Mass balance of fiprole pesticides over a conventional wastewater treatment train and engineered wetland

Samuel D. Supowit, Akash M. Sadaria, Edward J. Reyes, Rolf U. Halden The Biodesign Institute at Arizona State University, Center for Environmental Security 781 E. Terrace Mall, Tempe, AZ 85287-5904

Study Objectives

Determine the fiprole mass flow into and out of a conventional wastewater treatment train. Determine the distribution of fiproles in the various wastewater streams. Perform mass balances over primary treatment, secondary treatment, disinfection, the entire treatment train from primary to tertiary treatment, and the engineered wetland that serves as quaternary treatment. Assess the efficacy of conventional wastewater treatment in the removal of fiprole pesticides.

Calculations

Conclusions

•Conventional wastewater treatment is not efficient at removing fiproles.

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

•Fipronil conversion into fipronil sulfone most likely occurs primarily in aerobic reactors, including the aeration basins and engineered wetland.

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

Acknowledgements Support for this research comes from the National Institute for Environmental Health Sciences (NIEHS), award numbers R01ES015445 and R01ES020889. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS.

Abstract

Fipronil is a phenylpyrazole insecticide used in a variety of pest control products, including agricultural applications, termiticides, flea and tick treatment, and roach and ant bait. It has been implicated as a potential contributor to colony collapse disorder in honeybee populations.1 With an LD50 of 4-5 ng/bee, fipronil is extremely toxic to bees and other invertebrates. It is also toxic to non-target vertebrates, including fish and gallinaceous birds.2 A study in Madagascar indicated that birds and lizards are exposed to fipronil and its degradates (known collectively as “fiproles”) through the food chain, leading to various sub-lethal effects.3 Sub-lethal effects to birds, lizards, and most fish include genotoxic, and cytotoxic effects as well as growth and reproduction inhibition. Effective concentrations (96-hr EC50) for some sensitive non-target invertebrates, such as C. dilutus (mosquito) and H. azteca (pill bug), range from 30-730 ng/L, and sub-lethal effects are therefore expected among a proportion of these susceptible populations at lower concentrations still.4 Due to the persistence of fiproles, we hypothesized that treated municipal wastewater might be a source of inadvertent re-introduction of these compounds into the environment. This study investigated the efficacy of a conventional wastewater treatment train and engineered wetland for the removal of fiproles. An array of automated samplers programmed for flow-weighed sampling was deployed to sample the streams across the treatment train unit operations, including the primary clarifiers, secondary treatment (collectively, the aeration basins and secondary clarifiers), the disinfection basin, and engineered wetland downstream of the plant. Sampling was carried out over five consecutive days. In order to close a mass balance over the unit operations, the water from the various streams was extracted via solid phase extraction, the solids via solid-liquid extraction, and the extracts were subsequently analyzed by liquid chromatography-tandem mass spectrometry. Total fiprole influent concentrations were 29.2 ± 6.3 g/L. Mean influent and effluent mass loads of total fiproles from primary to tertiary treatment (chlorination) were not significantly different, although the mass of the parent compound, fipronil, was reduced at a rate of about 25%. Aqueous removal of total fiproles across the wetland (with combined influent flows from several treatment trains) ranged from 29-40%, with an influent concentration (from the treated wastewater stream) of 27.7 ± 8.9, levels that are within an order of magnitude of the EC50 for numerous non-target invertebrates. The results indicate that treated municipal wastewater is a source of inadvertent re-introduction of potent, recalcitrant pesticides to the environment.

Methodology – Sampling Campaign

Figure 1. Contiguous three week average daily flow pattern for a period prior to deploying samplers (n = 21). Samples are flow-weighed composites.

Flow-weighted sampling by predicting flow patterns.

Sampler placement for treatment train mass balance

Figure 2a. Layout of wastewater treatment train and control volume for a mass balance. Sampling locations are indicated by stars. Qx is the combined flow from other treatment trains.

Sampler placement for wetland mass balance

Figure 2b. Samplers were placed at the influent and outfall of an engineered wetland in order to perform a mass balance.

0.0

0.4

0.8

1.2

1.6

2.0

12 AM 4 AM 8 AM 12 PM 4 PM 8 PM 12 AM

Ave

rage

rat

io o

f h

ou

rly

flo

w

to d

aily

me

an f

low

Average hourly flow patterns WWTP influent Wetland influent Wetland effluent

Methodology – Analytics

500 mL Solvent switch to 1:1 H2O:MeOH

LC-MS/MS MeOH Water

Load 500 mL

Sorbent

Impurities Target

Wash

Elute 0.1% formic acid

in MeOH

Solid phase extraction (500 mg/3 mL Strata X/XL)

Figure 3. Water samples are spiked with labeled Fipronil-13C215N2, and subsequently extracted via large-volume SPE, using pyrrolidine-based resin. Eluate solvents are switched to 50% MeOH in water, and analyzed via LC-MS/MS.

ISCO samplers

20 ng surrogate spike

TREATMENT TRAIN MASS BALANCE

𝑄1′𝑖𝑛𝑓 𝑡 𝐶1′𝑖𝑛𝑓 𝑡 ∆𝑡

𝑡=5

𝑡=1

− 𝑄𝐷𝐼𝑒𝑓𝑓 𝑡 𝐶𝐷𝐼𝑒𝑓𝑓 𝑡 ∆𝑡

𝑡=5

𝑡=1

− 𝑄𝑃𝑆 𝑡 𝑓𝑃𝑆𝑠𝜌𝑃𝑆𝑠𝐶𝑃𝑆𝑠 + 𝐶𝑃𝑆𝑤 𝑡 ∆𝑡 −

𝑡=5

𝑡=1

𝑄𝑊𝐴𝑆 𝑡 𝑓𝑊𝐴𝑆𝑠𝜌𝑊𝐴𝑆𝑠𝐶𝑊𝐴𝑆𝑠 𝑡 + 𝐶𝑊𝐴𝑆𝑤 𝑡 ∆𝑡

𝑡=5

𝑡=1

= 𝑚𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑

WETLAND MASS BALANCE

𝑄𝑊𝐿𝑖𝑛𝑓 𝑡 𝐶𝑊𝐿𝑖𝑛𝑓 𝑡 − 𝑄𝑊𝐿𝑖𝑛𝑓(𝑡)𝐶𝑊𝐿𝑒𝑓𝑓 𝑡 ∆𝑡

𝑡=5

𝑡=1

= 𝑚𝑊𝐿𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑

Analytes – Fiproles Fipronil Fipronil sulfide Fipronil sulfone Fipronil amide

Results

0%

25%

50%

75%

100%

Primaryinfluent

Primaryeffluent

Secondaryeffluent

Disinfectioneffluent

Wetlandinfluent

Wetlandeffluent

Fip

role

mas

s fr

acti

on

Fipronil amide Fipronil sulfide Fipronil sulfone Fipronil

Flow direction

0

2

4

6

8

10

12

Primaryinfluent

Primaryeffluent

Secondaryeffluent

Disinfectioneffluent

Wetlandinfluent

Wetlandeffluent

Ave

rage

mas

s fl

ow

(g

/d)

Fipronil Fipronil sulfone Fipronil sulfide Fipronil amide Total fiproles

WWTP control volume Wetland control volume

Figure 5. Average daily fiprole masses detected in wastewater streams. Error bars represent standard deviation from a five day average (2 experimental replicates per day).

Figure 6. Average mass distribution of individual fiproles in wastewater streams. Averages derived from 2 experimental replicates per daily sample for 5 consecutive days’ samples.

Quantitation by isotope dilution and standard addition.

Fipronil reduction in the wastewater treatment train (primary treatment through disinfection) = 24 ±10% Total fiprole removal in the wastewater treatment train ≈ 0% Fipronil reduction in the wetland = 35 ±13% Total fiprole removal in the wetland ≈ 0%

References 1. Nicodemo, D., Maioli, M. A., Medeiros, H. C. D., Guelfi, M.,

Balieira, K. V. B., De Jong, D., & Mingatto, F. (2014). Fipronil and imidacloprid reduce honeybee mitochondrial activity. Environmental Toxicology and Chemistry / SETAC, 33(9), 2070-2075. doi:http://dx.doi.org/10.1002/etc.2655

2. Tingle, C. C.; Rother, J. A.; Dewhurst, C. F.; Lauer, S.; King, W. J., Fipronil: environmental fate, ecotoxicology, and human health concerns. Reviews of environmental contamination and toxicology 2003, 176, 1-66.

3. Peveling, R.; McWilliam, A. N.; Nagel, P.; Rasolomanana, H.; Raholijaona; Rakotomianina, L.; Ravoninjatovo, A.; Dewhurst, C. F.; Gibson, G.; Rafanomezana, S.; Tingle, C. C. D., Impact of Locust Control on Harvester Termites and Endemic Vertebrate Predators in Madagascar. Journal of Applied Ecology 2003, 40, (4), 729-741.

4. Ding, Y., Weston, D. P., You, J., Rothert, A. K., & Lydy, M. J. (2011). Toxicity of sediment-associated pesticides to chironomus dilutus and hyalella azteca. Archives of Environmental Contamination and Toxicology, 61(1), 83-92. doi:http://dx.doi.org/10.1605/01.301-0014658327.2011

Automated sampler

Grab sample

Control volume

HW ≡ Headworks AB ≡ Aeration Basin

GC ≡ Grit Chamber SC ≡ Secondary Clarifiers

PC ≡ Primary Clarifiers DI ≡ Disinfection Basin

WL ≡ Wetland AD ≡ Anaerobic Digesters

Passed through 74.6%

Adsorbed to WAS solids

0.9%

Degraded 24.5%

Fipronil mass balance over treatment train

Passed through 65.0%

Accumulated or degraded

35.0%

Fipronil mass balance over wetland

Figure 7. Fate of fipronil in a wastewater treatment train (left) and engineered wetland (right) over the course of five consecutive days.

Stream Characterization

Primary influent Disinfection effluent Primary sludge (effluent)

Waste activated sludge (effluent) Reacted

Reacted and accumulated Wetland influent Wetland effluent

Sampling Campaign

0%

20%

40%

60%

80%

100%

Fipronil Fipronilsulfide

Fipronilsulfone

Fipronilamide

Primary Influent

ND ND

Fipronil Fipronilsulfide

Fipronilsulfone

Fipronilamide

Waste Activated Sludge

Aqueous Particulate-bound

ND

Fipronil Fipronilsulfide

Fipronilsulfone

Fipronilamide

Primary Sludge

ND ND ND

Figure 4. Distribution of fiproles in three wastewater streams. ND ≡ not detected.