Cod: 2.1007
HIGH THROUGHPUT BIOANALYTICAL SCREENING OF INLAND WATERS OF SOUTHERNCALIFORNIA
A. Mehinto1, W. Lao1, D. Vandervort1, R. Mazor1, G. He2, M. Denison2, S. Vliet3, D. Volz3, K. Maruya1
Southern California Coastal Water Research Project2
University of California Davis
3
University of California Riverside
1Introduction
Monitoring water quality relies on measuring potentially harmful chemicals. As detection technology
advances and knowledge of impacts broadens, the chemical-specific risk assessment paradigm cannot
keep pace with the changing universe of chemicals. Moreover, this paradigm does little to address
mixtures that represent reality in drinking and receiving waters. High throughput bioanalytical techniques
have shown promise as screening tools for water quality monitoring. In vitro assays that respond tochemicals acting via common modes of action have been adapted for measuring bioactivity in water(Leusch et al. 2010). Several endpoints have been optimized for endocrine active chemicals (Jia et al.
2016), but hundreds of others have been investigated (Escher et al. 2014). High content in vivo methodshave also gained momentum for toxicity screening, including those utilizing embryonic life stages tovisualize and track developmental anomalies (e.g., Yozzo et al. 2013). These methods offer a morenimble, cost-effective alternative to whole animal testing. Currently, only a few in vitro assays are robustin differentiating among water quality (Mehinto et al. 2015). Benchmarking of water quality using highcontent in vivo methods is at an even less mature stage. This study applies high throughput in vitro
and in vivo bioanalytical methods to screen stream samples from southern California (USA), a highly
populated region impacted by multiple stressors.
Materials and methods
Surface water samples (4-L) from 31 streams and field blanks were collected during dry weather in2015. Sites were classified as urban, agricultural or open land based on predominant land use (Homer etal. 2001). Two 1-L aliquots from each sample were filtered and passed through Oasis HLC cartridges,
the cartridges dried and then eluted with methanol and acetone/hexane (10 mL ea). One aliquot was
exchanged to DMSO for bioanalytical screening, and the other exchanged to hexane for GC-MS analysis.
DMSO extracts were diluted in cell culture media and analyzed for estrogen and glucocorticoid receptor
activity (ER and GR, respectively) as described in Mehinto et al. (2015) and for aryl hydrocarbon
receptor (AhR) activity as described in He et al. (2013). ER and GR activity were expressed as equivalentconcentrations (ng/L) referenced to 17ß-estradiol (E2) and dexamethasone (DEX), respectively. AhR
activity was expressed relative to the maximum induction observed with 2,3,7,8-TCDD. Sample extracts
were further diluted and screened for potential impacts on embryonic development using transgenic
zebrafish (fli1:egfp) embryos as described in Yozzo et al. (2013). Newly fertilized embryos were exposedfrom 5 to 72 hours post fertilization (hpf) in 384 well plates at 28°C. At 72 hpf, survival, body lengthand pericardial area (a biomarker of cardiac defects) were quantified in live and hatched embryos with
an automated imaging system and custom software (Molecular Devices). The sample enrichment factors
for in vitro and in vivo testing were 10 and 1, respectively.
A subset of sample extracts in hexane that showed a range of in vitro responses were analyzed using anAgilent 7890/5975C GC-MS with a 30m DB-XLB column operating in the full scan (m/z 50-650) EI
mode. Resolved peaks were identified using the NIST mass spectral library.
Results and discussion
ER, GR and AhR activities in field blanks (n=7) were not detectable. ER activity was detectable in 8samples (maximum of 6.3 ng E2/L). GR activity was detectable in 2 samples at 31 and 37 ng DEX/L.
In contrast, AhR activity was detectable in 28 samples, with the 5 highest responses observed for urbansites (Fig. 1). The greater screening response observed with AhR is likely due to the greater structural
diversity of ligands that bind and activate the AhR compared to ER and GR (Denison et al. 2011). These
data provide a relative ranking of the net presence of ER, GR and AhR active chemicals in these samples.

Organohalogen Compounds

Vol. 78, (2016)835Based on endpoints measured in vivo, samples were not toxic to zebrafish embryos within the first 3 daysof development (Fig. 2). For all samples screened, 72-hpf embryo survival was >80% and no significant
effects on body length or pericardial area were detected following treatment from 5-72 hpf.
GC-MS analysis identified oxybenzone, 2,4-dichlorophenol; methyl-benzthiazole and the phosphate
flame retardants tris (2-chloroethyl)-phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCPP) andtris (1,3-dichloroisopropyl)phosphate (TDCPP) and tris (2-butoxyethyl)-phosphate (TBEP). These flameretardants were reported to be among the most abundant contaminants measured in effluent dominatedsouthern California waterways (Sengupta et al. 2014). No PAH were detected.
The mean score for the California Stream Condition Index (CSCI), an indicator of stream health basedon benthic macroinvertebrates, was negatively correlated with the AhR screening response (n=21;Rs=-0.84; p<0.01). Scatterplots of CSCI vs. in vitro screening results revealed that urban and agricultural
streams had the lowest CSCI, indicating more degraded conditions, whereas streams in open land werecharacterized by higher CSCI and lower in vitro screening responses. Together, the high throughput
in vitro and in vivo bioanalytical tools applied herein show promise as efficient screening elements inmonitoring of receiving waters.
Acknowledgements
This study was supported in part by the State of California (Agreement No. 14-090-270). We thank E.
Stein, and the members and field crews of the Southern California Stormwater Monitoring Coalition.
References:Denison, M. S., Soshilov, A. A., He, G., DeGroot, D. E., and Zhao, B. (2011) Toxicol Sci 124, 1-22.
Escher, B. I., et al. (2014) Environ Sci Technol 48, 1940–1956.
Jia, A., Wu, S., Daniels, K. D., and Snyder, S. A. (2016) Environ Sci Technol DOI: 10.1021/
acs.est.5b04893.
He, G., Zhao, J., Brennan, J. C., Affatato, A. A., Zhao, B., Rice, R. H., and Denison, M. S. (2013) In:
Optimization of Drug Discovery: In Vitro Methods, Second Edition (Yan, A. and Caldwell, G.W., Eds.),
pp. 221-235, Humana Press, New York, NY.
Homer, C., Dewitz, J., Fry, J., Coan, M., Hossain, N., Larson, C., Herold, N., McKerrow, A., VanDriel,J. N., and Wickham, J. (2007) Photogrammetric Engineering and Remote Sensing, 73, 337-341.
Leusch, F. D. L., de Jager, C., Levi, Y., Lim, R., Puijker, L., Sacher, F., Tremblay, L. A., Wilson, V. S.,and Chapman, H. F. (2010) Environ Sci Technol 44, 3853–3860.
Mehinto, A. C., et al. (2015) Water Res. 83, 303–309.
Sengupta, A., Lyons, J. M., Smith, D. J., Heil, A., Drewes, J. E., Snyder, S. A., and Maruya, K. A. (2014)
Environ Toxicol Chem 33, 350-358.
Yozzo, K. L., Isales, G. M., Raftery, T. D., and Volz, D. C. (2013) Environ Sci Technol 47, 11302-11310.
Fig. 1. In vitro response (% of max induction) for water samples using the aryl hydrocarbon receptor(AhR). Stream sites (n=31) were classified by watershed land use.
Fig. 2. High-content screening results using zebrafish (Danio rerio) embryos. A) survival; B) body length;C) pericardial area.

Organohalogen Compounds

Vol. 78, (2016)836

Organohalogen Compounds

Vol. 78, (2016)

837