es9022706 si 001

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Supporting Information for

Antidepressant Pharmaceuticals in Two U.S. Effluent-Impacted Streams:

Occurrence and Fate in Water and Sediment, and Selective Uptake

in Fish Neural Tissue

Melissa M. Schultza, Edward T. Furlong

b,*, Dana. W. Kolpin

c, Stephen L. Werner

b, Heiko L.

Schoenfussd, Larry B. Barber

e, Vicki S. Blazer

f, David O. Norris

g, Alan M. Vajda

g

aDepartment of Chemistry, The College of Wooster, Wooster, OH 44691

bNational Water Quality Laboratory, U.S. Geological Survey, Denver, CO 80225

cU.S. Geological Survey, Iowa City, IA 52244

dAquatic Toxicology Laboratory, St. Cloud State University, St. Cloud, MN 56301

eU.S. Geological Survey, Boulder, CO 80303

fNational Fish Health Research Laboratory, Kearneysville, West Virginia 25430

gDepartment of Integrative Physiology, University of Colorado, Boulder, CO 80309

*Corresponding author, phone (303) 236-3941, FAX (303) 236-3499;

email: [email protected]

For Environmental Science and Technology

13 pages, 3 figures, 4 tables

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Standards and Reagents. Standards of fluoxetine hydrochloride (pure material) and

paroxetine hydrochloride (pure material) were purchased from United States Pharmacopeia

(Rockville, Md.). Sertraline hydrochloride (>98%), norsertraline (also known as N-desmethyl

sertraline; >98%), duloxetine hydrochloride (>98%), D,L-venlafaxine hydrochloride (>98%),

bupropion hydrochloride (>98%), and the internal standard sertraline-d3 hydrochloride (>98%)

were acquired from Toronto Research Chemicals (Toronto, Ontario). Norfluoxetine

hydrochloride (97%), citalopram hydrobromide (>99%), fluvoxamine maleate (98%), and the

labeled surrogates fluoxetine-d5 hydrochloride (>98%) and norfluoxetine-d5 hydrochloride

(>98%) were obtained from Sigma Aldrich (St. Louis, Mich.).

Quality Assurance/Quality Control. Multiple quality assurance/quality control

measures were incorporated into this study to ensure data quality and are summarized below.

Sample Preservation: All samples were stored frozen until analysis to prevent degradation. The

sediment and water samples were frozen at -20!C. The brain tissues were stored at -80

!C until

analysis.

Blanks: Since the hypothesis of this study was that wastewater treatment is a point source of

antidepressants into the environment, the water, sediment, and brain tissue collected upstream of

the effluent discharge constitute, in essence, environmental “blank” samples, that is, negative

controls. At both study sites, physical controls (low-head dams) exist that effectively separate

unexposed and exposed fish populations. True field blanks consisting of nanopure water were

exposed to the same field conditions and were shipped with the collected environmental samples.

Method blanks were also run with each batch of water and sediment samples, with no

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antidepressants were detected. Instrument reagent blanks were also analyzed every five samples

within a batch to ensure no chromatographic carryover during instrumental analysis.

Analytical method performance: For water and sediment analyses, laboratory reagent spike

(LRS) samples were analyzed along with every 10 environmental samples to estimate method

performance during sample extraction and analysis. The LRS samples consisted of nanopure

water, or ashed quartz sand that were amended with known amounts of the antidepressants.

Recoveries from the LRS samplers provided an estimate of method performance in the absence

of sample matrix.

Replication: As stated in the manuscript, replicate samples were collected at each sampling site.

The 2005 water sampling campaign collected samples in triplicate at each site; duplicates were

collected in 2006. There was little bed sediment present in Boulder Creek, so duplicate samples

were collected. For fish tissue sampling at Boulder Creek in 2005, replicates were n=4 and n=6

upstream and downstream, respectively. The sample size ranged from 11 to 20 at each of the

Fourmile Creek sites. Note that these are all field replicates; the results of which encompass

combined field and analytical variation.

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F

F

F

O NH

F

F

F

O NH2

Cl

Cl

NH

Cl

Cl

NH2

O

O

NH

F

O

N

O

F

N

SNH

O

CH3

N

OH

O

F

F

F

O

N

O

NH2

Cl

O

NH

Fluoxetine Norfluoxetine

Sertraline Norsertraline

Paroxetine Citalopram

Duloxetine Venlafaxine

Fluvoxamine Bupropion

Figure S1. Chemical Structures of Target Antidepressants.

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Figure S2. The Boulder Creek Watershed, Colorado.

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Figure S3. The Fourmile Creek Watershed, Iowa.

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Table S1. Recommended Daily Dose of Studied Antidepressants

Drug Drug

Acronym

Daily Defined

Dose1

(mg/day)

Number of U.S.

Prescriptions

Dispensed in 20052

Estimated Annual

Mass Useda (g)

Fluoxetine FLX 20 N/Ab N/A

Norfluoxetine NFLX N/A N/A N/A

Sertraline SER 50 26 976 000 40 464 000

Norsertraline NSER N/A N/A N/A

Paroxetine PRX 20 3 609 000 2 165 400

Citalopram CIT 20 N/A N/A

Escitalopramc 10 24 788 000 7 436 400

Fluvoxamine FLV 100 N/A N/A

Bupropion BUP 300 11 044 000 99 396 000

Venlafaxine VEN 100 17 179 000 51 537 000

Duloxetine DLX 60 4 938 000 8 888 400 aEstimated annual mass was calculated by multiplying the daily defined dose by 30 days and the

number of prescriptions dispensed.

bNot available or not applicable: Numbers of U.S. prescriptions not available in reference 33, or

compound is a degradate. Thus, an estimated annual mass used could not be calculated. cEscitalopram cannot be differentiated from citalopram with this analytical method; thus, in this

study is reported as citalopram.

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Table S2. Individual Concentrations (ng/g) of Antidepressants in Boulder Creek White Sucker Brains Collected in 2005.

Sitea and

sample ID

FLX NFLX SER NSER PRX CIT FLV BUP VEN DLX

BC 1 fish 1 ND ND ND ND ND ND ND ND ND ND




BC 2 fish 1 0.454 0.342 2.010 2.279 <LOQ 0.025 ND 0.015 ND ND

BC 2 fish 2 0.292 0.216 0.987 1.426 <LOQ 0.024 ND <LOQ ND ND





limit of quantitation (LOQ) = 0.015 ng/g, ND = no detect aOfficial USGS station IDs and locations are shown in Figure S2

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Table S3. Individual Concentrations (ng/g) of Antidepressants in Fourmile Creek White Sucker Brains Collected in 2005. 6

7

Sitea and

sample ID


FMC 1 fish 1 <LOQ <LOQ ND ND ND ND ND ND ND ND

FMC 1 fish 2 ND ND ND ND ND ND ND ND ND ND




FMC 1 fish 6 <LOQ ND ND ND ND ND ND ND ND ND




FMC 1 fish 10 <LOQ ND ND ND ND ND ND ND ND ND

FMC 1 fish 11 ND <LOQ ND ND ND ND ND ND ND ND

FMC 1 fish 12 ND <LOQ ND ND ND ND ND ND ND ND


FMC 1 fish 14 <LOQ <LOQ ND ND ND ND ND ND ND ND

FMC 2 fish 1 0.472 3.567 4.149 28.902 0.113 0.068 ND 0.154 ND ND

FMC 2 fish 2 0.178 0.449 0.188 0.849 0.028 0.021 ND 0.073 ND ND

FMC 2 fish 3 0.140 0.984 1.007 3.998 0.047 ND ND 0.066 ND ND

FMC 2 fish 4 0.064 0.203 0.239 1.141 ND 0.016 ND <LOQ ND ND

FMC 2 fish 5 0.089 0.218 0.310 1.212 ND 0.020 ND 0.019 ND ND

FMC 2 fish 6 0.070 0.247 0.613 1.509 <LOQ <LOQ ND 0.022 ND ND

FMC 2 fish 7 0.050 0.124 0.456 0.967 0.021 0.033 ND 0.035 ND ND

FMC 2 fish 8 0.055 0.542 0.238 0.841 <LOQ 0.030 ND 0.040 ND ND

FMC 2 fish 9 0.115 0.513 0.404 1.399 0.018 0.051 ND 0.021 ND ND


FMC 2 fish 11 0.023 0.142 0.382 1.546 0.023 <LOQ ND 0.015 ND ND

FMC 2 fish 12 0.275 0.210 0.171 0.844 0.082 0.032 ND 0.018 ND ND

limit of quantitation (LOQ) = 0.015 ng/g, ND = no detect 8 aOfficial USGS station IDs and locations are shown in Figure S3 9

10

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Table S3. Individual Concentrations (ng/g) of Antidepressants in Fourmile Creek White Sucker Brains Collected in 2005—10

Continued. 11

12

Sitea and

sample ID FLX NFLX SER NSER PRX CIT FLV BUP VEN DLX

FMC 2 fish 13 0.221 0.587 0.445 2.449 0.015 0.022 ND 0.042 ND ND

FMC 2 fish 14 0.100 0.392 0.727 2.457 0.015 0.020 ND 0.026 ND ND

FMC 2 fish 15 0.193 1.261 0.685 2.687 0.015 0.054 ND 0.148 ND ND



FMC 2 fish 18 0.031 0.176 0.203 1.105 0.014 <LOQ ND 0.017 ND ND



FMC 4 fish 1 0.033 0.632 0.268 0.957 ND <LOQ ND 0.029 ND ND


FMC 4 fish 3 <LOQ 0.408 0.262 0.446 ND ND ND ND ND ND

FMC 4 fish 4 <LOQ 0.533 0.341 0.485 ND ND ND ND ND ND







FMC 4 fish 11 0.257 0.097 0.472 0.452 0.083 ND ND 0.348 ND ND


FMC 4 fish 13 0.266 0.243 0.285 0.728 0.041 0.072 ND 0.071 ND ND


FMC 4 fish 15 <LOQ 0.161 <LOQ 0.500 ND <LOQ ND <LOQ ND ND




15

16

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16

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Table S4. Individual Concentrations (ng/g) of Antidepressants in Fourmile Creek White Sucker Brains Collected in 2006. 18

19

Sitea and

sample ID







FMC 1 fish 6 0.049 ND ND ND ND ND ND ND ND ND



FMC 1 fish 9 0.021 ND ND 0.382 ND ND ND ND ND ND

FMC 1 fish 10 ND ND 0.099 ND ND ND ND ND ND ND

FMC 1 fish 11 0.160 ND ND 0.074 ND ND ND ND ND ND






FMC 1 fish 17 ND ND ND 0.030 ND ND ND ND ND ND




FMC 2 fish 1 0.491 0.132 2.190 2.626 0.026 0.195 ND ND ND ND

FMC 2 fish 2 0.223 0.095 1.805 2.298 ND 0.058 ND 0.053 1.118 ND

FMC 2 fish 3 0.150 0.092 1.890 1.759 ND <LOQ ND <LOQ ND ND



22

23

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Table S4. Individual Concentrations (ng/g) of Antidepressants in Fourmile Creek White Sucker Brains Collected in 2006—23

Continued. 24

25

Sitea and

sample ID



FMC 2 fish 6 0.233 0.137 2.360 2.532 <LOQ 0.045 ND 0.022 ND ND


FMC 2 fish 8 0.084 0.074 1.951 1.989 ND <LOQ ND ND ND ND



FMC 2 fish 11 0.175 0.287 1.282 1.110 ND 0.071 ND 0.023 0.048 ND


FMC 2 fish 13 0.188 0.061 2.327 2.478 0.044 0.149 ND 0.037 0.026 ND

FMC 2 fish 14 0.227 0.153 2.009 2.124 0.047 0.062 ND ND 0.036 ND


FMC 2 fish 16 0.109 0.144 1.351 1.724 ND ND ND ND ND ND

FMC 2 fish 17 0.194 0.311 0.995 1.355 0.045 0.074 ND 0.016 0.736 ND


FMC 2 fish 19 0.099 0.109 1.538 1.758 0.041 <LOQ ND ND ND ND


FMC 4 fish 1 0.082 <LOQ 1.579 2.068 ND ND ND ND ND ND

FMC 4 fish 2 <LOQ <LOQ 0.210 0.228 ND ND ND ND ND ND

FMC 4 fish 3 0.055 0.090 1.009 1.110 ND ND ND <LOQ ND ND

FMC 4 fish 4 0.173 0.128 1.708 2.348 ND <LOQ ND 0.178 0.069 ND

FMC 4 fish 5 0.109 0.062 1.746 2.047 ND ND ND 0.072 <LOQ ND





FMC 4 fish 10 0.391 0.195 1.410 1.950 ND ND ND ND 0.131 ND

FMC 4 fish 11 0.061 <LOQ 0.272 0.458 ND ND ND ND ND ND


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Literature Cited

1. World Health Organization ATC/DDD Index Database 2008.

http://www.whocc.no/atcddd/indexdatabase/index.php (accessed December 11, 2009).

2. Top 2005 Brand-Name Drugs by Units in 2005.

http://drugtopics.modernmedicine.com/drugtopics/Community+Pharmacy/Top-200-brand-name-

drugs-by-units-in-2005/ArticleStandard/Article/detail/311294 (accessed December 4, 2009).

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