, and gonad histopathology were used to study biological condition in longjaw mudsuckers (Gillichthys mirabilis). Metals, legacy organochlorine pesticides, PCBs, and contaminants of emerging concern were detected in sediments and whole fish. Overall gene expression patterns were characteristic to each of four sites investigated in this study. Differentially expressed genes belonged to several functional categories including xenobiotic metabolism, detoxification, disease, and stress responses. In general, plasma estradiol concentrations were similar among fish from all areas. Some fish gonads had pathologic changes (e.g., infection, inflammation) that could indicate weakened immune systems and chronic stress. The differential expression of some genes involved in stress responses correlated with the prevalence of histologic gonad lesions. This study indicates that gene expression is a promising tool for assessing the biological condition of fish exposed to environmental contaminants.

Annual and seasonal evaluation of reproductive status in hornyhead turbot at municipal wastewater outfalls in the Southern California Bight

Treated wastewater effluent containing endocrine-disrupting chemicals is discharged into the coastal waters of the Southern California Bight (SCB) daily. The present study investigated changes in indicators of reproductive health and environmental estrogen exposure in hornyhead turbot (Pleuronichthys verticalis) near wastewater outfalls. Fish were collected from discharge areas, farfield stations, and a reference location in the SCB to examine spatial and temporal patterns. Fish from the Orange County outfall farfield site were younger and less sexually mature than fish from other sites. The sex ratio was skewed in some fish from outfall sites as well as from the Dana Point reference site. However, no consistent pattern in sex ratio was present over time. Low-level induction of vitellogenin was frequently observed in male fish from all sites, suggesting widespread exposure to estrogenic compounds, but did not appear to impact reproductive function as there was no incidence of gonad abnormalities (ova-testis). Analysis of historical hornyhead turbot trawl data indicated that populations are either increasing or stable in the SCB; thus, environmental estrogen exposure was not adversely impacting fish abundance. Additional research is needed to determine the cause of the estrogenic response in hornyhead turbot and whether the source of the estrogenic compounds is a consequence of historical contamination or of ongoing sources or representative of baseline characteristic of this species.