Impact of irrigation with fipronil-contaminated waters on zucchini plants and their main insect pest, Aphis gossypii

doi:10.21203/rs.3.rs-5197843/v1

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Impact of irrigation with fipronil-contaminated waters on zucchini plants and their main insect pest, Aphis gossypii

https://doi.org/10.21203/rs.3.rs-5197843/v1

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Fipronil is a long-lasting, broad-spectrum insecticide with low residual activity and high efficacy at low concentrations. Due to its widespread use and long half-life, fipronil can persist during wastewater treatment and is found even in surface waters. When released into the environment, it can accumulate over time and can lead to concentrations in soil and plant tissues that are harmful to both target and non-target organisms. Effective removal of fipronil is crucial in agricultural settings. Prolonged environmental exposure to this insecticide may contribute to the development of pesticide resistance and cross-resistance to other insecticides used for pest control in agro-ecosystems. As the reuse of treated wastewater and sewage sludge presents challenges and opportunities for farmers, this study investigates the effects of irrigation with environmentally relevant doses of fipronil on zucchini plants and their main insect pest, Aphis gossypii. The fitness costs of A. gossypii reared on plants irrigated with fipronil and their resistance to higher doses of fipronil and to pyrethrins were investigated in the laboratory. Traces of fipronil were found in the zucchini leaves, but not in the flowers, after 35-days of cumulative exposure. A decrease in nymph-to-adult survival and a dose-dependent reduction in the fecundity of A. gossypii feeding on contaminated host plants was observed. Also, aphids that ingested fipronil from the host plant exhibited the same mortality rate as the control group when exposed to a sub-lethal dose of fipronil. However, when natural pyrethrins were used, there was a significant increase in resistance to this insecticide. Our results demonstrate the potential for fipronil to accumulate in plant tissues and highlight the risk of changes in insecticide susceptibility in insect pests. This suggests a need for a holistic approach to the complex dynamics of wastewater reuse in agriculture.

wastewater reuse

environmental risks

sustainable agriculture

pest control

aphids

life history traits

pyrethrins

pesticide resistance

cross-resistance

Fipronil is a phenyl pyrazole systemic insecticide developed by Rhone Poulenc Ag Company (now Bayer Crop Science) between 1985 and 1987 (Colliot et al. 1992) and launched in 1993 (Tingle et al. 2000). It is one of the most persistent lipophilic and toxic insecticides with contact and stomach action even at low field application rates. Foliar applications between 12.5 and 50 g of active ingredient/ha are effectively used against many insect pests (Tingle et al. 2000). Fipronil has a broad spectrum use, a long duration, and low residual activity even at low concentrations and can be used effectively against both chewing and piercing sucking insects (Colliot et al. 1992). Fipronil works by blocking the gamma-aminobutyric acid receptor in insects, disrupting the central nervous system, causing over-excitation of muscles and nerves, convulsions, paralysis, and ultimately death of the insect (Hainzl et al. 1998; Ikeda et al. 2004; Mohapatra et al. 2010; Singh et al. 2021).

Fipronil can cause severe damage to aquatic biota and can cause seizures, hyperactivity, cell damage, and neurotoxicity in fish at concentrations of 0.65 µg/L and lead to death in extreme exposure conditions (Moreira et al. 2021). Fipronil can be degraded in the environment by reduction, oxidation, hydrolysis or photolysis. For example, according to dissipation kinetics data in soil, fipronil concentrations decrease gradually, more rapidly in calcareous soil than in alluvial soil, and increase at lower temperatures (El-Aswad et al. 2024). The half-life of fipronil in soil can vary from 36 hours up to 194 days (Gunasekara et al. 2007) and its degradation is highly dependent on soil type, water content and the presence of microorganisms (Ying and Kookana 2002; Pei et al. 2004; Gunasekara et al. 2007; Bhatt et al. 2023). Metabolites can be formed, the most important of which are fipronil desulfinyl, fipronil sulfone, fipronil sulfide, and fipronil amide. These metabolites, except for fipronil amide, have been reported to be more toxic and persistent than the parent compound (Tingle et al. 2003; Gunasekara et al. 2007; Singh et al. 2021).

Fipronil has been used to protect plants against a wide range of insect pests, including Thrips tabaci, Diatraea saccharalis, Migdolus fryanus, Atta capiguara, and Oryzophagus oryzae (Ester et al. 1997; Cummings et al. 2006; Gonçalves et al. 2022). Fipronil has been shown to be effective against Pratylenchus zeae, a root lesion nematode associated with sugarcane (Kawanobe et al. 2019). This insecticide is also used to control the abundance of termites (Steinbauer and Peveling 2011) and cockroaches (Kaakeh et al. 1997). Fipronil is still used for flea and tick control on domestic animals, providing ectoparasiticidal activity for about one month (Case et al. 2016), but it is banned in animals destined for human consumption.

As a result of its long persistence and high toxicity, a wider range of non-target species are affected by fipronil in different environments. Despite being strictly regulated worldwide, fipronil affects pollinators, insect predators, parasitoids, and other non-pest insects (Stork 2018). The most studied non-target organisms affected by fipronil are honeybees (Zaluski et al. 2015; Kairo et al. 2017; Holder et al. 2018), aquatic invertebrates in surface waters, and various species of stream invertebrates, such as Chironomus dilutus and Hyalella azteca (Weston and Lydy 2014; Hapke et al. 2016). For example, exposure to less than 5.0 ng/L fipronil affected mysid growth, reproduction and survival (EPA 1996). Compared to its effect on aquatic organisms, fipronil has been shown to be extremely toxic to honeybees and even sub-lethal doses ingested throughout the foraging season can adversely affect colony development and maintenance (Zaluski et al. 2015; Kairo et al. 2017; Holder et al. 2018; Carter et al. 2020).

The European Commission published some restrictions on the use of fipronil (EUR-Lex 2014) after the European Food Safety Authority had identified a high acute risk to bees (EFSA 2013), and since 2014 fipronil has been banned on maize and sunflowers in Europe. Even without direct agricultural use, the levels of fipronil (and its main metabolites) continue to increase in the environment due to veterinary use, urban pest control, small area turf care, and indoor cockroach traps (Stork 2018; Sadaria et al. 2019). Fipronil has been found in various environmental media, including wastewater (Heidler and Halden 2009), runoff (Gan et al. 2012; Sengupta et al. 2014), and surface water (Stone et al., 2014). For example, it was recently found in treated wastewater in Korea at an approximately concentration 20 ng/L (Kim and Kim 2024). As to surface water, fipronil was recently found at high concentration (mean: 1.77 µg/L) in river waters from the Amazon basin (Cezarette et al. 2024) and at lower concentrations (0.03 µg/L) in the Aquidauana river (Finoto Viana et al. 2023).

Rapid urbanisation and water scarcity due to climate change are driving the development of sustainable water and fertiliser management in agriculture. Wastewater reuse and sewage sludge application present both challenges and opportunities for farmers, society and communities (Trotta et al. 2024a). Fipronil is persistent during conventional wastewater treatment and can be released to the environment through the recovery of treated wastewater and sewage sludge (Weston and Lydy 2014; McMahen et al. 2016; Supowit et al. 2016; Sadaria et al. 2017). The widespread presence of fipronil and its principal transformation products has been, in fact, consistently observed in wastewater (Sadaria et al. 2017, 2019). When present in irrigation water, fipronil and some of its metabolites are taken up by the roots and translocated to all parts of the plant (van der Sluijs et al. 2015). For example, 8 weeks old cabbage plants grown from fipronil-coated seeds showed concentrations of fipronil and its transformation products in their leaves less than or equal to 1 µg/kg (Gols et al. 2020). Pesticide residues can then contaminate consumed food, animal feed, soil, water and insects that feed on the plant (Assadpour et al. 2023; Trotta et al. 2024a). These chemicals accumulate in the environment and enter the food chain, with adverse effects on human health and on a wide range of non-target species.

The massive use of chemical pesticides in agriculture to increase yields is causing pests such as aphids to become resistant to their effects. This problem, together with adverse effects on non-target organisms, is mainly due to the indiscriminate use of persistent pesticides. The development of resistance is a major problem and the list of pesticides and chemicals to which pests have developed resistance is incredibly long (Devine et al. 2001; Siddiqui et al. 2023). For example, laboratory-induced resistance to fipronil has been reported in several insect like Oxycarenus hyalinipennis, Musca domestica, Plutella xylostella, and Sogatella furcifera (Sayyed and Wright 2004; Tang et al. 2010; Abbas et al. 2014; Wazir and Shad 2020, 2022).

Typically, pesticide resistance is the result of prolonged and persistent use of these substances, especially at high sub-lethal doses. The genetic variation that facilitates adaptive change in population resistant to a pesticide could be generated by gene mutation, or by epigenetic and epitranscriptomics mechanisms (Brevik et al. 2021; Chen et al. 2023). Insects can be exposed to various insecticides at concentrations as low as parts per billion through the irrigation of crops with wastewater or the application of sludge to soil. Moreover, the use of fipronil-contaminated wastewater for irrigation is a cause for concern also because fipronil accumulates in the soil (Margenat et al. 2019; Brienza and Garcia-Segura 2022; Trotta et al. 2024a). The European Parliament and the Council promote the sustainable use of wastewater to preserve water resources (EUR-Lex 2020). However, if polluted water sources are not adequately managed, prolonged environmental exposure to this insecticide could lead to the development of pesticide resistance (Bras et al. 2022) and, more problematically, cross-resistance to other insecticides used for pest control in agro-ecosystems, such as pyrethrins.

Natural pyrethrins are common insecticides widely used in organic farming against a wide range of insect pests, including aphids (Isman 2006). Pyrethrins affect both the peripheral and central nervous systems of insects by interfering with the function of sodium channels (Davies et al. 2007). One reason for the success of this group of insecticides is that the range of products registered for organic use is relatively limited. However, they have low mammalian toxicity and, unlike fipronil, are highly biodegradable in soil and water, do not leave toxic residues in plants and do not bioaccumulate in food chains (Jeran et al. 2021).

Aphis gossypii is a recurrent polyphagous pest of many cultivated crops worldwide and is considered the most important pest of cucurbits (Blackman and Eastop 2007). Its direct feeding on some plant species can cause severe damage to plant tissues (Ng and Perry 2004), but its main damage is associated with phytovirus transmission (Ebert and Cartwright 1997). This aphid is still largely controlled by a wide range of insecticides, but it is now one of the most insecticide-resistant aphid species (Mota-Sanchez and Wise 2020).

Although there are many detailed studies on the risks associated with fipronil contamination in aquatic environments, little is known about its potential effects in agricultural environments. This study examined the impact of irrigating zucchini plants with environmentally relevant doses of fipronil on their main insect pest, A. gossypii. The research focused on fipronil levels in the plants, as well as the fitness costs and changes in insecticide susceptibility of A. gossypii when reared on plants exposed to these concentrations of fipronil. As aphids feed on both zucchini flowers and leaves, the fipronil content was measured in these tissues. The detection of fipronil in the zucchini flowers is of particular interest because the flowers develop later in the plant's growth cycle (about a month after transplanting), are edible, and attract a variety of pollinators, predators and non-target insects.

Chemicals

The fipronil used in this study was obtained from the commercial product “Frontline® Spot On Gatti”, purchased from Boehringer Ingelheim Animal Health Italia S.p.A. (Milan), with the excipients as non-reactive compounds. The pyrethrins were obtained from a commercial product, “Piretro Compo®Bio”, an insecticide for organic farming, purchased from COMPO GmbH (Münster), with the excipients as non-reactive compounds.

To simulate a real-world scenario where wastewater reuse and sewage sludge are common agricultural practices, plants were irrigated with the environmentally relevant concentrations of 100 ng/L (coded as F100) of fipronil, and with a higher concentration of 1,000 ng/L (F1000) of fipronil. The F100 and F1000 concentrations were obtained by serial dilution of fipronil stock solutions in distilled water.

For the analysis of fipronil in the zucchini plants, analytical standards of fipronil and the internal standard fipronil-¹³C₂¹⁵N₂ were purchased from Cymit Quimica S.L. (Spain). Stock solutions were prepared in acetonitrile for fipronil and in methanol for fipronil-¹³C₂¹⁵N₂, with the fipronil stock solution used to prepare calibration curves through serial dilution.

Zucchini plant cultivation

Zucchini (Cucurbita pepo L.) seedlings of the cultivar ‘San Pasquale’ were used in this study. The seeds were kept for germination on wet cotton disks in sterile Petri dishes at 20 ± 2°C. After one week, the germinated seeds were sown in pots (14 cm diameter) filled with synthetic soil, consisting of a mixture of peat, perlite, sand and clay (see Trotta et al. 2024b for details). The pots were grown under controlled conditions in a climate chamber at 22 ± 1°C, 60 ± 5% relative humidity (RH) and an 18:6 Light:Dark (L:D) photoperiod until the end of the experiments. The lights used for plant growth consisted of 20-W, 130-lm/W white LED tubes (6500 K) coupled with 36-W, 100-lm/W full-spectrum LED tubes. The seedlings were subsequently watered with water plus 10 mL of 0.3% NPK (7.5-3-6 + Fe and microelements) nutrient solution PIANTE VERDI (Compo®, Ravenna, Italia). These conditions were maintained throughout the entire duration of the experiments, which lasted 35 days.

Insect rearing

The Aphis gossypii strain used in this experiment was collected from a zucchini field near Potenza, Italy (40°34′N, 15°45E) in August 2022 (Forlano et al. 2022) and reared in the laboratory on zucchini plants. No chemicals were used on this strain from the time of collection until the start of the experiment. Aphids were reared at room temperature under a 16:8 L:D photoperiod for two years. Aphis gossypii can show a wide range of colour variation, from yellow to dark green, as a response to developmental temperature, crowding conditions and host plant; it can also produce winged morphs in response to nutritional factors and crowding. Dark green apterous parthenogenetic females of A. gossypii were used for the following experiments.

To eliminate maternal and grandmaternal effects from aphids, 100 adult virginoparae females were isolated from the aphid culture, placed on a fresh host plant and allowed to reproduce for 24 h in a Binder KBF climatic chamber at 22 ± 1°C, 75 ± 5% RH and an 18:6 L:D photoperiod. The adults were then removed, and the cohort of new-born nymphs was reared on the plant for 7 days, corresponding to the beginning of the adult stage. This procedure was carried out for 2 generations and repeated independently for 3 groups of aphids, resulting in individuals from 3 independent replicates.

Experimental design of zucchini irrigation with fipronil

In the control treatment (Co), zucchini plants were watered with tap water. The two experimental treatments involved adding 100 ng/L of fipronil (F100) and 1,000 ng/L of fipronil (F1000) to the irrigation water. These concentrations were selected to reflect environmentally relevant levels (Sadaria et al. 2017, 2019; Zhang et al. 2023; Kim and Kim 2024), with the higher concentration used to explore potential degradation pathways. Experimental watering began one week after transplanting the seedlings into pots. At each watering, a single plant was watered with 150 mL of the respective water solution. To ensure consistent soil moisture between 30% and 90% of water-holding capacity, nine watering sessions were conducted over the 35-day period, with each plant receiving a total of 1,350 mL of experimental water by the end of the experiment. This resulted in a cumulative exposure of 135 ng of fipronil for plants in the F100 treatment and 1,350 ng for those in the F1000 treatment, while control plants received no fipronil.

The experiments were conducted in three separate periods, spaced 7 days apart, producing three independent replicates of 7 plants each, for a total of 21 plants per treatment group.

Aphid survival and fecundity

For each treatment, two adult female aphids of the same age (8–9 days old) were independently placed on 21 zucchini plants (7 plants per replicate) in the climatic chamber described above. After a few minutes, the two aphids were enclosed inside a clip cage (2 cm of diameter). The clip cages were placed on the adaxial side of the youngest fully developed leaf of each plant. After 24 h, adults and excess newborn nymphs were removed with a wet paintbrush, leaving only three nymphs per clip cage.

For the survival measures, these three nymphs were monitored to adulthood, with aphid mortality assessed on day 7. The offspring of the experimental adults was then assessed by daily counting and removal of new-born nymphs for 7 days (the same number of days as the pre-reproductive period). Aphid fecundity was estimated as the mean number of nymphs per aphid per day.

Acute toxicity bioassays

To assess the susceptibility of A. gossypii to the insecticides fipronil and pyrethrins at sub-lethal doses, adult aphids that had completed two generations on control (Co) and on plants treated with the F100 and F1000 doses of fipronil were used. The nymphs produced on day 6 and day 7 by the experimental adults used for the fecundity experiment were removed from the clip cages but left to become adults on another leaf of the same plant and then used for the experiments. The tests were performed according to the leaf dip method described in the Insecticide Resistance Action Committee Susceptibility Test Method No. 019 (IRAC 2024), with some modifications. The solutions used were 1 mg/L of fipronil (F-acute), 1 mg/L of pyrethrins (P-acute), and a control (tap water, TW). The doses of fipronil and pyrethrins were chosen based on previously determined acute lethal doses for A. gossypii (data not shown). The two insecticides and the tap water were applied to untreated zucchini disc leaves (3 cm of diameter) by dipping them in the test suspensions for 5 seconds with gentle agitation. Treated disc leaves were placed with their dorsal side on four layers of wet (saturated with distilled water) filter paper in a Petri dish, and 15 adult female aphids were placed on the surface of each treated leaf. Test and control discs leaves were maintained at 22°C in the thermostatic chamber described above.

For the insecticide susceptibility, nine experimental treatments were generated by combining high-dose insecticide treatments (TW, F-acute and P-acute) and the water treatment applied to the plants where the aphids developed (Co, F100 and F1000). Mortality was estimated after 48 hours as the number of live individuals relative to the initial number of aphids; an aphid was considered alive if it could walk or at least move when gently touched with a soft brush. Three replicates per treatment were used for this experiment, with each replicate consisting of at least 3 disc leaves containing 10–15 individuals, giving a total of at least 100 individuals per treatment.

Plant sample collection, fipronil extraction

Plant sampling included collection of leaves and flowers. At the end of the experiment (35 days after transplanting), leaf and flower samples were collected from three separate plants of each experimental treatment, weighed and lyophilised. The extraction of fipronil from the zucchini’s leaves and flowers was performed in triplicates, via QuEChERS extraction and dSPE PSA-C18 clean-up protocol (adapted protocol, Montemurro et al., 2020). In short, 1 g of freeze-dried and milled leaves or flowers was placed in a 50 mL falcon tube and hydrated with 9 mL HPLC water. The tubes were vortexed for 10 sec, shaken for 2 min at 2500 rpm and left to hydrate for 1 h. 10 mL of acetonitrile and 50 µL of formic acid were added in the tubes, vortexing was repeated and the extraction salts (1 g NaCl and 4 g MgSO₄) were added in the tubes. The mixture was instantly shaken to prevent the formation of crystalline agglomerates. Tubes were vortexed for 10 sec, shaken for 2 min at 2500 rpm and centrifuged at 4°C for 10 min at 4,000 rpm. The supernatant, containing the organic phase, was transferred into glass tubes, and left overnight at -20°C for the precipitation of fatty acids and waxes. The following day, the clean-up step involved the transfer of 6 mL of the supernatant into the PSA (primary secondary amine) tubes (150 mg PSA, 150 mg C18, 900 mg MgSO₄) and the mixture was vortexed and shaken, then centrifuged at 4°C for 5 min at 4,000 rpm. On the day of the analysis, 1 mL of the supernatant was spiked with the internal standard mixture at a concentration of 20 µg/L, the sample was evaporated until dryness under nitrogen at room temperature, reconstituted with 1 mL of methanol:water (2:8) solution and injected for UHPLC-MS/MS analysis.

The analysis of fipronil in the zucchini extracts was performed with a Waters Acquity Ultra-PerformanceTM liquid chromatography system coupled to a 5500 QTRAP hybrid triple quadrupole-linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA, USA) with a turbo Ion Spray source. Fipronil was analysed in negative ionization mode (method from Castaño-Trias et al., 2023) using an Acquity BEH C18 column (50 × 2.1 mm i.d., 1.7 µm particle size) for chromatographic separation. The mobile phase consisted of (A) ACN and (B) 5mM ammonium acetate/ammonia (pH = 8). The total run was 3.7 min with a flow rate was 0.6 mL/min, and the elution gradient was as follows: initial condition 95% B; 0-1.5 min 5–60% A, 1.5-2 min 60–100% A, 2–3 min 100% A, 3-3.2 min return to initial conditions, 3.2–3.7 min equilibration of the column. The column temperature was set at 30°C and an injection volume was set at 5µL. The quantification of fipronil analytes in zucchini flowers and leaves, as well as the detection (without possibility for quantification) of its metabolites, were performed by selective reaction monitoring by monitoring two mass transitions between the precursor ion and the most abundant fragment ions for each compound. The one at higher intensity was used for quantification purposes, while the second one was used for confirmation of the compound identification. Data acquisition and processing was performed with Analyst 1.5.1 software.

Statistical analysis

Data on nymph-to-adult survival and on the acute toxicity bioassays were analysed using generalized linear mixed-effects models with binomial error (logit link function), because a binomial distribution is appropriate for modelling binary data (dead / alive). P-values for differences among experimental treatments were obtained by analyses of deviance (type II Wald chi-squared tests). For the nymph-to-adult survival model, “water treatment” (three levels: Co, F100, and F1000) was the main factor and “replicate nested in treatment” was the random effect. For the acute toxicity bioassay model, “water treatment” and “insecticide treatment” (three levels: TW, F-acute, P-acute) were the fixed factors together with their interactions, and “replicate” nested within the fixed factors was the random effect.

The Shapiro-Wilk test was used to test the normality of the fecundity data and then analysed using a linear mixed-effects model fitted with restricted maximum likelihood, with “water treatment” (three leaves) and “reproductive interval” (seven levels) as a fixed factors and “replicate” nested within the fixed factors as random effect. The two main effects and the interaction term were fitted for this model.

A backward procedure was used to sequentially remove non-significant effects, allowing the identification of the most parsimonious model. All insect data were analysed using the statistical software R version “4.4.1-Race for Your Life” (R Core Team 2024) with the packages lme4 (Bates et al. 2015) and lmerTest (Kuznetsova et al. 2017).

The results of this study highlight the critical issue of environmental persistence of fipronil, particularly in agricultural systems where wastewater reuse is, or may become, common practice. While extensive research has documented the risks associated with fipronil contamination in aquatic environments, much less is known about its potential effects in agricultural ecosystems. This study addresses this knowledge gap by investigating the impact of irrigation with environmentally relevant doses of fipronil on zucchini plants and their primary insect pest, Aphis gossypii.

Fipronil content in zucchini flowers and leaves

Two types of zucchini samples, the leaves and the flowers, were analysed for fipronil. For the detection and quantification of fipronil, matrix-match calibration curves for both the leaves and flowers were used, exhibiting very good linearity (R² > 0.9997), in the range 1-100 µg/L. The limits of detection and quantification for fipronil were 29 and 9 ng/g (dry weight, d.w.) in leaves and 15 and 5 ng/g (d.w.) in flowers. Average recoveries of fipronil and standard deviations from the triplicate samples in the two matrices were calculated as 102.4 (± 3.7)% and 105.4 (± 5.0)% in leaves and flowers, respectively. The analytical method permitted for the detection of fipronil metabolites as well, and the spectrometry parameters used for the detection of all compounds in zucchini samples are given in Table 1.

Table 1

Spectrometry parameters used for the detection of fipronil and its metabolites in zucchini samples. Q1-Q3 = precursor and fragment ions in the first and third quadrupole respectively; DP = declustering potential; CE = collision energy; CXP = collision cell exit potential.
Compound	Retention time (min)	Q1–>Q3 transitions	DP (volts)	CE (volts)	CXP (volts)
Fipronil	2.06	435.126 / 330.000	-50.000	-22.000	-15.000
Fipronil	2.06	435.126 / 249.800	-50.000	-38.000	-29.000
Fipronil sulfone	2.13	450.868 / 414.900	-125	-24	-23
Fipronil sulfone	2.13	450.868 / 281.900	-125	-36	-17
Fipronil sulfide	2.13	418.900 / 382.900	-130	-20	-19
Fipronil sulfide	2.13	418.900 / 261.900	-130	-34	-11
Fipronil desulfinyl	2.08	452.884 / 347.800	-90	-16	-17
Fipronil desulfinyl	2.08	452.884 / 303.800	-90	-44	-11
Fipronil-¹³C₂¹⁵N₂ (I.S.)	2.05	438.906 / 333.900	-75	-22	-15
Fipronil-¹³C₂¹⁵N₂ (I.S.)	2.05	438.906 / 251.900	-75	-36	-11

Even though fipronil peaks were visible in the chromatograms of the zucchini leaves samples, they were below the detection limit based on the applied calibration curve. Nevertheless, both fragments of the parent ion and the main fragment were identified in those samples. For the leaves, the areas of the peaks in the samples irrigated with the spiked waters were on average 1.3-2x larger compared to the lowest calibration curve point (0.1 µg/L), suggesting some presence of fipronil in the former, albeit in trace amounts. The areas of the samples spiked at 1,000 ng/L and at 100 ng/L did not differ, suggesting that at such low levels it would be difficult to differentiate between the two concentrations (Fig. 1). For the flowers, the peaks were either lower than the controls or absent, therefore fipronil was not present in the flower parts of the plant. The method for the detection of fipronil allows for the detection of three of its metabolites as well (fipronil sulfone, fipronil sulfinyl and fipronil desulfinyl), and peaks only for fipronil sulfone (the primary metabolite of fipronil) were detected in the zucchini leaves for both spiked concentrations (Fig. 2); same as with fipronil, both fragments were present, verifying the presence of this metabolite. The area of the fipronil sulfone peak for the sample spiked at 1,000 ng/L was on average 1.2x larger than the area of the sample spiked at 100 ng/L.

Fipronil is recognized for its long half-life in soil and water, leading to its accumulation over time and potentially reaching concentrations in soil and plant tissues that may harm both target and non-target organisms (Bhatt et al. 2023). The potential detection of fipronil in zucchini flowers, which develop later in the plant’s growth cycle, should further emphasize the widespread contamination and potential risks associated with its use. This aligns with the broader environmental concerns highlighted by the Worldwide Integrated Assessment (WIA) on systemic insecticides, which stress the persistent and pervasive contamination of ecosystems with neonicotinoids and fipronil (van der Sluijs et al. 2015). According to the WIA, these compounds, due to their systemic nature, are absorbed by plants and can persist in the environment for extended periods, leading to chronic exposure of non-target organisms across various ecosystems, including agricultural lands (Pisa et al. 2014). Zucchini flowers attract a variety of pollinators and beneficial insects, which could inadvertently be exposed to the insecticide. During the experiments, the total volume of water received by the plants was 1.35 L, resulting in a cumulative exposure over 35 days of 135 and 1,350 ng fipronil for plants in the F100 and F1000 treatments, respectively. Traces of fipronil were detected in the zucchini leaves, but none were found in the flowers. Moreover, none of the fipronil transformation products included in the analytical protocol (fipronil sulfone, fipronil sulfinyl, and fipronil desulfinyl) was detected in either the leaves or the flowers. It is possible that the total water received by the plants during this short irrigation period was not sufficient for fipronil to accumulate to quantifiable levels in the plant tissues. Prolonged irrigation, which is common in field or greenhouse cultivation, could have resulted in higher levels. It should be remembered that the water uptake of zucchini plants grown under open field conditions is about 250 L/m² (Rouphael et al. 2005), depending on the length of the growing cycle (3–5 months). As for the flowers, this experiment cannot determine whether the absence of peaks was due to low levels of fipronil in the irrigation water or other parts of the plant, or whether it is a mechanistic issue where fipronil is retained only in the leaves and not in the zucchini flowers.

Aphid survival and fecundity

The occurrence of fipronil in plant tissues has direct implications for Aphis gossypii. Aphids engage in a feeding process that involves several short intracellular punctures into parenchyma cells before reaching the phloem. During these punctures, they ingest small amounts of cytoplasm, which contributes to their diet (Alvarez and Griffing 2023). It is plausible that aphids pick up fipronil through phloematic feeding and also during these brief punctures, which may accumulate in the cytoplasm of the plant cells. This uptake of fipronil present in the host plant, even at low doses, affects the life history traits of A. gossypii developed on it.

Nymph-to-adult survival was significantly affected by water treatment (χ² = 10.01, d.f. = 2, P < 0.01), with F100 and F1000 decreasing aphid survival if compared to the control treatment (Fig. 3).

As expected, the age of the aphids had a significant effect on their fecundity (Fig. 4), with very low values on the first day, peaking around the third day and then gradually decreasing from the fourth day (F_6,278 = 13.14, P < 0.001). Similar to survival, aphids developed on plants treated with the F100 and F1000 doses of fipronil had a significantly lower fecundity than the control (F_2,278 = 4.67, P < 0.05). The interaction between the two factors was not significant. The cumulative values fecundity over 7 days of the F1000 treatment (21.8 ± 0.42 nymphs/individual) was lower than that of the F100 (25.8 ± 0.43) and control (27.38 ± 0.44) groups.

Unlike other insecticides such as imidacloprid, fipronil has been shown to cause time-reinforced toxicity in honey bees at trace levels (2.9 ng/bee), resulting in mass mortality of adult individuals (Holder et al. 2018). Fipronil ingested by a honey bee in a single meal was also still present 6 days later (Holder et al. 2018), highlighting the issue of bioaccumulation in living organisms, which can lead to lethal and sub-lethal effects (Zaluski et al. 2015). Our results in aphids are consistent with those found in honeybees and provide evidence of the potentially serious impact of dietary fipronil bioaccumulation on insect life history traits and pest management. Fipronil uptake reduced the fitness of aphids feeding on contaminated host plants, measured as a reduction in nymph-to-adult survival and a dose-dependent reduction in their fecundity. The lowest aphid fecundity observed at the highest dose of fipronil in the irrigation water (1,000 ng/L) strongly suggests that the presence and bioaccumulation of the active substance in the mothers diverts resources away from nymph production towards cellular detoxification mechanisms, so that the fecundity of these individuals declines over time. However, this apparent beneficial effect of fipronil at environmental doses for pest control raises the question of adverse effects on human health, the environment, and non-target beneficial organisms, particularly predators and parasitoids commonly used for pest control.

Acute toxicity bioassays

The environmental persistence of fipronil could potentially lead to the development of resistance in pest populations at realistically low doses of the chemical. The acute toxicity bioassays (Fig. 5) showed that mortality at 48 h of A. gossypii was strongly influenced by fipronil and pyrethrins at the concentration of 1 mg/L (χ² = 298, d.f. = 2, P < 0.001). Fipronil was more effective than pyrethrins against aphids at the same concentration, independently of the water treatment applied to the plants where the aphids developed.

Mortality was not affected by the water treatments (χ² = 2.4, d.f. = 2, P = 0.29) but, more interestingly, there was a significant interaction between the water treatment and insecticide (χ² = 12.34, d.f. = 4, P < 0.05), indicating the occurrence of differential susceptibility to a particular insecticide as a function of the presence of fipronil in the plants. Aphids developed on plants irrigated with the F100 or the F1000 doses had the same mortality as the control when exposed to the acute dose of fipronil. When the aphids were exposed to the acute dose of pyrethrins, the mortality of the individuals that developed on the F1000 plants was statistically lower than the control and similar to the F100 (although a decreasing mortality trend was observed).

Laboratory studies of fipronil resistance have been reported in a wide range of insects (Sayyed and Wright 2004; Tang et al. 2010; Abbas et al. 2014; Wazir and Shad 2020), suggesting that this insecticide should be applied at high doses where refuges of the insects are located, using chemicals rotation and environmentally friendly cultural practices to delay the development of resistance and prolong its efficacy (Wazir and Shad 2020, 2022). In the present study, contact acute toxicity bioassays showed no change in aphids mortality when exposed to a sublethal dose of fipronil (1 mg/L), regardless of whether they developed on plants irrigated with environmentally relevant doses of fipronil or on control plants. This result is not surprising, as pesticide resistance is the result of prolonged and persistent use of these substances at sub-lethal doses and longer experiments might be needed. Aphids that ingest fipronil by feeding on contaminated host plants may activate biomolecular mechanisms that allow them to partially overcome the stress caused by fipronil at sub-lethal doses, as with other abiotic stresses (Hoffmann et al. 2003). However, under our experimental conditions, this effect could have been masked by the high efficacy of fipronil against A. gossypii at a concentration of 1 mg/L. When natural pyrethrins were used in the contact acute toxicity bioassays, a significant increase in resistance to pyrethrins was observed in aphids that developed on plants irrigated with fipronil. Although fipronil and pyrethrins have two different modes of action, they may share some common detoxification enzyme system (Li et al. 2019). The results obtained from the pyrethrins bioassays support our hypothesis that low doses of fipronil may activate molecular mechanisms that enhance the activity of detoxification enzymes, thereby increasing aphid survival to higher doses of pyrethrins via a “hardening” effect. Our study highlights that if fipronil is not adequately removed from non-conventional irrigation water, prolonged environmental exposure to this insecticide could lead to the development of pesticide resistance and cross-resistance (Bras et al. 2022).

The observed increased resistance to pyrethrins was most likely due to epigenetic mechanisms, as the aphids had only spent two generations on the treated plants, making it unlikely that these changes were due to gene mutation. The rapid development of insecticide resistance in other species, such as the Colorado potato beetle (Leptinotarsa decemlineata), which has evolved resistance to over 50 insecticides, illustrates the potential for sublethal insecticide exposure to cause heritable changes due to decreased global DNA methylation (Brevik et al. 2018, 2021). It has been hypothesized that the aphid Acyrthosiphon pisum may respond to selective pressure through a transgenerational epigenetic adaptive mechanism that confers plasticity mediated by transposition (De Fabrizio et al. 2024). A similar response has been observed in Myzus persicae, where exposure to the insecticide imidacloprid induced reproductive hormesis, accompanied by intermittent changes in the expression of detoxification and stress-coping genes, enabling the insects to better handle subsequent stress (Rix et al. 2016).

Fipronil is still considered an effective pesticide because of its high efficacy in pest prevention and control, and its minimal risk to crop safety. However, it degrades slowly in the natural environment, and although several countries have restricted (but not banned) its agricultural use due to its high toxicity to aquatic organisms and bees, it remains widely used for urban pest control and as an ectoparasiticide for fleas and ticks on pets. For all these reasons, fipronil is frequently detected in various environments around the world, making its effective removal from agricultural and domestic wastewater of high concern, particularly in areas where wastewater is reused.

The current study investigated the persistence and effects of fipronil in an agricultural setting, in particular its impact when present in zucchini irrigation water. Our results demonstrate that fipronil had the potential to accumulate in plant tissues, posing risks to non-target organisms. Additionally, the study highlights the risk of fipronil contributing to the development of pesticide resistance and cross-resistance in insect pests like Aphis gossypii. Understanding the ecological impact of fipronil in agro-ecosystems, particularly its role in the emergence of pesticide resistance, shifts in pollinators population dynamics, and effects on other non-target organisms, is crucial. These insights are vital for guiding the implementation of sustainable agricultural practices that minimize environmental contamination and protect biodiversity. However, it is important to highlight the need for detailed biochemical studies to further explore the mechanisms underlying the observed insecticide interactions, as well as the wider ecological implications of the environmental persistence of fipronil. The findings of this study advocate for a more cautious and informed approach to fipronil use in agriculture and emphasize the importance of ongoing monitoring and research to safeguard both ecosystem health and agricultural productivity.

Ethics approval
This study did not involve human participants, human material, or human data, so it does not need an ethical approval document.

Consent to participate
Not applicable.

Consent for publication
All authors have agreed to co-author this manuscript.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research was partially funding from MUR under the umbrella of the PRIMA- Partnership for Research & Innovation in the Mediterranean Area through the research project SAFE "Sustainable water reuse practices improving safety in agriculture, food and environment". ID project: 1826, and, partially, by under the umbrella of the WATER4ALL Partnership, granted project NeWater. The Ultra-Performance Liquid Chromatography Triple Quadrupole Mass spectrometry (UPLC-MS) hybrid Linear Ion Trap (LIT), Acquity UPLC-MS QTRAP 5500, Waters-SCIEX. facility received support from the CERCA Institute through the CERCAGINYS programme, funded by the Spanish Ministry of Science and Innovation. The authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through a Consolidated Research Groups (ICRA-ENV-2021 SGR 01282 and ICRA-TECH − 2021 SGR 01283).

Author contributions

Study design: VT, MB. Methodology: VC, SS, RR, VT. Data curation: VT, SS. Formal analysis and investigation: VC, MB, SS, VT. Validation: VC, MB, SS, GB, RR, PF, DB, VT. Writing – original draft preparation: VC, VT. Writing – reviewing and editing: VC, MB, SS, GB, RR, PF, DB, VT. Resources: MB, DB, GB. Supervision: VT, MB, GB.

Data Availability

All data presented in this article will be available in a single Excel file as Supplementary File.

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Impact of irrigation with fipronil-contaminated waters on zucchini plants and their main insect pest, Aphis gossypii

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Chemicals

Zucchini plant cultivation

Insect rearing

Experimental design of zucchini irrigation with fipronil

Aphid survival and fecundity

Acute toxicity bioassays

Plant sample collection, fipronil extraction

Statistical analysis

Results and Discussion

Fipronil content in zucchini flowers and leaves

Aphid survival and fecundity

Acute toxicity bioassays

Conclusions

Declarations

Ethics approval
This study did not involve human participants, human material, or human data, so it does not need an ethical approval document.

Consent to participate
Not applicable.

Consent for publication
All authors have agreed to co-author this manuscript.

Competing interests

Funding

Author contributions

Data Availability

References

Supplementary Files

Status:

Version 1

Impact of irrigation with fipronil-contaminated waters on zucchini plants and their main insect pest, Aphis gossypii

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Chemicals

Zucchini plant cultivation

Insect rearing

Experimental design of zucchini irrigation with fipronil

Aphid survival and fecundity

Acute toxicity bioassays

Plant sample collection, fipronil extraction

Statistical analysis

Results and Discussion

Fipronil content in zucchini flowers and leaves

Aphid survival and fecundity

Acute toxicity bioassays

Conclusions

Declarations

Ethics approval This study did not involve human participants, human material, or human data, so it does not need an ethical approval document.

Consent to participate Not applicable.

Consent for publication All authors have agreed to co-author this manuscript.

Competing interests

Funding

Author contributions

Data Availability

References

Supplementary Files

Status:

Version 1

Ethics approval
This study did not involve human participants, human material, or human data, so it does not need an ethical approval document.

Consent to participate
Not applicable.

Consent for publication
All authors have agreed to co-author this manuscript.