Organic contaminants found in local streams

environment SADE J. RHODES1*, JOMANI CHEESEMAN3, DAMIAN CANADY4, PORCHÈ L. SPENCE2, SAUNDRA DELAUDER5, TONYA M. GERALD-GOINS1* * Corresponding authors 1. North Carolina Central University, Department of Chemistry, 3202 Mary M. Townes Science Building, North Carolina Central University Durham, NC 27707 2. North Carolina Central University, Department of Environmental, Earth and Geospatial Sciences, 2202 Mary M. Townes Science Complex, Durham, NC 27707 3. North Carolina Central University, Department of Biology, 2222 Mary M. Townes Science Building, North Carolina Central University Durham, NC 27707 4. North Carolina Central University, Department of Math and Physics, 3222 Mary M. Townes Science Complex, Durham, NC 27707 5. Delaware State University, School of Graduate Studies, Dover, DE 19901

Organic contaminants found in local streams:

Using gas chromatography-mass spectrometry approach KEYWORDS: Organic containments; estrogen; gas chromatography-mass spectroscopy; freshwater; endocrine disruptor compounds; hormones.

Abstract

Monitoring the levels of organic contaminants (OCs) in local freshwater systems is essential for sustaining healthy ecosystems and communities. OCs found in freshwater systems are suspected of disrupting normal biological pathways and mimicking endocrine disruptor compounds. The purpose of this study is to analyze OC samples from eight streams located in Durham, North Carolina. Preliminary gas chromatography-mass spectrometry results suggest that OCs are present in these streams. OCs detected includes phenol, 2, 4-bis (1,1-dimethylethyl), the antioxidant used for cosmetic formulations known as butylated hydroxytoluene (BHT), and Astaxanthin. Several detected OCs contain phenolic and methyl substituent groups, which are essential for binding estrogen receptors. Findings suggest the need to further study the effects OC’s may have on gene expression.

INTRODUCTION Clean water is valuable for sustaining healthy communities and ecosystems. Water is essential for conducting several household activities that incorporate the usage of personal care products, endocrine disrupting compounds (EDCs), and other organic compounds. When these personal care products and EDCs enter into the water supply from either direct or indirect sources, they become organic contaminants (OCs). Studies discussing the detection personal care products and endocrine disrupting compounds in drinking water throughout the United States include atenolol, atrazine, carbamazepine, estrone, gemfibrozil, meprobamate, naproxen, phenytoin, sulfamethoxazole, tris(2-carboxyethyl)phosphine (TCEP), and trimethoprim (1). Both atrazine and estrone are known as potential EDCs and are used as indicators for other organic contaminates by water treatment plants (1). Atrazine is a widely used herbicide and has been detected in surface waters containing effluent from wastewater (1). Estrone is a natural reproductive hormone that is excreted by women, men and wild animals via urine (2), and is a contributor to postmenopausal women (3). Discharged effluent from water treatment plants is a direct major source of these OCs to the environment (4). However a study conducted by Boyd et al. (5) suggest that when water is properly treated, personal care products and EDCs are removed from treated sewage and drinking water effluents. There are many methods and techniques used to analyze water samples. The EPA has developed a number of new

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methods for the detection of unregulated contaminants which includes; EPA Method 522, 537 and 538. EPA Method 522 uses gas chromatography and mass spectrometry (GC-MS), to detect the presence of 1,4-dioxane, a chemical that can irritate the eyes and digestive tract (6) and EPA Method 537 uses liquid chromatography and mass spectrometry (LC-MS), taken together to test water for perfluorinated alkyl acids (7). EPA Method 538 can be used to detect pesticides using direct aqueous injection liquid chromatography and mass spectroscopy (8). In contrast, a study was undertaken considering the determination of steroid hormones and other related compounds using gas chromatography with tandem mass spectroscopy (4). These methods and studies have proven GC-MS to be one of the most common instrumentation used for analysis of organic compounds and steroid hormones. Organic compounds (OCs, pharmaceuticals, EDCs) and site factors both have a statistically significant influence on groundwater pollution. A study was undertaken to explain the correlation between the two, resulting in the interaction between site and chemical factors, which represents the most important control on the occurrence of pesticides in groundwater (9). Some of the organic contaminants found in the streams located in Durham, NC include, Astaxanthin, butylated hydroxytoluene (BHT), ethyl iso-allocholate and calcitriol. Astaxanthin is a reddish pigment that belongs to a group of chemicals called carotenoids and has been shown to have antioxidant benefits as well as display neuroprotection against the pathological causes of Alzheimer’s disease (10). butyrate hydroxytoluene (BHT), is a common food additive and as an antioxidant at

H&PC Today - household and Personal Care today, Vol. 8(4) July/August 2013

concentrations from 0.0002% to 0.5% for cosmetic formulations (11, 12). BHT is also used to “produce proliferative pulmonary changes characterized by increased DNA, RNA, and lung weight.” Calcitriol is a bioactive form of vitamin D and is a steroid hormone known for regulating body levels of calcium, phosphorus, and mineralization of bone (13). The main sources of contamination for watersheds in the city of Durham, North Carolina is raw sewage from sanitary sewer breaks and overflows, erosion and sediment control violations, and petroleum spills and discharges (City of Durham Stormwater Services Division, 2008), The purpose of this research project is 1. to determine if OCs are present in freshwater streams located in Durham North Carolina, 2. to test if these OC’s potentially pose a threat to human health and the possible cellular responses mediated by exposure to OCs. If the stream samples indicate the presence of OCs, a second study will be conducted to determine the effects of the suspected organic contaminants on the protein expression of estrogen receptor beta expression in the neuroblastoma cell line, SH-SY5Y and breast cell line, MCF7. The significance of this research will help determine if organic contaminants measured in local streams can elicit changes in protein expression that correlate with mRNA levels. More specifically, the project will evaluate the protein level expression of estrogen receptor beta in SH-SY5Y and MCF7 cells after treatment with the suspected organic contaminants.

between June and July 2012. Samples were transported in 1L amber glass bottles and paraffin was used to secure the bottle top. Samples were acidified using sulfuric acid at a pH of 2.5. These samples were stored at 4°C until sample analysis. The samples (1L) were filtered to separate the debris from the sample and extracted twice by liquid/liquid extraction with a mixture of n-heptane and diethyl ether (80mL, 80:20 v/v). The organic extracts were dried (anhydrous sodium sulfate), rotavaporated to a volume of 2mL, and transferred into 2mL glass vials with screw caps. The stream samples were analyzed using gas chromatography-mass spectroscopy (GC-MS). GC-MS is used for the qualitative analysis of volatile compounds (organics) by comparing the GC retention time and the background corrected characteristic spectral masses with those of authentic standards (15). Analysis was carried out using an Agilent 7890A gas chromatograph directly coupled with an Agilent 5975C mass selective detector. Chromatographic separation was performed on a HP-5ms column (30 m x 0.25 mm x 0.25mm). The GC-MS method used for the samples parameter included temperature range (75-220°C), hold time (6 minutes at 220°C), injections (5mL), rate (20°C/min) and a runtime of 14.25 minutes. Quantification of organic compounds was carried out using an external standard, 17β estradiol (10-6, 10-8, 10-9M) using the previous GC-MS method. After samples were analyzed, the program “National Institute of Standards” (NIST) was used to determine the structure at each peak on the chromatogram.

MATERIALS AND METHODS RESULTS AND DISCUSSION The study area consists of two urbanized watersheds (Sandy Creek and Third Fork Creek) located in the portion of the city Onsite visualization of the Sima site exhibited lots of trash of Durham, North Carolina that drains into the Cape Fear (soda cans, potato chip bags, furniture) and little to no trees River Basin. These watersheds do not receive treated effluent surrounding the site. Elmira site contained lots of trash, from wastewater treatment plants, but have a history of vegetation, live habitation (frogs, tadpoles) and stream excessive fecal coliform levels (14). Water samples were movement was very slow. Forest Hills and Anderson site was collected from eight streams; Elmira, Sima, MLK, Forest Hill also very clean, with lots of trees surrounding the site. Park, Cornwallis, Hope Valley, Garrett, and Anderson. Criteria Cornwallis, site was very clean while Garrett, MLK and Hope for the selection of local surface sites were as follows: Valley contained vegetation but little to no pollution. availability of water samples, polluted/non-polluted sites, GC-MS results showed that organic contaminants are habitation, low-income/high-income areas in Durham NC, present in all the water sites excluding Cornwallis. surrounding North Carolina Central University (NCCU) and Duke University. Onsite visual observations were determined and the variables we chose to observe were vegetation by the water sources, pollution, and live habitation. Vegetation was considered dependent on whether the water site was shaded or not by trees; diversity of population income was geared to either low income or high income areas; pollution is whether trash was observed in the water or around the site; live habitation was considered if there was mobility in the water source by an organism. A total of 24 grab water samples were collected from 8 different Figure 1. Representative chromatograph of 17β Estradiol standard (10-8M) streams located in Durham, NC


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sites at an approximate peak area of 7.221. Shown in Figure 3, is a list of suspected organic contaminants found in the drinking and local surface water sites in Durham, NC. Of the eight sampling sites, the highest number of OC’s was detected at the Sima site. GC-MS analysis for the Cornwallis site showed no activity, this could be due to an environmentally clean site. butylated hydroxytoluene (BHT) is found in cosmetic products, this phenolic compound was seen in the Anderson and Hope Valley water sites. BHT is an Figure 2. The chromatogram is a representative GC-MS data for the sample site, Anderson. antioxidant at concentrations The major peak at 7.326 is a phenolic compound and around 12.247 are compounds that from 0.0002% to 0.5% for resemble estrogens structure cosmetic formulations (11, 12). Calcitriol, also known as Vitamin D, was found in the local surface site Hope Valley Cornwallis chromatograph showed no peaks. Elmira, Sima, and drinking water sample. It is also used for the treatment Forest Hill and Anderson contained a great number of of dermatological conditions such as rashes, acne, organic compounds, hydrocarbons and phenolic eczema, or psoriasis (18). compounds with structures that resemble estrogen. Hydrocarbons (petrochemical and rubber factories), and phenolic (gas and coke manufacture, synthetic resin manufacture, textiles), compounds are commonly found in industrial discharges 816) (16). Figure 1 shows a representative chromatograph of 17β estradiol standard (10-8M), used as an external standard in comparison to suspected organic contaminants found in water sources. In Figure 2, Anderson showed a phenolic compound (BHT) with a peak area of 9.042 minutes and compounds resembling estrogen’s structure peak area 12.122 minutes. Hope Valley also contained compounds resembling estrogen’s structure around 12.156 minutes and compounds such as calcitriol. The Elmira local surface water had vegetation surrounding the area and there was live habitation. Ethyl iso-allocholate is an antioxidant and bioactive chemical of the ethanolic extract of leaves and bark of Naringi crenulata with a peak area of 2.26 on the mass spectrum (17). This OC was also present in MLK, Anderson, Forest Figure 3. Drinking and Local Streams Organic Contaminants (OCs) Hill, Sima and Hope Valley water

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