Samuel D. Supowit, Akash M. Sadaria, Edward J. Reyes, Rolf U. Halden

Mass balance of fipronil in a wastewater treatment train and engineered wetland

GLOBAL SECURITY INITIATIVE

Fiproles

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Fipronil Sulfide Sulfone Amide Desulfinyl

Rationale • Fipronil is a high production chemical

• Banned for use on rice in China, 2009

• It has been banned for most agricultural uses in the E.U., 2013

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• Implicated in colony collapse disorder

• Highly toxic to bees

LD50 = 1-6 ng/bee

Rationale

Compound

Procambarusa Hyalella aztecab Diphetor hagenib 33 OC urban

water conc.

(µg/L)

Half-life

31 LC50 (µg/L) 30 LC50 (µg/L)

30 EC50 (µg/L)

30 LC50 (µg/L)

30 EC50 (µg/L)

34 Silt loam (d)

35 Facultative conditions (d)

Fipronil 14.3-19.5 1.3-2.0 0.65-0.83 0.20-0.57 0.11-0.21 0.05-0.39 21±0.15 -

-desulfinyl 68.6 - - - - 0.05-0.13 - 217-497

-sulfide 15.5 1.1-1.7 0.007-0.003 - - ND >200 195-352

-sulfone 11.2 0.35-0.92 0.12-0.31 0.19-0.54 0.055-0.13 0.05-0.19 >200 502-589

aProcambarus species were clarkii and zonangulus. bValues for H. azteca and D. hageni are the 95% confidence interval. OC – Orange County, California

ND – non detect

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Rationale

5 http://www.actbeyondtrust.org/wp-content/uploads/2013/07/IUCN2013sympo03_sluijs.pdf

Rationale

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• Plants uptake and translocate pesticides through their xylem, providing an indirect route of exposure to non-target foragers and pollinators

Rationale

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• Plants uptake and translocate pesticides through their xylem, providing an indirect route of exposure to non-target foragers and pollinators

Rationale • Fiprole

degradate fate in WWTPs not assessed in literature.

• Only one study assessed fipronil in influent, effluent, biosolids.

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Background

• In a prior study, Heidler & Halden (2009) determined 18 ± 22 % aqueous removal of fipronil in a conventional WWTP.

• Are similarly toxic degradates formed?

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Objective

• Perform a mass balance for fiproles over a wastewater treatment train and engineered wetland, screening for heretofore unexamined metabolites.

– Use isotope dilution and standard addition for quality control to produce high prec. data.

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Specific Aims 1. Develop analytical methods for assessing

fiproles in WWTP matrices (influent, effluent, sludge).

2. Design a sampling campaign in order to determine the fate of fiproles across primary, secondary, and tertiary treatment.

3. Perform a mass balance for fiproles over a WW treatment train and engineered wetland.

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• Fiproles are largely resistant to degradation in treatment.

Hypothesis

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• Fiproles are largely resistant to degradation in treatment.

• If parent compound “disappears,” degradates form in treatment.

• Biosolids have more sulfide.

• WAS has more sulfone.

• Wetland has more amide.

Hypothesis

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WWTP

Sampling plan • Locations

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PP

Wetland

River

= =

Primary sedimentation

basins

Secondary sedimentation

basins

Headworks Aeration

basins

PS Thickening Centrifuge

WAS Thickening Centrifuge

Acid Phase

Methane Phase

DS Thickening Centrifuge

Centrate Treatment

Disinfection

ISCO 6700 and 6712 • Incremental sampling

program to approximate flow pattern

20 mL increments at designated times

2.5 L composites

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Experimental design • Extraction (water)

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1000 mL

WAS & PS

500 mg/3 mL Strata XL 4 mL eluate x 2

LC-MS/MS

Concentrations calculated by both standard addition and isotope dilution

Experimental design

• Extraction (solids)

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Surrogate addition

Acetone extraction

Shake Centrifuge Solvent

switch to hexane

Cleanup on Florisil

Analyze by

LC-MS/MS

Method performance

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Chemical

Wastewater Solids

Spiking

level

(pg/L)

MDL

(pg/L)

Relative

recovery

(%)

Absolute

recovery

(%)

Spiking

level

(pg/g)

MDL

(pg/g)

Relative

recovery

(%)

Absolute

recovery

(%)

Fipronil 100 46 116 ± 14 60 ± 14 50 19 120 ± 13 55 ± 18

-Sulfide 300 159 N/A 67 ± 13 150 144 N/A 48 ± 18

-Sulfone 200 72 N/A 101 ± 19 100 98 N/A 89 ± 32

-Amide 500 304 N/A 87 ± 22 250 88 N/A 90 ± 21

-Desulfinyl 1000 773 N/A 78 ± 15 500 242 N/A 85 ± 15

N/A ≡ Not applicable

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Table 1. Spike levels, detection limits, and recoveries of fiproles extracted from surrogate wastewater and sludge matrices (n = 7).

Figure 1. (Right) Chromatograms of five fiproles extracted from spiked (20 ng/g nominal) and unspiked dewatered sludge, after cleanup on Florisil and elution with 4 mL DCM. Primary ion transitions are shown at top, and secondary (qualitative) transitions at bottom. *Fipronil-desulfinyl was analyzed by GC-MS/MS.

ESI negative mode C8 column

Sampling

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Sampling

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Sampling

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Results

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Figure 2. Concentrations of fiproles in (A) WWTP influent, (B) WWTP effluent (wetland influent), (C) wetland effluent, and (D) biosolids. Biosolids concentrations are normalized to 1 g dry weight. Error bars represent max and min values for water streams (n = 2), and standard deviation for biosolids (n = 3).

Co

nce

ntr

atio

n (

ng/

L)

WWTP influent

WWTP effluent

Wetland effluent

Biosolids

Results

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Figure 3. Fiprole mass distribution in three WW streams. The most abundant congener in all three streams is fipronil. The amide and desulfinyl degradates were not detected in these streams.

Results – parent compound mass balance

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1.1 ± 0.1% adsorbed to WAS

25 ± 3% transformed 74 ± 3% passed

through to disinfection

basin effluent

Fipronil mass balance over treatment train Fipronil mass balance over wetland

44 ± 4% transformed or

accumulated

56 ± 4% passed through

Figure 4. Fipronil mass balance over treatment train from primary treatment to disinfection (left) and engineered wetland (right).

Accounted for by degradates

Not accounted for by degradates

Results – total fiproles over treatment train

77 ± 11 73 ± 11 83 ± 24

0.09 68 ± 6 1.4 ± 0.003

Qx ≡ Combined flow from other treatment trains

Figure 5. Treatment train total 5-day fiprole load in mmol.

Results – individual fiproles

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Figure 6. Fiprole mass loads (in mmol) in wastewater streams over the course of five days. Direction of water flow is from left to right, (primary influent to disinfection basin effluent). Error bars represent high and low values from two experimental replicates. The bars on top are enlarged portions of the histogram on the bottom, in order to make fipronil-desulfinyl masses visible. Fipronil-desulfinyl concentrations are estimated, near the detection limit. Sludge streams are omitted, as their mass contributions are negligible (n = 2 ). m

mo

l

Results

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Figure 7. (A) Average daily mass loads of fiproles over five days, where error bars represent standard deviations (n = 10). (B) Daily mass loads of wetland (WL) influent and effluent streams on days 1 and 5, respectively, where error bars represent max/min values (n = 2); the hydraulic retention time of the wetland was 4.7 days. The right-hand y-axis is expressed as grams of fipronil per day.

47 ± 13% total fiprole reduction

No discernable change

Discussion • Total fiprole mass discharge = 7.9 Σf g/day (into wetland)

= 6.3 lb/yr

Calculating annual mass discharge

20𝑛𝑔𝐿

× 3.785 𝐿𝑔𝑎𝑙

× 106𝑔𝑎𝑙𝑀𝐺

× 75𝑀𝐺𝑑

× 365𝑑

× 10−12𝑘𝑔𝑛𝑔

× 2.2 𝑙𝑏𝑘𝑔

= 𝟒. 𝟔 𝒍𝒃

Discussion

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• The entire volume of AG fipronil in the U.K. during peak use was about 124 kg/yr (273 lb/yr)

• The estimated, extrapolated discharge by US WWTPs is 520 kg/yr (1140 lb/yr)

Discussion

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While the amount of fipronil inadvertently discharged into the environment in the form of treated wastewater is alarmingly high, it is unclear how wastewater contributes to the fiprole pollen loads in angiosperms, the body burdens of aquatic organisms, or the toxicological effects for other non-target organisms. Further research is needed to link the fiprole load in wastewater effluents to plant uptake and non-target organism exposure and effects.

Conclusions

•Conventional wastewater treatment is not efficient at removing fiproles.

•Reduction in parent compound mass may coincide with degradate formation (sulfone, in particular).

•Total fiprole levels re-entering the environment from wastewater treatment are toxicologically relevant and may impact biota.

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Future research needed

• Modeling uptake of fiproles in plants and food chain

• Risk assessment needed in order to determine ecotoxicological effects

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Acknowledgements

• Dr. Rolf Halden, PI

• Dr. Arjun Venkatesan

• Akash Sadaria

• Edward Reyes

• Top secret collaborators

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Questions

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