- Conventional wastewater treatment is not efficient at removing fiproles like fipronil, with total fiprole levels remaining similar between influent and effluent. - Fipronil is reduced by about 25% during treatment, while its metabolite fipronil sulfone forms, likely in aerobic reactors. - Total fiprole levels re-entering the environment from wastewater treatment effluent are toxicologically relevant and may impact aquatic life.

1. 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
Averageratioofhourlyflow
todailymeanflow
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-13C2
15N2, 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
− 𝑄 𝐷𝐼𝑒𝑓𝑓 𝑡 𝐶 𝐷𝐼𝑒𝑓𝑓 𝑡 ∆𝑡
𝑡=5
𝑡=1
− 𝑄 𝑃𝑆 𝑡 𝑓𝑃𝑆 𝑠
𝜌 𝑃𝑆 𝑠 𝐶 𝑃𝑆 𝑠
+ 𝐶 𝑃𝑆 𝑤
𝑡 ∆𝑡 −
𝑡=5
𝑡=1
𝑄 𝑊𝐴𝑆 𝑡 𝑓 𝑊𝐴𝑆 𝑠
𝜌 𝑊𝐴𝑆 𝑠 𝐶 𝑊𝐴𝑆 𝑠
𝑡 + 𝐶 𝑊𝐴𝑆 𝑤
𝑡 ∆𝑡
𝑡=5
𝑡=1
= 𝑚 𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑
WETLAND MASS BALANCE
𝑄 𝑊𝐿 𝑖𝑛𝑓 𝑡 𝐶 𝑊𝐿𝑖𝑛𝑓 𝑡 − 𝑄 𝑊𝐿 𝑖𝑛𝑓(𝑡)𝐶 𝑊𝐿𝑒𝑓𝑓 𝑡 ∆𝑡
𝑡=5
𝑡=1
= 𝑚 𝑊𝐿𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑
Analytes – Fiproles
Fipronil Fipronil sulfide Fipronil sulfone Fipronil amide
Results
0%
25%
50%
75%
100%
Primary
influent
Primary
effluent
Secondary
effluent
Disinfection
effluent
Wetland
influent
Wetland
effluent
Fiprolemassfraction
Fipronil amide Fipronil sulfide Fipronil sulfone Fipronil
Flow direction
0
2
4
6
8
10
12
Primary
influent
Primary
effluent
Secondary
effluent
Disinfection
effluent
Wetland
influent
Wetland
effluent
Averagemassflow(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
accumulatedWetland influent Wetland effluent
Sampling Campaign
0%
20%
40%
60%
80%
100%
Fipronil Fipronil
sulfide
Fipronil
sulfone
Fipronil
amide
Primary Influent
ND ND
Fipronil Fipronil
sulfide
Fipronil
sulfone
Fipronil
amide
Waste Activated Sludge
Aqueous Particulate-bound
ND
Fipronil Fipronil
sulfide
Fipronil
sulfone
Fipronil
amide
Primary Sludge
NDNDND
Figure 4. Distribution of fiproles in three wastewater streams. ND ≡ not detected.