Multiresidue analysis of bat guano using GC-MS/MS.

Bats are the second largest mammalian order and are an endangered species group with a strong need for contamination monitoring. To facilitate non-invasive monitoring of the ecological burden in bat populations, a multiresidue method for the simultaneous quantification of 119 analytes including pesticides, persistent organic pollutants (POPs), active pharmaceutical ingredients (APIs), polycyclic aromatic hydrocarbons (PAHs), UV blockers, plasticizers, and other emerging pollutants in bat guano with gas chromatography tandem mass spectrometry (GC-MS/MS) was developed. Sample preparation and clean-up were performed with a modified QuEChERS approach based on DIN EN 15662. The method uses 1.00 g bat guano as sample with acetonitrile and water for liquid-liquid extraction. Phase separation is assisted by citrate-buffered salting out agent. For clean-up of the extract, primary secondary amine (PSA) was combined with graphitized carbon black (GCB). The lower limits of quantification (LLOQ) ranged between 2.5 and 250 µg kg-1. Linearity was shown in a concentration range from the respective LLOQs to 1250 µg kg-1. The median of the mean recovery was 102.4%. Precision was tested at three concentrations. Method and injection precision were adequate with a relative standard deviation (RSD) below 20%. Furthermore, the comparative analysis with LC-MS/MS demonstrated the reliability of the results and provided a valuable extension of the analytical scope. As proof of concept, three guano samples from a German nursery roost of Myotis myotis were analysed. The results show a time-dependent change in contaminant concentration, highlighting the strong need for non-invasive contamination monitoring of whole bat populations.

Effects of a Systemic Pesticide Along an Aquatic Tri-Trophic Food Chain.

Systemic pesticides, such as the neonicotinoid imidacloprid, can be introduced into aquatic ecosystems through contaminated plant material, which is the basis for detrital (brown) aquatic food-webs. With the aim of exemplarily assessing for indirect effects on the level of predators, we first offered imidacloprid contaminated and uncontaminated alder leaves to the stonefly shredder Protonemura sp. for 72 h. Shredder survival, leaf decomposition, body length and biomass were all between 20% and 50% lower under imidacloprid exposure compared to uncontaminated conditions, indicating physiological implications. Subsequently, these shredders were provided as prey to stonefly predators (Isoperla sp.) kept in cages in a stream. Predator biomass and length decreased by up to 11% and 4.3%, respectively, when feeding on imidacloprid exposed prey. Our study hence suggests that plant material contaminated with systemic pesticides can exert adverse effects in aquatic predators when preying on shredders consuming such leaves, which warrants a further consideration of this pathway.

Modeling Remobilization of Neonicotinoid Residues from Tree Foliage in Streams-A Relevant Exposure Pathway in Risk Assessment?

Systemic neonicotinoid insecticides are increasingly used as a crop protection measure to suppress insect pests on trees. However, senescent foliage falling from treated trees represents a rarely studied pathway through which neonicotinoids may enter nontarget environments, e.g., surface waters. To estimate risk posed by this pathway, neonicotinoid residues were analyzed in foliage from black alder trees treated with one of three neonicotinoid insecticides (imidacloprid, thiacloprid, or acetamiprid) at five concentrations, each ranging from 0.0375-9.6 g active ingredient/cm trunk diameter at breast height (n = 3). Foliar residues measured at the time of leaf fall were used as input parameters for a model predicting imidacloprid water concentrations over a 100-m-long stream stretch as a consequence of remobilization from introduced foliage (input: 600 g foliage/m2 containing 80 µg imidacloprid/g). The water concentration (up to ∼250 ng/L) predicted by the model exceeded the recently proposed Maximum Permissible Concentration of 8.3 ng/L for ∼6.5 days. Moreover, dietary uptake was identified as an additional exposure route for aquatic organisms. The alternative pathway (i.e., introduction via leaf fall) and exposure route (i.e., dietary uptake) associated with the systemic nature of neonicotinoids should be accounted for during their registration process in order to safeguard ecosystem integrity.

The Landau Stream Mesocosm Facility: pesticide mitigation in vegetated flow-through streams.

Vegetated treatment systems have the ability to reduce the risk of adverse effects of nonpoint source pesticide pollution in agricultural surface waters, however, flow-through systems have rarely been evaluated. Peak concentrations of a mixture of two insecticides and two fungicides (Indoxacarb, Tebuconazole, Thiacloprid and Trifloxystrobin) were reduced by more than 90% in 45 m experimental stream mesocosms. Plant density and solubility had the highest explanatory power for the response variable reduction of peak concentration (R² = 0.70, p < 0.001). Optimized vegetated streams can be highly effective in reduction of runoff related pesticide peak concentrations.

A new sample preparation approach for the analysis of 98 current-use pesticides in soil and herbaceous vegetation using HPLC-MS/MS in combination with an acetonitrile-based extraction.

A simple acetonitrile-based extraction method for the determination of 98 current-use pesticides (CUPs) in soil and herbaceous vegetation using HPLC-ESI-MS/MS is reported. The method was optimized in terms of extraction time, buffer (ammonium formate) ratio, and graphitized carbon black (GCB) ratio for the clean-up of vegetation. The validated method yielded accuracy in terms of percentage recovery of 71-125% (soil) and 70-117% (vegetation) for the majority of 98 CUPs. The precision in terms of relative standard deviation was at 1-14% (soil), and 1-13% (vegetation). Matrix-matched calibration curves exhibited good linearities (R2 > 0.99). The limits of quantitation ranged between 0.008 and 21.5 µg kg-1 in soil and vegetation. The reported method was applied to soils and vegetation from 13 agricultural sites across Germany. Overall, 44 of the 98 common CUPs were detected in our samples and the qualitative load is well above the average for arable soils in the EU.

Direct pesticide exposure of insects in nature conservation areas in Germany.

In Germany, the decline of insect biomass was observed in nature conservation areas in agricultural landscapes. One of the main causal factors discussed is the use of synthetic pesticides in conventional agriculture. In a Germany-wide field study, we collected flying insects using Malaise traps in nature conservation areas adjacent to agricultural land. We used a multi-component chemical trace element analysis to detect 92 common agricultural pesticides in ethanol from insect traps sampled in May and August 2020. In total, residues of 47 current use pesticides were detected, and insect samples were on average contaminated with 16.7 pesticides. Residues of the herbicides metolachlor-S, prosulfocarb and terbuthylazine, and the fungicides azoxystrobin and fluopyram were recorded at all sites. The neonicotinoid thiacloprid was detected in 16 of 21 nature conservation areas, most likely due to final use before an EU-wide ban. A change in residue mixture composition was noticeable due to higher herbicide use in spring and increasing fungicide applications in summer. The number of substances of recorded residues is related to the proportion of agricultural production area in a radius of 2000 m. Therefore, a drastic pesticide reduction in large buffers around nature conservation areas is necessary to avoid contamination of their insect fauna.

Mixture effects of a fungicide and an antibiotic: Assessment and prediction using a decomposer-detritivore system.

Antimicrobials, such as fungicides and antibiotics, pose a risk for microbial decomposers (i.e., bacteria and aquatic fungi) and invertebrate detritivores (i.e., shredders) that play a pivotal role in the ecosystem function of leaf litter breakdown. Although waterborne toxicity and diet-related effects (i.e., dietary exposure and microorganism-mediated alterations in food quality for shredders) of fungicides and antibiotics on decomposer-detritivore systems have been increasingly documented, their joint effect is unknown. We therefore assessed waterborne and dietary effects of an antimicrobial mixture consisting of the fungicide azoxystrobin (AZO) and the antibiotic ciprofloxacin (CIP) on microbial decomposers and the shredder Gammarus fossarum using a tiered approach. We compared effect sizes measured in the present study with model predictions (i.e., independent action) based on published data. During a 7-day feeding activity assay quantifying waterborne toxicity in G. fossarum, the leaf consumption of gammarids was reduced by ∼60 % compared to the control when subjected to the mixture at concentrations of each component causing a 20 % reduction in the same response variable when applied individually. Moreover, the selective feeding of gammarids during the food choice assay indicated alterations in food quality induced by the antimicrobial mixture. The food selection and, in addition, the decrease in microbial leaf decomposition is likely linked to changes in leaf-associated bacteria and fungi. During a long-term assay, energy processing, growth and energy reserves of gammarids were increased in presence of 15 and 500 µg/L of AZO and CIP, respectively, through the dietary pathway. These physiological responses were probably driven by CIP-induced alterations in the gut microbiome or immune system of gammarids. In general, model predictions matched observed effects caused by waterborne exposure on the leaf consumption, energy processing and growth of gammarids during short- and long-term assays, respectively. However, when complex horizontal (bacteria and aquatic fungi) and vertical (leaf-associated microorganisms and shredders) interactions were involved, model predictions partly over- or underestimated mixture effects. Therefore, the present study identifies uncertainties of mixture effect predictions for complex biological systems calling for studies targeting the underlying processes and mechanisms.

The role of organic matrices in the fate of hydrophobic pesticides: An outdoor stream mesocosm study.

To assess potential aquatic pesticide risks, environmental monitoring strategies often focus on water and sediment. However, knowledge gaps with regard to the pollution status of organic matrices important for the structure and functioning of aquatic ecosystems do exist. The present study assessed the dissipation of the triazole fungicide tebuconazole (TEB; KOW = 5.01 × 103) and the pyrethroid insecticide etofenprox (ETO; KOW = 7.94 × 106) as model hydrophobic pesticide compounds among aquatic plants, vertical layers of allochthonous leaf litter, and detritus within flow-through outdoor stream mesocosms. During a 3-h pesticide exposure and a subsequent 24-h post-exposure period, retention was higher for ETO (max concentration: Myriophyllum spicatum > Elodea nuttallii > Ranunculus fluitans > Potamogeton perfoliatus â‰« leaf litter > detritus) and depended amongst other factors on surface area, while in the water compartment the pesticides reached concentration levels < LOQ 2 h after exposure. Desorption was observed for both pesticides in plants, and for TEB in detritus, while in leaves the ETO levels even increased over time, suggesting leaf litter to be a suitable additional sampling matrix for transient hydrophobic pesticide peaks, yet also a potential source of contamination for invertebrate shredders. The upper layer of leaf material contained higher ETO levels than those situated further in the sediment, which implies short-term positive effects for species inhabiting the deeper leaf layers, yet again pinpoints to a potential pesticide exposure pathway via organic matter in aquatic systems.

Sampling rates for passive samplers exposed to a field-relevant peak of 42 organic pesticides.

Pesticide concentrations in agricultural streams are often characterised by a low level of baseline exposure and episodic peak concentrations associated with heavy rainfall events. Traditional sampling methods such as grab sampling, which are still largely used in governmental monitoring, typically miss peak concentrations. Passive sampling represents a cost-efficient alternative but requires the additional determination of sampling rates to calculate time-weighted average (TWA) water concentrations from the accumulated pesticide mass in the sampler. To date, sampling rates have largely been determined in experiments with constant exposure, which does not necessarily reflect field situations. Using Empore styrene-divinylbenzene (SDB) passive sampler disks mounted in metal holders, we determined sampling rates for 42 organic pesticides, of which 27 sampling rates were lacking before. The SDB disks were in an artificial channel system exposed to a field-relevant pesticide peak. We used an open-source algorithm to estimate coefficients of equations for the accumulated pesticide mass in disks and to determine exposure time-dependent sampling rates. These sampling rates ranged from 0.02 to 0.98 L d-1 and corresponded to those from previous studies determined with constant exposure. The prediction of sampling rates using compound properties was unreliable. Hence, experiments are required to determine reliable sampling rates. We discuss the use of passive sampling to estimate peak concentrations. Overall, our study provides sampling rates and computer code to determine these under peak exposure designs and suggests that passive sampling is suitable to estimate peak pesticide concentrations in field studies.

Reduction of Pesticide Toxicity Under Field-Relevant Conditions? The Interaction of Titanium Dioxide Nanoparticles, Ultraviolet, and Natural Organic Matter.

In surface waters, the illumination of photoactive engineered nanomaterials (ENMs) with ultraviolet (UV) light triggers the formation of reactive intermediates, consequently altering the ecotoxicological potential of co-occurring organic micropollutants including pesticides due to catalytic degradation. Simultaneously, omnipresent natural organic matter (NOM) adsorbs onto ENM surfaces, altering the ENM surface properties. Also, NOM absorbs light, reducing the photo(cata)lytic transformation of pesticides. Interactions between these environmental factors impact 1) directly the ecotoxicity of photoactive ENMs, and 2) indirectly the degradation of pesticides. We assessed the impact of field-relevant UV radiation (up to 2.6 W UVA/m²), NOM (4 mg TOC/L), and photoactive ENM (nTiO2, 50 µg/L) on the acute toxicity of 6 pesticides in Daphnia magna. We selected azoxystrobin, dimethoate, malathion, parathion, permethrin, and pirimicarb because of their varying photo- and hydrolytic stabilities. Increasing UVA alone partially reduced pesticide toxicity, seemingly due to enhanced degradation. Even at 50 µg/L, nano-sized titanium dioxide (nTiO2) reduced but also increased pesticide toxicity (depending on the applied pesticide), which is attributable to 1) more efficient degradation and potentially 2) photocatalytically induced formation of toxic by-products. Natural organic matter 1) partially reduced pesticide toxicity, not evidently accompanied by enhanced pesticide degradation, but also 2) inhibited pesticide degradation, effectively increasing the pesticide toxicity. Predicting the ecotoxicological potential of pesticides based on their interaction with UV light or interaction with NOM was hardly possible, which was even more difficult in the presence of nTiO2. Environ Toxicol Chem 2020;39:2237-2246. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Retention of plant protection products (PPPs) by aquatic plants in flow-through systems.

Understanding fate and transport of plant protection products (PPPs) that enter vegetated streams from agricultural fields is important for both exposure assessment and risk attenuation, yet limited knowledge is available. The present laboratory study investigated sorption processes governing mass transfer of three common PPPs between water and aquatic plant phases at flow-through exposure conditions (transient aqueous-phase PPP-peak of 4 h 25 min) using three temperature regimes. The exposure produced rapid sorption of PPPs to plants, followed by a gradual depuration from plants. Dynamic sorption kinetics depended on temperature, plant species, and physicochemical properties of the PPPs. Sorption to plants contributed to a 10% reduction of the water-phase peak concentrations of the PPPs. However, being reversible, the attenuation effect was limited to the residence time of the PPPs in the systems. Results of the present study highlight that effectivity of aquatic plants in the attenuation of PPP loads may vary greatly depending on hydrodynamic properties of aquatic systems.

Structural and functional effects of a short-term pyrethroid pulse exposure on invertebrates in outdoor stream mesocosms.

Agricultural land-use frequently results in short pulse exposures of insecticides such as pyrethroids in river systems, adversely affecting local invertebrate communities. In order to assess insecticide-induced effects, stream mesocosms are used within higher tier aquatic risk assessment. Regulatory acceptable concentrations (RACs) derived from those studies are often higher compared with tier 1 RACs. Hence, the present mesocosm study evaluates this aspect using a pulse exposure scenario typical for streams and the pyrethroid insecticide etofenprox. A 6-h pulse exposure with measured concentrations of 0.04, 0.3 and 5.3µgL-1 etofenprox was used. We considered abundance, drift and emergence of invertebrates as structural endpoints and the in situ-measured feeding rates of the isopod Asellus aquaticus as functional endpoint. Most prominent effects were visible at 5.3µgL-1 etofenprox which caused adverse effects of up to 100% at the individual and population level, as well as community structure alterations. Transient effects were observed for invertebrate drift (effect duration ≤24h) and for the invertebrate community (9 days after exposure) at 0.3µgL-1 etofenprox. Furthermore, 0.04µgL-1 etofenprox affected the abundance of the mayfly Cloeon simile (decrease by 66%) and the feeding rate of A. aquaticus (decrease by 44%). Thus, implications for the functional endpoint leaf litter breakdown in heterotrophic ecosystems may be expected. A hypothetical RAC derived from the present mesocosm study (0.004µgL-1) is in line with the official tier 1 RAC (0.0044µgL-1) and thus shows that the present mesocosm study did not result in a higher RAC.

Response and recovery of the macrophytes Elodea canadensis and Myriophyllum spicatum following a pulse exposure to the herbicide iofensulfuron-sodium in outdoor stream mesocosms.

Interest in stream mesocosms has recently revived for higher tier aquatic macrophyte risk assessment of plant protection products mainly because 1) the highest predicted environmental concentrations for the assessment of effects are frequently derived from stream scenarios, and 2) they allow an effect assessment using stream-typical pulse exposures. Therefore, the present stream mesocosm study used an herbicide pulse exposure and evaluated the responses of Elodea canadensis and Myriophyllum spicatum. Macrophytes were exposed for 24 h to 1 µg/L, 3 µg/L, 10 µg/L, and 30 µg/L of the herbicide iofensulfuron-sodium with a subsequent recovery period of 42 d. Biological endpoints were growth rates of the main, side, and total shoot length, the shoot number, the maximum root length, and the dry weight. The total shoot length was identified as the most sensitive endpoint; the growth rate of the total shoot length was inhibited by up to 66% and 45% in M. spicatum and E. canadensis, respectively. The lowest no observed effect concentrations (NOECs) were observed at day 7 and/or day 14 after herbicide treatment and were 1 µg/L for M. spicatum and 3 µg/L for E. canadensis. The no-observed-ecologically-adverse-effect concentrations (NOEAECs) were 10 µg/L and 30 µg/L for M. spicatum and E. canadensis, respectively. Such or similar mesocosm designs are useful to simulate typical stream exposures and estimate herbicide effects on aquatic macrophytes in stream systems. Environ Toxicol Chem 2017;36:1090-1100. © 2016 SETAC.

Transient effects following peak exposures towards pesticides - An explanation for the unresponsiveness of in situ measured functional variables.

Invertebrate-mediated leaf litter decomposition is frequently used to assess stress-related implications in stream ecosystem integrity. In situ measures such as the mass loss from leaf bags or the feeding of caged invertebrates deployed for days or weeks may, however, fail to detect transient effects due to recovery or compensatory mechanisms. We assessed the relevance of transient effects using the peak exposure towards an insecticide (i.e., etofenprox) as a model scenario at three levels of complexity. These were 1) the assessment of the decomposition realised by invertebrate communities in stream mesocosms over 21 days via leaf bags, 2) 7-days lasting in situ bioassays quantifying the leaf consumption of Gammarus fossarum, and 3) a laboratory experiment determining the daily feeding rate of the same species over 7 days. Etofenprox did not trigger a significantly altered decomposition by invertebrate communities during the leaf bag assay, while in situ bioassays detected a significant reduction in gammarids' feeding rate at the highest tested concentration. The laboratory bioassay suggests that observed mismatches might be explained by recovery and post-exposure compensation. As leaf-shredding invertebrates are likely in a vulnerable state following transient effects, biomonitoring for implications of peak exposures and other pulsed stress events must happen at an adequate temporal resolution.

Experiments in water-macrophyte systems to uncover the dynamics of pesticide mitigation processes in vegetated surface waters/streams.

Knowledge on the dynamics and the durability of the processes governing the mitigation of pesticide loads by aquatic vegetation in vegetated streams, which are characterized by dynamic discharge regimes and short chemical residence times, is scarce. In a static long-term experiment (48 h), the dissipation of five pesticides from the aqueous phase followed a biphasic pattern in the presence of aquatic macrophytes. A dynamic concentration decrease driven by sorption to the macrophytes ranged from 8.3 to 60.4% for isoproturon and bifenox, respectively, within the first 2 h of exposure. While the aqueous concentrations of imidacloprid, isoproturon, and tebufenozide remained constant thereafter, the continuous but decelerated concentration decrease of difenoconazole and bifenox in the water-macrophyte systems used here was assumed to be attributed to macrophyte-induced degradation processes. In addition, a semi-static short-term experiment was conducted, where macrophytes were transferred to uncontaminated medium after 2 h of exposure to simulate a transient pesticide peak. In the first part of the experiment, adsorption to macrophytes resulted in partitioning coefficients (logK D_Adsorp) ranging from 0.2 for imidacloprid to 2.2 for bifenox. One hour after the macrophytes were transferred to the uncontaminated medium, desorption of the compounds from the macrophytes resulted in a new phase equilibrium and K D_Desorp values of 1.46 for difenoconazole and 1.95 for bifenox were determined. A correlation analysis revealed the best match between the compound affinity to adsorb to macrophytes (expressed as K D_Adsorp) and their soil organic carbon-water partitioning coefficient (K OC) compared to their octanol-water partitioning coefficient (K OW) or a mathematically derived partitioning coefficient.

Reference scenarios for exposure to plant protection products and invertebrate communities in stream mesocosms.

Higher tier aquatic risk assessment for plant protection products (PPPs) is often based on pond-like mesocosm studies in which transient and dynamic PPP exposure scenarios as observed in lotic systems are hardly achievable. Thus, the present study presents dynamic PPP exposure scenarios at different time scales under flow-through conditions as typical for streams in agricultural landscapes. The stream mesocosm setup allows testing the influence of spatial gradients of exposure over the length of themesocosms. The use of the fluorescent tracer uranine revealed the hydraulic processes generally underlying peak- and hour-scale exposure scenarios and demonstrated an optimized application technique to achieve stable day-scale exposures. Furthermore, to account for potential reactions of invertebrates to PPP exposures in streams (e.g. avoidance behavior and drift), the present study thus aimed at a comprehensive evaluation on how PPP exposure and the establishment of invertebrates can be advanced within streammesocosm testing. For both, peak- and hour-scale exposure as well as the experiments considering the establishment of invertebrates, the presented compilation of experiments was able to highlight the influence of aquatic macrophyteswithin streammesocosms. Since the field relevance of the higher tier aquatic risk assessment for PPPs relies qualitatively on the presence of potentially sensitive or vulnerable species, those species were especially considered. Thus, the establishment of aquatic invertebrates in nondosed streams was evaluated with respect to (i) the presence of different aquatic macrophytes and (ii) the duration of the pre-experimental period. The present study highlights the beneficial influence of complex-structured macrophytes and prolonged pre-experimental periods on the abundance of invertebrate taxa. Furthermore, population dynamics were evaluated statistically by simulating PPP-related declines of 30, 50 and 70%. Thereby,