Marco Conceptual

3.4.1 Reservoir Analysis

The contents of the reservoirs were analyzed on a number of occasions (See

Appendix E). As expected, there was variability in the constituency of the reclaimed water collected form the reclaimed water from the Integrated Water Strategies system at the Jordan Lake Business Center. Unfortunately the contents of the reservoirs could not be analyzed as regularly as would have been ideal and, thus while the volume of water applied to each plant is known (35.1L for each sweet potato plant, 8.3L for each lettuce plant; Supporting Material Z1, part of an electronic labbook available on file at UNC), the mass of each analyte applied within these volumes cannot be well estimated. It was observed, however, that samples collected from the spiked-reclaimed water reservoir one to two weeks after the spike was delivered, still had elevated levels of each compound as compared to the unspiked reservoir. The amount of elevation observed also varied and was not always as large as expected from the spike administered (designed to increase the concentration of each analyte within the

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reservoir by 10µg/L). This observation is not entirely surprising, however, for even though the reservoirs were shielded from light and moderately temperature-controlled within the refrigeration unit, they were not expected to provide a highly stable, degradation-resistant environment. Despite these limitations, an extremely crude estimate for the mass of each analyte applied to the sweet potato and lettuce plants is provided in Table 25. These

estimates are based on the average responses for the reservoir samples shown in Appendix E.

Irrigation Source Caffeine (µg) Triclosan (µg) Estradiol (ng) Caffeine (µg) Triclosan (µg) Estradiol (ng) Tap Water 3.6E+00 3.1E+00 0.0E+00 8.5E-01 7.4E-01 0.0E+00 IWS Reclaimed 1.7E+01 2.8E+01 2.4E+02 4.0E+00 6.7E+00 5.7E+01 Spiked IWS Reclaimed 1.5E+02 1.2E+02 2.7E+04 3.5E+01 2.8E+01 6.5E+03

Estimated Mass Delivered to Sweet Potatoes (35.1L)

Estimated Mass Delivered to Lettuce (8.3L)

Table 25: Estimated mass of target analytes delivered to sweet potato and lettuce based on limited reservoir analysis (Appendix E) and volume applied.

Additionally, with crop and soil samples only being taken at the end of the growing seasons, there was considerable time and opportunity for degradation processes to occur within the plant-soil system prior to harvest and before extraction. As a result of these limitations, mass balance was not pursued as a goal in this study. Rather, the extracts from the crop tissues and growing matrices were compared across treatment groups using the methods developed to determine whether significant differences in the matrix concentration and total analyte mass accounted for within each matrix were observable.

3.4.2 Crop Tissue and Growing Matrices Analysis

Extraction of crop and growing matrices, and storage of matrix extracts, were performed in the months following the crop harvests in the fall of 2011 (refer to Supporting Materials series X and Y for all details available, part of electronic labbook on file at UNC, including schedule, for the extraction of each matrix). Analysis of stored extracts was

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performed on multiple occasions during the fall of 2011 and spring of 2012, with the strengths, weaknesses and results from all analysis presented below.

3.4.2.1 Fall 2011 Trend and “Concentration” Approach

Having limited supplies, and not knowing what to expect for analyte concentrations within each matrix, it was determined that preliminary ELISA analysis would be run without full duplicate standard curves in order to investigate appropriate dilution factors and

determine trends between extracts (i.e. is extract X more or less concentrated than extract Y). Therefore, only the nil, highest and lowest analyte calibration solutions were analyzed by the ELISA kits in tandem with the experimental samples; and the responses of the experimental extracts judged on the basis of being between/above/below the quantification range of each ELISA kit. As a result of this, extracts of matrix homogenates processed for ELISA analysis can only be compared to other samples processed by the ELISA kit on the same day (i.e. without the full calibration curve we cannot make observations that definitely compare samples from one day to the next).

ELISA analysis was complete in this manner (three calibration points rather than a full calibration curve) on three days in the fall of 2011: November 28, November 29, and December 23. All “concentrations” associated with the fall 2011 findings (described in sections 3.5 and 3.6, and shown in and in the Z Appendices) were approximated by

comparing the nil, high and low standards analyzed to the suite of full calibration curves run during the entire research period, and plugging the absorbance responses for the samples into the curve that most closely matched. These “concentrations” then are not presented as hard values, but provide numerical approximations to give a slightly more thorough scrutiny to the trends observed.

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To emphasize of the limitations of this strategy, consider the following example from the analysis of triclosan run on November 29, 2011. The response of the nil, low and high calibration points on this day were equally well matched to either the full calibration curve from an analysis run on June 6, 2011 or the calibration curve run on March 8, 2012 (Figure 17).

Figure 17: Triclosan ELISA full calibration curves from June 6, 2011 and Mach 8, 2012 and high/low calibration points from November 29, 2011.

Using the three calibration point strategy, one or both of these curves would have been used to estimate the concentration of triclosan within samples run on November 29, 2011. Figure 17 demonstrates how the calibration curves from different ELISA kits, run on different days, even with nearly identical absorbance values for the highest and lowest calibration points, will exhibit different shapes within the quantitation range. As a

consequence of the different shapes, concentration estimates returned by the two curves for a sample within the quantitation range can vary significantly. For example, the absorbance value for the calibration solution #4 used to make the full calibration curve on June 6, 2011 (with known concentration 0.5µg/L) was 0.53. A sample with this absorbance on March 8, 2012, based on the calibration curve, would be expected to have a concentration of

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lowest concentration calibration solution (furthest most left) on November 29, 2011, had a lower absorbance value than either best matching curve; yet for the highest concentration calibration solution (furthest most right) on November 29, 2011 had the greatest absorbance value of all three curves. Thus it would be expected that had a full calibration curve been created on November 29, 2011, it would cross over the calibration curves from June 6, 2011 and March 8, 2012 at some unknown absorbance values “X.” Thus, the June and March calibration curves will overestimate the concentration within a sample from November with absorbance values greater than “X,” and will underestimate the concentration from a

November sample with absorbance values less than “X.” Ultimately, without knowing the shape of the kit-specific calibration curve between the high and low calibration points, concentration estimates using “best matching” curves are made very tentatively.

3.4.2.2 Spring 2012 Trend Approach

In spring 2012, as method validation studies were being further investigated (see Phase 3 investigations, section 3.2.3) stored extracts from the greenhouse experiment

(extractions executed in the fall/winter of 2011) were also analyzed. These extracts have the benefit of having been run in tandem with full duplicate standard curves, however by the time the extracts were analyzed, many had been in storage for 3-5 months and the stability of the extracts is not well known. Some samples were run both in the fall and in the spring, and while the concentration values cannot truly be compared apples to apples (recall the fall samples were not run in tandem with full calibration curves) it appears that most of these extracts gave responses of similar magnitude during both fall and spring analysis. For a few of these extracts, however, when analyzed in the fall of 2011 the analyte concentration appeared to be within the quantitation range of some ELISA kits (albeit very near the LOD),

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but when analyzed in the spring of 2012 the same extracts gave responses below the LOD, indicating that stability may be an issue.

All of the tables referring to the analysis of experimental crops grown in the

greenhouse use a 4 unit coding separated by colons to refer to the samples. Use of the key in Table 26 will facilitate an understanding of the origin of the samples being referenced in the subsequent text:

Crop Irrigation Source Growing

Matrix

Matrix Extracted and

Replicate #

(P) Sweet

Potato (G) Greenhouse tap (SA) Sand (P#) Potato

(L) Lettuce (F) Field composition

reclaimed IWS water (SO) Soil (L#) Leaves (S) Spiked-reclaimed IWS

water (S#) Sand/Soil

Table 26: Coding used to identify sample extracts from the greenhouse experiments.

Example: P:G:SO:P1 => Sweet Potato Crop, Greenhouse Tap Irrigated, Grown in Soil, Potato Tissue Extracted, Replicate #1. Recall that during extraction, replicate extracts were prepared from each homogenate. (Refer to Z and Y Series Appendices)