Applications of Bioassays for Recycled Water...Effluent RO permeate RO brine Effluent Ozone:TOC 0.25...
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Applications of Bioassays for Recycled Water Shane Snyder, Ph.D. Professor & Co-Director University of Arizona Visiting Professor National U. of Singapore California Water Boards: Recycled Water Research Workshop 27 th October 2015 – Costa Mesa
Transcript of Applications of Bioassays for Recycled Water...Effluent RO permeate RO brine Effluent Ozone:TOC 0.25...
Applications of Bioassaysfor Recycled Water
Shane Snyder, Ph.D.Professor & Co-Director
University of Arizona
Visiting ProfessorNational U. of Singapore
California Water Boards: Recycled Water Research Workshop27th October 2015 – Costa Mesa
J. Robert Oppenheimer
(1904-1967)
“It is a profound and necessary truth that the deep things in science are not found because
they are useful; they are found because it was
possible to find them.”
A Shift in Paradigm is Imminent
US EPA Already uses In Vitro Bioassays for Water Safety
Targeted vs. Untargeted
Targeted Analytical Mechanistic BioassayKnown compoundsQuantitativeIndividual compounds
Knowns/unknownsSemi‐quantitativeSynergism/Antagonism
Why Bother With Bioassays?
• The problem …– Too many chemicals– Mixture toxicity
– additive, synergistic, antagonistic
• Bioassay tools– Toxicity testing– Adverse outcome pathway– Bioanalytical tools– Water treatment monitoring
– Especially potable reuse
Why Bother With Bioassays?
• There are too many chemicals out there to quantify them one by one analytically…
• Far too many to conduct animal testing one by one… 33,000,000 animals were used in the USA alone
in a single year (1988 NAS Report) Animals not always good to mimic effects in
humans (i.e., stomach and pharmacokinetics) EPA and WHO use large uncertainty (safety)
factors because of difficulties in extrapolating
Something from nothing …
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
55.0
0.0
25.0
50.0
75.0
100.0
% effe
ct
Backhaus et al. (2008) in Kummerer (Ed.) Pharmaceuticals in the Environment, pp. 257-276
10 similarly acting toxicants on bacterial luminescence
Bioassay Endpoints
Las Vegas – 1985 Return Flow Credit Established:
Water returned to the Colorado earns credit to withdraw an equivalent amount of drinking water
1999 Environ. Sci. Tech. 33(16) 2814-2829
2001 Environ. Sci. Tech. 35(18) 3620-2625
Next Generation Bioassays
Cellular Bioassays
Glucocorticoids (GR)
Glucocorticoids (GR)
• Natural & Synthetic
• Used for human diseases such as severe allergies, skin problems, asthma, and arthritis
• Used as veterinary medicine to restore muscle strength and as growth promoters to increase muscle size
Santa Cruz River Sampling Sites
01020304050607080AN
Outf.
Cam. d. Cer.
Belm
ont
Belm
ont
Belm
ont
Ina Outf.
Cortaro
Twin P.
FBAv
ra Valley
Sand
ers
Trico M.
LB
DexaEQ (n
g/L)
Sample Site
Site Triplicate
Flow Direction
GR Cellular Activity
GR Mass Spectrometric Analysis5x10
0
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0.9
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1.1
1.2
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2.9
Cpd 32: Fluocinonide: -ESI MRM Frag=380.0V CF=0.000 DF=0.000 [email protected] (553.2000 -> 375.2000) 20140927_010_Cal_50ppb+IS.d
14.892
1 1
Counts vs. Acquisition Time (min)1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5
Triamcinolone
Aldosterone
Prednisolone
Hydrocortisone
Prednisone
Cortisone
Methylprednisolone
Betamethasone
Dexamethasone
Corticosterone
Flumethasone
Beclomethasone
Triamcinoloneacetonide
Flunisolide
Fluocinoloneacetonide
Fludrocortisoneacetate
Fluorometholone
Deflazacort
BudesonideR (+), S (−)
Spironolactone
Fluocinonide
Deoxycorticosteroneacetate
Amcinonide
Clobetasolpropionate
Fluticasonepropionate
Mometasonefuroate
Beclomethasonedipropionate
Clobetasonebutyrate
Baseline Separation for Epimers:Betamethasone vs Dexamethasone
Separation for Chiral Isomers:R (+) and S (−) Budesonide
PrednisoloneHydrocortisonePrednisoneCortisoneMethylprednisoloneBetamethasoneDexamethasoneFlumethasoneTriamcinolone acetonideFluocinolone acetonideFludrocortisone acetateBudesonideClobetasol propionateFluticasone propionate
0
1
2
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GV Raw eff GV O3 GV Cl2 GV UV GV O3/UV GVUV/H2O2
Con
cent
ratio
n (n
g/L
)Distribution of GRs
Prioritize GCs monitoring effort
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1-1 1-2 2-1 2-2 3 4 1-1 1-2 2-1 2-2 3 4
Chemical concentration (ng/L) Dex-EQ concentration (ng/L)
WWTP secondary effluents Fluticasone propionateClobetasol propionateFluocinonideBudesonideFluocinolone acetonideTriamcinolone acetonideDexamethasoneBetamethasone
6α-methylprednisoloneCortisoneHydrocortisonePrednisolone
Bioassay based prioritization monitoring effort help selecting the most important and health related chemicals
Mass-Activity Balance
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1-1 1-2 2-1 2-2 3 4
Glucocorticoid Concentrationsin WWTP effluents (ng/L)
Fluticasone propionate
Clobetasol propionate
Fluocinonide
Budesonide
Fluocinolone acetonide
Triamcinolone acetonide
Dexamethasone
Betamethasone
Methylprednisolone
Cortisone
Hydrocortisone
Prednisolone -5
10
25
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100
115
1.E-13 1.E-12 1.E-11 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06
% D
EX
Max
Concentration (M)
Cortisone
Prednisolone
Hydrocortisone
Methylprednisolone
Betamethasone
Fluocinonide
Dexamethasone
Triamcinolone acetonide
Budesonide
Fluocinolone acetonide
Clobetasol propionate
Fluticasone propionate
Glucocorticoid activity potency
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Efflu
ent
RO
per
mea
te
RO
brin
e
Efflu
ent
Ozo
ne:T
OC
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Ozo
ne:T
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Ozo
ne:T
OC
1.0
Pre-
Chl
orin
atio
n
Post
-Chl
orin
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Dec
hlor
inat
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Efflu
ent
UV
20
mJ/c
m2
UV
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mJ/c
m2
UV
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mJ/c
m2
UV
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mJ/
cm2
Efflu
ent
MF
RO
UV
Ozo
ne
Efflu
ent
WWTP1-1 WWTP1-2 WWTP2-1 WWTP2-2 WWTP3 WWTP4
Dex
amet
haso
n E
quiv
alen
t Con
cent
ratio
n (n
g/L
)
Chemi-EQBioassay-EQ
Chemical analysis match with toxicological effect
Concentrations Activities
Metabolomic Approach
F. Zhao et al. / Toxicology and Applied Pharmacology 271 (2013) 72–77
Our Approach – Beas 2B w/wo H1F1A knockout
Where does this leave us?
1. Attempts to reduce all substances to less than detectable are FUTILE and energy intensive.
2. There is ZERO possibility that the current paradigm used for risk assessment/regulatory determination will be able to address those substances already identified, let alone the unknowns.
3. Chemicals will ALWAYS exist as complex mixtures, yet toxicity tests are conducted with single chemicals and high‐doses.
4. Animal models often do not model human health.5. Rapid bioassays is the ONLY answer for comprehensive evaluation of water toxicity.
Critical Research Needs1. Most procedures rely on SPE/concentration
a) Highly polar, volatile, and hydrophobic difficult
b) pH of sample impacts ionization/extractionc) 1,4-dioxane, perchlorate, and NDMA
examples2. More QA/QC required for bioassays
a) Lack of surrogates for bioassaysb) Nontargeted analytical limited by
ion/volatility/conditions in generalc) Conducting automated SPE with
microfluidic transfers should be explored
Critical Research Needs1. More validation of S&I approach at full-scale2. More consideration of sampling bias
a) Diurnal, location, holding times, etc.b) Especially in regard to bioassay methods
3. Rational discussion regarding appropriate detection limits to better optimize methods
4. Screening of RO Brines for initial characterization of contaminant occurrencea) ≈5-10x concentration of source waterb) Passive samplers for bioaccumulation
studies and composite samples for hydrophilic contaminants
Contact: [email protected] Us: snyderlab.arizona.edu
