Science of the Total Environment 784 (2021) 147167

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Science of the Total Environment

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Ecological effects of imidacloprid on a tropical freshwater ecosystem and subsequent recovery dynamics

Lemessa B. Merga a,b,PaulJ.VandenBrinka,c,⁎ a Aquatic Ecology and Water Quality Management group, Wageningen University, P.O. Box 47, 6700 AA Wageningen, the Netherlands b Department of Chemistry, Ambo University, P.O. Box 240, Ambo, Ethiopia c Wageningen Environmental Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands

HIGHLIGHTS GRAPHICAL ABSTRACT

• Effects of imidacloprid (IMI) to aquatic organisms was assessed in Ethiopia. • Mayfly nymphs are more sensitive to IMI than other macroinvertebrates. • We found lower toxicity values of IMI for tropical species relative to temperate ones. • Community structure was affected at TWA112d concentrations ≥0.02 μg/L. • Low, chronic, levels of IMI may cause long term structural alterations.

article info abstract

Article history: This study aimed to investigate the effect of imidacloprid on structural (invertebrates and primary producers) Received 16 December 2020 and functional (organic matter decomposition and physicochemical parameters) characteristics of tropical fresh- Received in revised form 11 April 2021 waters using acute single species and mesocosm studies performed in Ethiopia. The recovery of affected end- Accepted 12 April 2021 points was also studied by using a mesocosm study period of 21 weeks. Our acute toxicity test showed that Available online 18 April 2021 Cloeon dipterum (96-h EC50 = 1.5 μg/L) and Caenis horaria (96-h EC50 = 1.9 μg/L) are relatively sensitive arthro- Editor: Sergi Sabater pods to imidacloprid. The mesocosm experiment evaluated the effects of four applications of imidacloprid with a weekly interval and the results showed that the macroinvertebrate and zooplankton community structure changed significantly due to imidacloprid contamination in mesocosms repeatedly dosed with ≥0.1 and ≥ 0.01

Keywords: μg/L, respectively (time weighted average concentrations of 112 days (TWA112d)of≥0.124 and ≥≈0.02 μg/L, re- Arthropods spectively). The largest responses were found for C. dipterum, C. horaria, Brachionus sp. and Filinia sp. Chlorophyll- Ethiopia a concentrations of periphyton and phytoplankton significantly increased in the ≥0.1 μg/L treatments levels Indirect effect which are indirect effects as a result of the release of grazing pressure. A significant, but quantitatively small, de- Invertebrates crease of organic matter decomposition rate was observed in mesocosms treated with repeated doses of 1 μg/L Insecticide (TWA of 2.09 μg/L). No recovery was observed for the macroinvertebrates community during the study pe- Mesocosms 112d riod of 21 weeks, but zooplankton recovered after 9 weeks. We observed spatio-temporal related toxicity differ- ences between tropical and temperate aquatic taxa, with tropical taxa generally being more sensitive. This suggests that use of temperate toxicity data for the risk assessment of imidacloprid in tropical region is not recommended. © 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

⁎ Corresponding author at: Aquatic Ecology and Water Quality Management group, Wageningen University, P.O. Box 47, 6700 AA Wageningen, the Netherlands. E-mail address: [email protected] (P.J. Van den Brink).

https://doi.org/10.1016/j.scitotenv.2021.147167 0048-9697/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

1. Introduction et al., 2019) and cosm studies (Pestana et al., 2009; Colombo et al., 2013; Cavallaro et al., 2018; Rico et al., 2018; Sumon et al., 2018)have Fulfilling the food demand of the growing world population is the demonstrated impacts of imidacloprid on aquatic organisms, particu- key driver for the intensive use of agrochemicals by the agricultural sec- larly on arthropods, at field relevant concentrations. The majority of tor (Sánchez-Bayo, 2010; Schäfer et al., 2010). The annual use of pesti- the studies are from temperate climatic regions with two exceptions cides in agriculture has increased markedly over the years. For from (sub-)tropical (Sumon et al., 2018) and Mediterranean (Rico example, the Food and Agriculture Organization of the United Nations et al., 2018) regions. (FAO, 2019) showed that the agriculture global use of the pesticides in Geographical origin may possibly affect species sensitivity to 2017 (5.9 million tonnes of active ingredient (a.i.)) was 92% higher com- chemicals (Kwok et al., 2007; Diepens et al., 2014). Rico et al. (2018) pared to the usage in 1991 (3.1 million tonnes, a.i.). and Sumon et al. (2018) reported differences in toxicity of imidacloprid Imidacloprid, 1-(6-chloro-3- pyridylmethyl)-N-nitroimidazolidin-2- to freshwater arthropods between temperate, Mediterranean and ylideneamine, is a neonicotinoid insecticide introduced into the market (sub)-tropical species, where (sub)-tropical and Mediterranean aquatic in the 1990s by Bayer CropScience (Anderson et al., 2015). It is a systemic arthropods were more sensitive than temperate species. Therefore, we pesticide with agonistic effect to the acetylcholine receptor. Imidacloprid investigated the effects of the insecticide (imidacloprid) to aquatic life causes effects on target pests and non-target species such as insects that under tropical conditions such as in Ethiopia to further investigate this possess the receptor through contact and stomach action (Capowiez reported variation of results between climatic regions. Furthermore, mi- and Berard, 2006; Rico et al., 2018). Of the neonicotinoid insecticides, crocosm and mesocosm studies performed with imidacloprid studying whichhaveashareof24%oftheglobalinsecticidemarket,imidacloprid the recovery of affected populations are scarce (Hayasaka et al., 2012; has been reported as the most widely used insecticide around the world Cavallaro et al., 2018; Rico et al., 2018), despite the importance of study- in various sectors including agriculture to control pests such as sucking in- ing recovery as part of ecological risk assessment to evaluate the accept- sects (Anderson et al., 2015; Cavallaro et al., 2018; Rico et al., 2018). Its ability of observed effects (Gergs et al., 2016). Recovery studies are high toxicity to target pests and low toxicity to mammals compared to important to evaluate how fast an ecosystem can return to a state com- other insecticides increased the acceptance of imidacloprid among users parable to an untreated control (Van den Brink et al., 1996; Brock et al., (Capowiez and Berard, 2006; Colombo et al., 2013). Imidacloprid is ap- 2000; Beketov et al., 2008; Gergs et al., 2016), while they can also enable plied as seed dressing, soil treatment, and foliar treatment to a variety evaluation of delayed effects (Hayasaka et al., 2012). of crops and orchards (Capowiez and Berard, 2006). Moreover, Given these data gaps, the objectives of this study were: to 1) assess imidacloprid has domestic uses such as the control of cockroaches, ter- the effects of imidacloprid on structural and functional endpoints of mites and flea treatment for pets, and to garden and lawn care aquatic ecosystems under tropical conditions; 2) investigate post expo- (Capowiez and Berard, 2006; Duchet et al., 2018). Currently, use of sure recovery of aquatic communities and ecosystem function, by using imidacloprid is highly controversial due to its potential effects on non- outdoor mesocosms. target species such as bees (Lundin et al., 2015) and aquatic organisms (Smit et al., 2015), leading to a ban on outdoor use in Europe in 2018 2. Materials and methods (European Commission, 2018). Although imidacloprid is not intentionally applied to aquatic ecosys- 2.1. Acute toxicity tests tems, it can enter water bodies through airborne (e.g. spray drift during aerial application) and waterborne (e.g. runoff from agricultural during Acute toxicity tests to evaluate the single species toxicity of precipitation, leaching and irrigation) pathways (Schäfer et al., 2010; imidacloprid were performed with Cloeon dipterum, Caenis horaria, Rico et al., 2018). Its high solubility in water (0.610 mg/L at 1 atm and Plea minutissima and Corixidae sp. The species were collected from un- 25 °C) and persistence in soil (soil half-live = 9–1250 days) increases contaminated streams proximate to Ambo University located in Ambo, the widespread occurrence of imidacloprid in surface waters Ethiopia, a city to the west of Addis Ababa with an altitude of 2102 m. (Anderson et al., 2015; Morrissey et al., 2015; Duchet et al., 2018). The organisms were transferred into a bucket containing 50% pre- Only a few monitoring studies are available showing surface water conditioned test water and 50% stream water. The species were accli- residual concentration of imidacloprid in Africa, probably due to a lack mated for three days to laboratory conditions at room temperature of available analytical facilities. Imidacloprid was reported in agricul- prior to the experiment with aeration and feeding using substrates col- tural river watersheds (Krom River, Berg River and Hex River) of lected from the stream. The tests were carried out between 04 April South Africa (Curchod et al., 2020), and in the effluent from floriculture 2019 and 12 May 2019. The experimental setup was established accord- enterprises in the area discharging into Lake Ziway of Ethiopia (Jansen ing to Roessink et al. (2013) with minor modifications. Briefly, 1 L jars and Harmsen, 2011) with detectable water concentrations (mean and filled with 900 mL pre-conditioned test water were used as test systems range) of 2.3 (0.73–7.2) μg/L and 0.16 (0.04–0.3) μg/L, respectively. and 10 individuals and a stainless steel mesh were introduced into each Mean concentrations of imidacloprid ranging from <0.002–16 μg/L replicate with the mesh serving as additional surface for the animals to were reported in surface waters with agricultural land use in developed minimize additional stress. Including control, seven treatments (0, 0.1, countries such as USA, Japan, Canada, Sweden and Australia (Morrissey 0.3, 1, 3, 10, 30 μg/L for C. dipterum C. horaria and Corixidae sp., and 0, et al., 2015). Our survey about farmers' pesticide use performed in 2017 1, 3, 10, 30, 100, 300 μg/L for P. minutissima)wereused,withthreerep- (Merga, 2021) indicated that imidacloprid formulation with Gain 20 SL licates per treatment. The test jars were placed in a water bath, which trade name is widely used by smallholder vegetable farmers in the cen- had continuous water in- and out-flow to regulate the temperature of tral Ethiopian rift valley region of Ethiopia where Lake Ziway is located. the test systems (21 ± 0.5 °C) and a photoperiod of 12:12 h (h) dark- Similarly, Negatu et al. (2016) reported the use of the neonicotinoid in- light was employed to mimic the photoperiod of the region (Fig. SI1 c secticides thiamethoxam and imidacloprid in this region. Imidacloprid and d). Imidacloprid formulation (CON-FIDENCE, China) containing is also among the recommended pesticides to control thrips in 350 g imidacloprid a.i./L was used to prepare 90 mg/L, 9 mg/L, 0.9 Ethiopia, particularly in the central Ethiopian rift valley region mg/L and 0.09 mg/L stock dosing solutions. Appropriate volumes that (Negash et al., 2020). Thus, the aquatic ecosystems in Ethiopia, particu- ranged from 1 mL to 3 mL from the appropriate stock were spiked larly those found in the central Ethiopian rift valley region (e.g., Lake into the system using a micropipette. No aeration of the system and Ziway), are contaminated by residual levels of neonicotinoid insecti- feeding of the test animals occurred during the 96-h (h) experimental cides, of which imidacloprid is mostly used and detected. period. After 24-h, 48-h, 72-h and 96-h of imidacloprid exposure time Many single species toxicity tests (Roessink et al., 2013; Morrissey in each test system, the mortality and immobility of individual animals et al., 2015; Van den Brink et al., 2016; Sumon et al., 2018; Bartlett was assessed. Individual test organisms were scored as dead when no

2 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167 response of any kind was observed for 3–5 s and immobile when there phytoplankton chlorophyll-a concentrations were expressed in mg/m2 was no observed movement over a time period of 20 s after a tactile and μg/L, respectively. stimulation using a Pasteur capillary pipette. The toxicity test was con- For zooplankton sampling, a depth-integrated water sample of 5 L sidered invalid when control immobility exceeded 15% (OECD, 2011). was collected from each cosm and passed through a 55 μm mesh plank- Water quality parameters including pH, dissolved oxygen (DO), tem- ton net and concentrated to 50 mL. The samples were collected on 7 and perature and electrical conductivity (EC) were measured daily from 1 d pre-treatment and on 2, 9, 16, 23, 30, 44, 58, 86, 114, and 142 d after the start until the end of the experiment in all test systems. the first imidacloprid application. The zooplankton samples were treated with 10% buffer formalin solution and stored at 4 °C until further 2.2. Mesocosm experiment analysis. A binocular microscope was used for taxa identification and as- sessment of the abundance of the zooplankton taxa from a 1 mL sub- 2.2.1. Experimental setup sample. Macrozooplankton species were not found in our cosms. Back The mesocosm study was conducted at Ambo University, Ambo, calculation was employed to obtain the number of zooplankton per vol- Ethiopia (8°58′57.48“ N and 37°50’44.54” E). The experimental area ume of water (#/L). was fenced, and roofed using transparent plastic sheets used for green- house roofing (Fig. SI1 a and b). Locally available cylindrical polyvinyl 2.2.3. Macroinvertebrates chloride (PVC) containers (height: 85 cm and diameter: 110 cm) were Two pebble baskets (volume = 0.0123 m3)thatservedasartificial used to establish the mesocosm ponds. Twelve (12) PVC containers substrates were introduced into each cosm and put on the brick two were buried (about 60 cm of its height) into the ground to minimize weeks before the first imidacloprid application. The baskets were sam- temperature fluctuations between day and night (Daam and Van den pled alternatively on 7 and 1 d pre-treatment and on 7, 14, 21, 28, 42, Brink, 2011) (Fig. SI1 a). The cosms were filled with natural sediment 56, 84, 112, 140 and 168 d after the first application of imidacloprid. to about 15 cm height, which was collected from an unpolluted pond During the sampling, one of the pebble baskets was gently retrieved near Ambo University. Subsequently, 600 L pre-conditioned water from the brick and directly enveloped with a net (500 μm mesh net). from which the residual chlorine was removed, was added to each con- Macroinvertebrates were carefully collected from the substrate and tainer. After one week of water addition to the systems, the cosms were the net into a white tray after which the pebble basket was returned stocked with macroinvertebrate species and concentrated plankton, to the cosm to its original position. To collect free swimming inverte- which were collected from natural and unpolluted ponds and streams brates, the 500 μm mesh net was passed through water column and located near to the experimental sites. Following invertebrate and one-fourth of the walls surface (Sumon et al., 2018). On each sampling plankton stocking, but prior to the application of imidacloprid, the sys- date Chironomid larvae were collected from the sediment using a poly- tems were left for seven months to allow the establishment of biological vinyl chloride (PVC) pipe (about 4 cm internal diameter). The collected communities. During this 7 month establishment period any reduction macroinvertebrates were taxonomically identified to family level ex- of water volume due to evaporation was compensated by adding pre- cept for C. dipterum, C. horaria and P. minutissima,andcounted.The conditioned water. In addition, 20% of the water (volume) was ex- alive macroinvertebrates were released back to their original cosms changed between the cosms every two weeks to promote homogeneity (Rico et al., 2014; Sumon et al., 2018). among the systems (Sumon et al., 2018). During the experimental pe- riod, nitrogen (1.4 mg/L nitrogen, urea) and phosphorus (0.18 mg/L P, 2.2.4. Organic matter decomposition trisodium phosphate) were added every three weeks to the systems Nylon litter bags (300 μm mesh size) were used to investigate any (Rico et al., 2014; Sumon et al., 2018). After the establishment time of effects on the microorganism community by assessing the organic mat- seven months (30-05-2019), the model ecosystems were weekly ter decomposition rate. Banana leaves were leached in pre-conditioned dosed for four weeks with three imidacloprid treatment levels (treat- tap water for 48-h to remove easily leachable humic substances (Sumon ment-1 (0.01 μg/L), treatment-2 (0.1 μg/L) and treatment-3 (1 μg/L)) et al., 2018). Then the conditioned leaves were dried at 70 °C for 48-h in and control (i.e., not dosed with the insecticide) with three replicates an oven. About 2 g of the dried banana leaves was introduced into a lit- each, using freshly prepared stock imidacloprid solutions from the ter bag. Eight bags were introduced into each cosm 1 d before the first imidacloprid formulation containing 350 g a.i./L. The treatment-1 (T- imidacloprid application. The litter bags were placed at a depth of ap- 1), treatment-2 (T-2) and treatment-3 (T-3) cosms were dosed with proximately 30 cm below the water surface. One of the 8 litter bags 10 mL of the 0.6 mg/L, 6 mg/L and 60 mg/L stock solutions, respectively. was retrieved on 2, 16, 28, 42, 56, 70, 84 and 98 d after the first imidacloprid application. The remaining leaf material from the retrieved 2.2.2. Phytoplankton, periphyton, and zooplankton bag was carefully collected into clean glass petri-dish and dried at 70 °C Phytoplankton samples were collected at 7 and 1 day (d) before the for 48 h and weighed. The percentage of weight loss per day was calcu- first pesticide application and 2, 9, 16, 23, 30, 44, 58, 86, 114 and 142 d lated and recorded as the organic matter decomposition rate. post first application. To measure phytoplankton chlorophyll-a, water samples (100 mL) were collected from each cosm at a depth of 30 cm 2.2.5. Physicochemical parameters and filtered using a GF/C glass-fibre filter. The GF/C glass-fibre filter Water quality variables including dissolved oxygen (DO), pH, elec- were wrapped in aluminium foil and stored at −20 °C until analysis. Pe- trical conductivity (EC) and temperature were measured in-situ using riphyton chlorophyll-a was assessed by introducing three glass slides a portable multi-meter (HQ40d, HACH). For the variables total hard- (surface area = 37.5 cm2) into each cosm at 30 cm depth below water ness, alkalinity, ammonia, nitrate and phosphate concentrations, water surface 7 days before the first application of imidacloprid. The slides samples of about 500 mL were collected from each cosm and analysed from each cosm were retrieved on 0 d (before pesticide application) in the laboratory according to USEPA (1983). Water quality measure- and 7, 14, 21, 28, 42, 56, 84, 112 and 140 d after the first imidacloprid ments were performed on 7 and 1 d pre-treatment, and 0, 7, 14, 21, application. At each sampling date, the attached periphyton was 28, 42, 56, 84, 112, 140 and 168 d after the first application of scraped into a glass beaker containing 100 mL tap water and filtered imidacloprid. using a GF/C glass-fibre filter and also stored at −20 °C until analysis. Af- terwards, the glass slides were put back in the cosms in their original 2.3. Concentration verification of imidacloprid exposure position. Chlorophyll-a of periphyton and phytoplankton was quanti- fied using ultraviolet–visible (UV-VIS) spectrophotometric technique As discussed, an imidacloprid formulation (trade name: CON- (Rico et al., 2014) after extraction of the chlorophyll-a from the GF/C FIDENCE, China) containing 350 g imidacloprid a.i./L was used to pre- glass-fibre filter using acetone solvent. The periphyton and pared stock dosing solutions, which were 90 mg/L, 9 mg/L, 0.9 mg/L

3 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167 and 0.09 mg/L for single species toxicity tests, and 60 mg/L, 6 mg/L and 3. Results and discussion 0.6 mg/L for the mesocosm study. Samples (10 mL) from the stock dos- ing solutions were collected into amber glass for verification and stored 3.1. Acute single species toxicity tests at 4 °C until analysis. Samples were also collected from acute and cosm test systems to in- The average concentration of imidacloprid measured in the dosing vestigate the fate and verify the exposure test concentrations. In the stock solutions used for single species toxicity test was 105 acute toxicity tests water samples (approximately 100 mL) were (91.3–123)% of the intended concentrations. The average exposure con- taken from the control and the lowest and the highest test concentra- centrations of imidacloprid in treatments at the start and end of the tions 1 h after the application and at the end of the experiment (96 h). experiment were 104 ± 18% and 107 ± 19% of the nominal concentra- Similar volumes of water were collected from the cosms just after the tions, respectively. This indicates that each treatment in the acute tests application and just before the next application, i.e. on 1 h, 1, 7, 7.1, received an appropriate amount of imidacloprid. We, therefore, used 14, 14.1, 21 and 21.1 days. Samples were also taken during the post ap- the nominal concentrations for the data analysis. plication period on 28, 42, 56, 84 and 112 days after the first Van den Brink et al. (2016) reported a recovery of 86–94% of the imidacloprid application. intended concentration of imidacloprid after 4 days of an acute toxicity Solid phase extraction (SPE) using Oasis HLB cartridges was used for experiment performed under 12:12 photoperiod and 18 ± 1 °C. The re- the extraction of imidacloprid from the collected water samples. Prior to sult reported by Van den Brink et al. (2016) is comparable to our result. the sample loading, the cartridge was conditioned with 3 mL methanol This is likely due to the similarity in employed light conditions as degra- followed by 2 × 3 mL ultrapure water. The water sample was loaded to dation of imidacloprid in water is mainly due to photolysis (Bonmatin the conditioned SPE under vacuum and the loaded SPE was washed im- et al., 2015). Low recovery (47%) was reported by Sumon et al. (2018) mediately with ultrapure water (3 mL). The cartridge was dried under after 4 days of acute exposure under high temperatures (27 ± 0.6 °C). vacuum for 1 min before being wrapped with aluminium foil, placed The photoperiod was not specified by Sumon et al. (2018), but they de- in a plastic bag and kept in the freezer (−20 °C) till further analysis. Fol- scribed it as a high intensity, explaining the relatively large loss. lowing defrosting of the loaded SPE, the residual imidacloprid was No significant increasing or decreasing trend in the levels of the eluted from the cartridge using 2 × 0.5 mL acetonitrile, then bring the monitored water quality variables (DO, EC, pH and temperature) was eluate to 2 mL by adding ultrapure water. observed during the experimental period of the acute single species Imidacloprid quantification was performed using liquid tests. As also reported by Roessink et al. (2013), a concentration depen- chromatography-tandem mass spectrometry (LC-MS) as described by dent slight increase in EC value was observed in this study, most possi- Roessink et al. (2013). bly a direct result of the addition of the test substance. The observed value range of the parameters were 6.67–8.91 mg/L, 19.5–22.4 °C, 2.4. Data analysis 215–238 μs/cm, 7.42–8.04 for DO, temperature, EC and pH, respectively (Table SI1). The acute toxicity test results for C. dipterum, C. horaria, During the acute tests, a low control immobility ranging from 3.3 to P. minutissima and Corixidae sp. were used to calculate lethal concentra- 6.7% was observed in tests with C. dipterum, C. horaria and P. minutissima tions (LC) and effect concentrations (EC) at which 10%, 50% and 90% of after 96-h of exposure (Table 1). The highest control immobility (16.7% the tested species were affected. Log-logistic regression as programmed at 96-h) was observed for the test with Corixidae sp., which is slightly in the software GenStat 11th (VSN International Ltd., Oxford, UK) ac- higher than the accepted threshold value (15%) according to OECD cording to Rubach et al. (2011) was used to calculate the LC10, LC50 (2011) for the standard test organism Chironomus sp. with 48-h expo- and LC90, and EC10, EC50 and EC90 values. sure time. The 48-h control immobility for Corixidae sp. was 3.3%, and The Williams test (Williams, 1972), as available in the Community thus significantly lower than the OECD threshold value. Analysis computer program, version 4.3.05 (Hommen et al., 1994), Our acute toxicity results showed that C. dipterum (96-h LC50 = 2.7 was used to calculate no-observed-effect-concentration (NOEC) values μg/L; EC50 = 1.5 μg/L) is the most sensitive species, followed by for the mesocosm variables including water quality parameters, C. horaria (96-h LC50 = 3.4 μg/L; EC50 = 1.9 μg/L), Corixidae sp. (96- chlorophyll-a levels of phytoplankton and periphyton, abundance of h LC50 = 6.8 μg/L; EC50 = 3.6 μg/L) and P. minutissima (96-h LC50 = zooplankton and macroinvertebrate taxa and organic matter decompo- 68 μg/L; EC50 = 36 μg/L) (Table 1). P. minutissima was found to be the sition rates. Prior to the analyses, the abundance data of zooplankton most tolerant species compared to the other tested arthropods with and macroinvertebrates were ln(Ax+1) transformed. Detailed informa- 96-h LC50 and EC50 values being 23–25 times higher than the effect tion about the determination of A and the rationale behind this transfor- values calculated for the most sensitive species, C. dipterum. The de- mation is available in Van den Brink et al. (2000). tailed effect concentrations for 24-h to 96-h exposure days and the Datasets of zooplankton and macroinvertebrates were analysed by raw toxicity datasets are presented in Table SI2 and Table SI3, the principal response curve (PRC) method using the CANOCO Software respectively. package, version 5 (Van den Brink and Ter Braak, 1999; Ter Braak and The 96-h LC50 values we observed for C. dipterum were lower by a Smilauer, 2012). The PRC method is a specific type of redundancy anal- factor of 10 and 13 compared to the values reported for temperate ysis (RDA) that extracts the variation in community composition which summer (Roessink et al., 2013) and winter generations (Van den can be attributed to the stressor from the total variation. It does so by in- Brink et al., 2016), respectively (Table 2a). The 96-h EC50 of the species cluding the treatment regime and its interaction with time as explana- (C. dipterum) calculated in this study was also lower by a factor of 17 tory variables, and the sampling date as a covariate. The overall compared to a temperate winter generation (Van den Brink et al., significance of the effect of imidacloprid treatment on the variation in 2016), but equivalent with the value reported by Roessink et al. community composition (p ≤ 0.05) was tested by performing Monte (2013) for a summer generation. Moreover, compared to the effect Carlo permutations (Van den Brink and Ter Braak, 1999). As the amount values (96-h LC50 = 1152 μg/L and 96-h EC50 = 23.1 μg/L) reported of replication was not enough to test each treatment against the control for overwintered Cloeon sp. generation sampled in early spring (May) for each sampling date using Monte Carlo permutation tests under the in Canada (Raby et al., 2018), our results are lower by a factor of 427 RDA option, the community data were reduced to a single number per and 15 for 96 h- LC50 and EC50, respectively (Table 2a). cosm for each sampling date using principal component analysis Similarly, Sumon et al. (2018) reported lower effect values (96-h LC50 (PCA). The PCA coordinates were subsequently analysed by the Wil- = 0.024 μg/L and EC50 = 0.0055 μg/L) for Cloeon sp. from the sub- liams test to calculate a NOECcommunity for each sampling date (Van tropical climate in Bangladesh compared to the temperate regions den Brink et al., 1996). (Table 2a).

4 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

Table 1 Results of single species acute toxicity studies of imidacloprid to four freshwater arthropods. Effect concentrations are expressed as 96-h L(E)C50 and 96-h L(E)C10 values in μg/L.

Test species Mortality Immobility Control mortality/immobility (%)

96-h LC10 96-h LC50 96-h EC10 96-h EC50

Cloeon dipterum 0.54 (0.2–1.4) 2.7 (1.7–4.3) 0.3 (0.11–0.83) 1.5 (0.96–2.4) 6.7 Caenis horaria 1.4 (0.81–2.4) 3.4 (2.5–4.5) 1.2 (0.65–2.2) 1.9 (1.4–2.7) 6.7 Corixidae sp. 1.5 (0.59–4.0) 6.8 (4.3–11) 0.96 (0.4–2.2) 3.6 (2.4–5.6) 16.7 Plea minutissima 15 (5.9–40) 68 (43–107) 9.6 (4.1–22) 36 (24–56) 3.3

LC50 = median lethal concentration; CI = confidence interval; LC10 = lethal concentration for 10%; EC50 = median effective concentration; EC10 = effective concentration for 10%.

The 96-h lethal (LC50) and immobility (EC50) toxicity values we es- outdoor microcosm study under sub-tropical climatic conditions in timated for C. horaria and P. minutissima were lower than the values re- Bangladesh, a dissipation of about 47–55% of the compound after 7 ported for temperate winter populations (Van den Brink et al., 2016)by days was reported, with an average cosm temperature of 28 ± 2 °C a factor ranging from 4.2–8.2 and 3.2–5.2, respectively. Our results for under sunny circumstances with the sunlight filtered by a transparent these species are comparable with the values reported by Roessink plastic slate (Sumon et al., 2018). Under Mediterranean climatic condi- et al. (2013) for the temperate summer generations of the same species. tions with a cosm temperature ranging from 16 to 25 °C, a half-life of 10 Furthermore, our 96-h LC50 and EC50 values for Corixidae sp. were days was reported by Rico et al. (2018) in a mesocosm study performed lower by a factor of 4 and 3, respectively, compared to the effect values between April and June. In both studies (i.e., by Rico et al., 2018; Sumon of summer generation of Micronecta sp. (Roessink et al., 2013)andbya et al., 2018), the authors stated the presence of particulate matter, con- factor of 66 and 17, respectively, relative to the values reported for win- tinuous growth of algae and macrophytes as mitigating factors for the ter generation Trichocorixa sp., also belonging to the Corixidae family observed low dissipation of imidacloprid. (Raby et al., 2018)(Table 2a). The mesocosms established in this study were roofed with transpar- These differences in sensitivity between summer and winter gener- ent plastic slates similar to the cosms used by Sumon et al. (2018) and ations, and temperate and tropical populations (see discussion below in the temperature range (16.6–20.9 °C) of our cosms overlaps with the section 3.3) could point towards an effect of metabolic state on the sen- range reported by Rico et al. (2018). However, imidacloprid was applied sitivity of species (e.g., Cloeon sp.), as a result of differences in in the wet season (June – July) in our study, while the experiments by toxicokinetics, e.g. increased uptake (Van den Brink et al., 2016). How- Sumon et al. (2018) and Rico et al. (2018) were performed during ever, further experiments are needed to find the mechanistic links caus- sunny periods. Thus, based on a comparison of experimental setup ing the differences in sensitivity and spatio-temporal backgrounds of and climatic conditions between our cosm experiment and the cosm the tested populations. studies by Rico et al. (2018) and Sumon et al. (2018), we hypothesised a half-life of imidacloprid in our mesocosm study of ≥10 days. Therefore, 3.2. Mesocosm experiment a build-up of the concentration is to be expected, meaning that the max- imum concentration after 4 applications on day 21 could have been be- 3.2.1. Verification of imidacloprid concentrations tween a factor of 2 (DT50 = 10 d) and 4 (DT50 = ∞) higher than the The measured average concentration of imidacloprid in the dosing concentration after the first application. The TWA estimation result stock solutions used for mesocosm experiment was 118 (110–128)% (Fig. SI2) also indicated the slow dissipation of imidacloprid and con- of the intended concentrations. The estimated time weighted average firms the build-up the insecticide in the cosms of the T-2 and T-3

(TWA) concentrations over 112 days (TWA112d) are 0.124 μg/L for treatments. treatment-2 (nominal 0.1 μg/L) and 2.09 μg/L for treatment-3 (nominal 1 μg/L) (Fig. SI2). The higher concentrations than expected is a result of a 3.2.2. Invertebrates response slight overdosing on day 14 (Fig. SI2). Unfortunately, the TWA of the Overall, our cosm results showed that the effect threshold values for lowest treatment (T-1) and control cannot be reported as we observed invertebrates seem to be lower than the reported values in temperate contamination of the samples after storage. The contamination likely region. Such variations can be due to higher temperature in tropical ex- occurred by spillage of a dosing stock solution while the samples were periments (Kwok et al., 2007; Macaulay et al., 2020) and differences in transported to the Netherlands for quantification, as the dosing solu- life cycle of the test species (Van den Brink et al., 2016). The sensitivity tions containing vials and SPE cartridges were packed in one container. differences observed between this and other studies are discussed in Based on the amount spiked from dosing solution, the test volume and section 3.3. the calculated TWA, the result showed that each cosm belonging to the T-2 and T-3 treatments received the appropriate amount of 3.2.2.1. Zooplankton. Our pond mesocosms were dominated by five imidacloprid. Based on these results, the TWA112d of the 0.01 μg/L treat- Rotifera taxa (Brachionus sp., Filinia sp., Keratella sp., Polyarthra sp. and ment (T-1) is expected to be between 0.0124 and 0.0209 μg/L. For clarity Trichocerca sp.) and two Copepoda taxa (nauplii and Afrocycops sp.). we used the TWA112d concentration of 0.02 μg/L to define the T-1 treat- Cladocerans were not found during the whole experimental period in ment level, any of the cosms. The PRC shows that the imidacloprid treatment ex- Light and temperature are important environmental conditions af- plained a significant (P–value = 0.002) part of the variation in the zoo- fecting the dissipation of imidacloprid in aquatic ecosystem (Lu et al., plankton community composition between the different treatments 2015; Hayasaka et al., 2019). Therefore, a relatively fast dissipation of during the experimental time (Fig. 1). The maximum effect of the compound is expected in (sub)-tropical climates. Indeed, fast dissi- imidacloprid on the zooplankton community was observed on day 23 pation of imidacloprid has been reported for a pond microcosm (half- and day 30 after the first imidacloprid application (NOECcommunity life = 28 ± 8 h; cosm temperature = 10–24 °C) exposed to high levels below the concentration of T-1 (< 0.02 μg/L)), coinciding with the of ultraviolet radiation (Colombo et al., 2013), as well as for experimen- first week after the last application (Table 3). tal rice paddies (half-life 0.9–3 days; rice mesocosm temperature = According to the estimated species weight (kb), the rotifer 20–31 °C) established in summer (July) in Portugal (Daam et al., Brachionus sp. showed the largest response to the imidacloprid treat- 2013; Pereira et al., 2017). However, the presence of suspended partic- ments followed by nauplii and Filinia sp., while Trichocerca sp. showed ulate matter, e.g. due to a high abundance of algae and macrophytes, can a small response compared to the other species (Fig. 1). For all zoo- reduce the dissipation of imidacloprid by photolysis. For instance, in an plankton species, except Afrocyclops sp., we found consistent significant

5 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

Table 2 Acute and chronic toxicity, and cosm study no observed effect concentration (NOEC) values of imidacloprid to invertebrates reported in the literature.

Species Acute toxicity Chronic toxicity Cosm NOEC values Country

96 h LC50 (μg/L) 96 h EC50 (μg/L) Toxicity endpoint L(C)x(μg/L) Exposure duration NOEC (μg/L)

a. Macroinvertebrates Cloeon dipterum (SG) 26.3a 1.0a 28-d EC10 0.033a ––Netherlands Cloeon dipterum (WG) 34b 25b ––––Netherlands Cloeon dipterum ––– –28-d < 0.09f Spain Cloeon dipterum 2.7e 1.5e ––21-d 0.02e Ethiopia Cloeon sp. (WG) 1152c 23.1c ––––Canada Cloeon sp. 0.024d 0.0055d ––9-d < 0.03d Bangladesh Epeorus sp. ––– –20-d 0.3n Canada Caenis horaria (SG) 6.68a 1.8a 28-EC10 0.024a Netherlands Caenis horaria (WG) 28b 6.0b ––––Netherlands Caenis sp. (WG) < 21.8c < 21.8c ––––Canada Caenis sp. ––– –– 2.3g,p Germany Caenis horaria 3.4e 1.9e ––14-d <0.02e Ethiopia Chironomidae sp. ––– –49-d 5.2g Germany Chironomus dilutus ––14-d LC20 0.47k ––Canada Chironomus riparius ––10-d LC10 1.64l ––Czech Republic Chironomid larvae ––– –28-d 0.3d Bangladesh Chironomini ––– –10-d < 0.2f Spain Chironomidae sp. ––– –21-d 0.02e Ethiopia Culicidae sp. ––– –21-d 0.02e Ethiopia Notonecta tiguttata ––– –140-d 157h Japan Notonecta sp. ––– –16-d 0.03d Bangladesh Notonectidae sp. ––– –14-d 0.124e,p Ethiopia Micronecta sp. (SG) 28.2a 10.8a ––––Netherlands Trichocorixa sp. (WG) 450c 63.1c ––––Canada Corixidae sp. 6.8e 3.6e ––42-d 0.02e Ethiopia Gerris sp. ––– –16-d 0.03d Bangladesh Gerris latiabdominis ––– –120-d 49i Japan Gerridae sp. ––– –42-d 0.02e Ethiopia Plea minutissima (SG) 37.5a 36a 28-d LC10 2.03a ––Netherlands Plea minutissima (WG) 287b 189b ––––Netherlands Plea minutissima 68e 36e ––21-d 0.124e,p Ethiopia Hydaticus sp. ––– –28-d < 60j Portugal Dytiscidae sp. ––– –14-d 0.02e Ethiopia Planorbella pilsbryi ––28-d EC10 15.4m ––Canada Physidae sp. ––– –21-d 0.124e,p Ethiopia Planorbidae sp. ––– –28-d 0.124e,p Ethiopia

b. Zooplankton Diaptomus sp. 6.5d 0.0386d ––16-d < 0.03d Bangladesh Keratella sp. ––– –9-d < 0.03d Bangladesh Keratella quadrata ––– –42-d 1f Spain Keratella sp. ––– –23-d 0.02e Ethiopia Polyarthra sp. ––– –16-d < 0.03d Bangladesh Polyarthra sp. ––– –16-d 0.02e Ethiopia Brachionus sp. ––– –28-d 0.03d Bangladesh B. calyciflorus ––10-NOEC 6220o ––Iran Brachionus sp. ––– –30-d 0.02e Ethiopia Filinia sp. ––– –23-d 0.3d Bangladesh Filinia sp. ––– –23-d 0.02e Ethiopia Trichocerca sp. ––– –23-d 0.3d Bangladesh Trichocerca sp. ––– –30-d 0.02e Ethiopia Cyclops sp. ––– –9-d 0.3d Bangladesh Cyclopoida ––– –17-d 1f Spain Afrocyclops sp. ––– –30-d < 0.02e Ethiopia nauplii ––– –9-d 0.03d Bangladesh nauplii ––– –3-d 5f Spain nauplii ––– –23-d 0.02e Ethiopia

Note: WG = winter generation; SG = summer generation. Reference f and j are from Mediterranean climate, d is from sub-tropical and e from tropical. The others are from temperate climate. a Roessink et al. (2013). b Van den Brink et al. (2016). c Raby et al. (2018). d Sumon et al. (2018). e This study. f Rico et al. (2018). g Colombo et al. (2013). h Kobashi et al. (2017). i Hayasaka et al. (2012). j Pereira et al. (2017). k Cavallaro et al. (2017). l Chandran et al. (2018). m Prosser et al. (2016). n Alexander et al. (2008). o Gharaei et al. (2020). p Indicates Time Weighted average (TWA) values. 6 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

1.5 Brachionus spp. imidacloprid treatments in the study of Sumon et al. (2018) in Bangladesh (Table 2b). As only scarce data are available on the toxicity of imidacloprid to rotifers, it is difficult to draw a conclusive statement nauplii for the observed treatment related decline in abundance of Rotifera Filinia sp. taxa in this study and other cosm studies from the Mediterranean 1 (Rico et al., 2018) and sub-tropical (Sumon et al., 2018)regions Afrocyclops sp. Keratella sp. (Table 2b). Polyarthra sp. The magnitude of effect on the zooplankton community decreased Trichocerca sp. after 30 days after the first application and showed a full recovery

0.5 after 86 days (i.e. 65 days after the last application). However, some spe- cies were not recovered yet at the population level (Table 3). Similarly, for mesocosm ponds treated with a single dose of ≤25 μg/L imidacloprid, Rico et al. (2018) reported a recovery time of 56 days for the zooplank- 0.25 kb ton community. Moreover, Hayasaka et al. (2012) reported a recovery 0 0 time of 63 days for the zooplankton community of a mesocosm paddy 0 -0.25 mimicking a rice field in Japan initially treated with 49 μg/L of μ -0.5 imidacloprid which quickly (i.e., in 3 days) declined to 1 g/L dt

C (Table SI4). This suggests that for tropical aquatic ecosystems repeat- -0.75 edly exposed to environmentally relevant low concentrations of -1 imidacloprid, the zooplankton community can be affected for a long pe- -1.25 riod of time before the group returns to its untreated condition. -1.5 -25 0 25 50 75 100 125 150 3.2.2.2. Macroinvertebrates. Eleven taxa of macroinvertebrates were Days post first application identified in our mesocosm ponds. The taxa included Ephemeroptera Control 0.01 µg/L 0.1 µg/L 1 µg/L (C. dipterum and C. horaria), Diptera (Culicidae sp. and Chironomidae sp.), Hemiptera (Corixidae sp., Pleidae sp., Gerridae sp. and Fig. 1. Principal Response Curve (PRC) depicting the effect of imidacloprid contamination on zooplankton community over the course of experimental period. The sample weights Notonectidae sp.), Coleoptera (Dytiscidae sp.) and Gastropoda

(Cdt) indicate differences between the treatments and the control at the different (Planorbidae sp. and Physidae sp.). In abundance, the gastropods were sampling dates. The affinity of each taxon of the community with the response indicated the dominant taxa during the experimental period. The PRC (Fig. 2)in- by the PRC is provided by the species weight (kb). dicates that the macroinvertebrate community composition was signif- icantly (P-value = 0.01) altered by the imidacloprid treatments, as is treatment related effects on their abundance in the two highest also reported in many other cosm studies (Table SI4; Table 2a) imidacloprid treatments (i.e., at least on two consecutive samplings) (Alexander et al., 2008; Pestana et al., 2009; Pereira et al., 2017; Rico (Table 3). Consistent effects for Afrocyclops sp. were found in all treat- et al., 2018; Sumon et al., 2018). The estimated no observed effect con- ments (Table 3). Imidacloprid treatment-related responses of rotifers centration for the macroinvertebrate community (NOECcommunity)was and copepods were reported in cosm studies performed in Bangladesh 0.02 μg/L (Table 4), indicating that the two highest imidacloprid concen- (Sumon et al., 2018)andinSpain(Rico et al., 2018). The NOECs reported trations significantly changed the community composition of the mac- by Sumon et al. (2018) in Bangladesh for the rotifers Keratella sp., roinvertebrate community. Polyarthra sp., Brachionus sp., Filinia sp. and Trichocerca sp. were < In this study, C. dipterum (7-d NOEC = 0.02 μg/L), C. horaria (14-d 0.03 (23-d), < 0.03 (16-d), 0.03 (28-d), 0.3 (23-d) and 0.3 (23-d) μg/L, NOEC <0.02 μg/L) and Culicidae sp. (21-d NOEC = 0.02 μg/L) showed respectively (Table 2b). The values were comparable to our results for the largest response to the imidacloprid treatments (i.e., higher species the rotifer species, except for Filinia sp. and Trichocerca sp. for which weight, kb values) (Fig. 2) relative to the other macroinvertebrates. Our our results are lower by about one order of magnitude (Table 3; NOEC for C. dipterum was in agreement with the effect values reported Table 2b). Rico et al. (2018) reported a NOEC of 1 μg/L for the rotifer in the cosm studies performed in Spain (Rico et al., 2018; 28-d NOEC Keratella quadrata, which is about 50 times higher than the NOEC <0.09 μg/L (TWA) for C. dipterum) and Bangladesh (Sumon et al., (0.02 μg/L) estimated in this study (Table 2b). Sumon et al. (2018) re- 2018; 9-d NOEC <0.03 μg/L for Cloeon sp.) (Table 2a). However, the ported <0.03 (28-d), 0.03 (9-d) and 0.3 (9-d) μg/L NOECs for the cope- chronic 28-d EC10 values for C. dipterum (0.033 μg/L) and C. horaria pods Diaptomus sp., nauplii and Cyclops sp., respectively, which are (0.024 μg/L) reported for temperate populations (Roessink et al., comparable with our results for Afrocyclops sp. (30-d NOEC <0.02 2013) are above our NOECs as is also supported by our acute toxicity re- μg/L) and nauplii (30-d NOEC = 0.02 μg/L) (Table 2b). However, com- sults (Table 2a). Moreover, in a stream mesocosm study under temper- pared to the NOEC values reported by Rico et al. (2018) for Cyclopoida ate conditions with continuous imidacloprid application, Alexander (17-d NOEC = 1 μg/L), and nauplii (3-d NOEC = 5 μg/L), our results et al. (2008) also reported a higher effect threshold (20-d NOEC = 0.3 for copepods are lower by about two orders of magnitude. This indicates μg/L) for Epeorus sp. nymphs relative to our values for mayflies that the abundance of rotifers and copepods were severely affected by (i.e., C. dipterum and C. horaria)(Table 2a). imidacloprid upon chronic exposure to environmentally relevant low The consistent NOEC concentration estimated in our study for the concentrations. The toxicity of imidacloprid to Diaptomus sp. (copepod) dipteran Chironomidae sp. and Culicidae sp. of 0.02 μg/L are comparable was demonstrated by Sumon et al. (2018) in the sub-tropical region of with the cosm 10-d NOEC (< 0.2 μg/L) reported for Chironomini in the Bangladesh (96-h LC50 = 6.5 μg/L; EC50 = 0.0386 μg/L) (Table 2b). Mediterranean cosm experiment (Rico et al., 2018)(Table 2a). How- This suggests that the effect observed on copepods in our cosm study ever, relative to the sensitivity of dipteran species from the temperate is likely due to direct effects of imidacloprid. region such as Chironomus dilutus (14-d LC20 = 0.47 μg/L) (Cavallaro Toxicity studies of imidacloprid with rotifer species for comparison et al., 2017)andChironomus riparius (10-d LC10 = 1.64 μg/L) to our results are not available in the literature. The exception is for (Chandran et al., 2018), our observed NOECs are low (Table 2a). the rotifer Brachionus calyciflorus for which high effect values (i.e., 24- For the Hemipteran taxa of Gerridae sp. and Corixidae sp. the largest h NOEC (mortality) = 17,500 μg/L; 10-d NOEC (population growth) significant effect was observed after the 3rd application of imidacloprid =6220μg/L) were reported (Gharaei et al., 2020)(Table 2b). However, with 42-d NOECs of 0.02 μg/L (Table 4). Similarly, the largest effects on similar to our cosm result, Brachionus sp. showed a response to the abundances of Notonectidae sp. (14-d NOEC = 0.124 μg/L) and

7 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

Table 3 The No Observed Effect Concentration (NOEC) values (μg/L) for water quality and periphyton (a), and zooplankton and phytoplankton (b) variables. The arrow between brackets indicate an imidacloprid treatment related significant increase (↑)ordecrease(↓) of a variable (Williams test; p<0.05). The NOEC values greater than 1 μg/L is marked by > symbol. The shed with light grey indicates the application period of imidacloprid, while the −7and− 1 are pre-application and > 21 are post application periods. An – indicates that the endpoint was not eval- uated on that sampling date. a. Monitored Treatment me (day)

variables -7 -1 0 7 14 21 28 42 56 84 112 140 168

Water Quality

Dissolved oxygen > > > > > > > > T-1(↓) > > T-2(↑) >

Conducvity > > > > > >>>>>>T-2(↓)>

Temperature > > > > > >>>T-1(↓)>>

pH > > > > > >>>>>>>>

Alkalinity > > > > > T-2 (↑) > > > T-2(↓) > T-2(↑) >

Total hardness > > > > > >>>>>>T-1(↑)>

Ammonia > > > > > >>>>>>>>

Nitrate T-2(↑) T-2(↑) > > > > >T-2(↑)>>>>>

Phosphate > > > > > > >T-2(↓)>>>>>

Periphyton

Chlorophyll-a - - > T-2 (↑) T-1 (↑) T-1 (↑) > T-1 (↑) T-1 (↑) < T-1 (↑) T-2(↓) > -

b. Monitored Treatment me (day)

variables -7 -1 2 9 16 23 30 44 58 86 114 142

Zooplankton

Community > > > > T-1 < T-1 < T-1 T-1 T-1 > > >

Brachionus sp. > > > > T-1 (↓) < T-1 (↓) T-1 (↓) T-1 (↓) T-2 (↓) T-2 (↓) T-2(↓) >

Filinia sp. > > > > T-2(↓) T-1 (↓) T-1 (↓) T-1 (↓) < T-1 (↓) T-1 (↓) > >

nauplii > > > > T-1 (↓) < T-1 (↓) T-1 (↓) T-1 (↓) T-1 (↓) T-1 (↓) > >

Keratella sp. > > T-2 (↑) > T-2 (↓) T-1 (↓) T-1 (↓) T-1 (↓) > > > >

Afrocyclops sp. > > > > T-1 (↓) T-1 (↓) < T-1 (↓) < T-1 (↓) T-1 (↓) T-1 (↓) > >

Trichocerca sp. > > > > > < T-1 (↓) T-1 (↓) T-1 (↓) T-2 (↓) > > >

Polyarthra sp. > > > > >

Phytoplankton

Chlorophyll-a >> > > T-1 (↑) T-1 (↑) T-2 (↑) T-2 (↑) > < T-1 (↑) > >

*Note that the concentration of T-1 = 0.02 μg/L (based on the analytical results obtained for T-2 and T-3), T-2 = 0.124 μg/L (TWA), T-3 = 2.09 μg/L (TWA).

P. minutissima (21-d NOEC = 0.124 μg/L) were observed in the highest the temperate population is 16 times higher compared to the NOEC treatment on 14 days and 21 days after the first application of (21-d NOEC = T-2 concentration (0.124 μg/L)) measured in our study imidacloprid, respectively (Table 4). For Gerris sp. and Notonecata sp., for the species in Ethiopia (Table 2a). Sumon et al. (2018) reported a 16-d NOEC of 0.03 μg/L in sub-tropical Pereira et al. (2017) demonstrated significant effects of imidacloprid cosms, which is comparable to our result for Gerris sp. but for (applied once at 60 μg/L which quickly dissipated with a half-life of 2.7 Notonectidae sp. our result is higher by one order of magnitude days) on Hydaticus sp. (Coleoptera, Dytiscidae family) using rice (Table 2a). Rico et al. (2018) also reported a NOEC of 0.2 μg/L for mesocosms under Mediterranean climatic condition (Table 2a). Simi- Notonetidae sp. from a mesocosm study performed in Mediterranean larly, we observed effects on Dytiscidae sp. (14-d NOEC = 0.02 μg/L) climatic conditions (Table 2a). However, for Gerris latiabdominis (Table 2a), although we tested much lower concentrations with re- (Hayasaka et al., 2012) and Notonecta tiguttata (Kobashi et al., 2017) peated application. no significant effects of imidacloprid (at 49 μg/L for G. latiabdominis The snails (i.e., Physidae sp. and Planorbidae sp.) showed the and at 157 μg/L for N. tiguttata) were reported in a mesocosm study smallest response to the imidacloprid treatments. For both species using rice paddies in Japan (Table 2a). Furthermore, the 28-d EC10 NOECs of 0.124 μg/L (TWA) was found (Fig. 2; Table 4). There are studies value (2.03 μg/L) reported for P. minutissima (Roessink et al., 2013)for from the temperate region indicating that imidacloprid is toxic to

8 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

2.5 aquatic snails (e.g., Planorbella pilsbryi). Prosser et al. (2016) reported a Cloeon dipterum 28-d EC10 (on growth), EC50 (on growth), LC10 and LC50 of 15.4, 39.4, 45.7 and 646 μg/L, respectively for the freshwater snail, P. pilsbryi 2 Caenis horaria (Table 2a). This result indicates that the effects observed on abundance of the Physidae sp. and Planorbidae sp. snails in this study are possibly

1.5 due to direct toxicity of imidacloprid. However, the NOECs we measured for both species herein are one order of magnitude lower compared to the 28-d EC10 value reported by Prosser et al. (2016) for P. pilsbryi 1 (Table 2a). Culicidae Full recovery of the macroinvertebrate community did not occur Corixidae Plea minutissima during the 21 weeks recovery period (i.e. starting after the last Notonectidae 0.5 Chironomidae imidacloprid application) (Table 4). The differences in community com- Dytiscidae position between the treatment and the control decreased 42 days after Gerridae 0.25 fi kb Planorbidae the rst imidacloprid application (Fig. 2). Compared to the zooplankton 0 0 Physidae 0 community, we found that the macroinvertebrate community required -0.25 a longer recovery period. This is probably due to the longer generation -0.5 times of macroinvertebrate taxa compared to zooplankton taxa which dt

C -0.75 have a relatively short life cycle (Beketov et al., 2008; Rico and Van

-1 den Brink, 2015). In small and static tropical ecosystems repeatedly ex-

-1.25 posed to imidacloprid, effects could occur at chronic concentrations >0.02 μg/L and full recovery of the macroinvertebrate community, es- -1.5 -25 0 25 50 75 100 125 150 175 pecially of mayflies and mosquitos, could take over five months. Com- Days post first application paring the recovery of the populations of individual species, except for Control 0.01 µg/L 0.1 µg/L 1 µg/L the most sensitive species (C. dipterum, C. horaria and Culicidae sp.), 21 weeks after the last imidacloprid application all other species com- Fig. 2. Principal Response Curve (PRC) depicting the effect of imidacloprid contamination pleted their recovery (i.e., no significant differences between treatments on the macroinvertebrate community over the course of experimental period. The sample and control) (Table 4). Similar delayed recovery results were reported weights (Cdt) indicate differences between the treated macroinvertebrate community and by Beketov et al. (2008) for the neonicotinoid thiacloprid insecticide the control in the different sampling dates. The affinity of each taxon of the community studied using mesocosms designed to mimic temperate steams. The with the PRC is indicated by the species weight (Kb). authors (Beketov et al.,2008) reported that recovery of the

Table 4 The No Observed Effect Concentration (NOEC) values (μg/L) for macroinvertebrates and organic matter decomposition parameters. The arrow between brackets indicate an imidacloprid treatment related significant increase (↑)ordecrease(↓) of a variable (Williams test; p<0.05). The NOEC values greater than 1 μg/Lismarkedas>.Theshedwithlightgreyindicatestheap- plication period of imidacloprid, while the −7and− 1 are pre-application and > 21 are post application periods. An – indicates that the endpoint was not evaluated on that sampling date.

Monitored Treatment me (day)

variable -7 -1 2 7 14 16 21 28 42 56 70 84 98 112 140 168

Macroinvertebrates

Community > > - T-1 T-1 - T-1 T-1 T-1 T-1 - T-1 - T-1 T-1 T-1

Physidae sp. > > - > > - T-2 (↓) T-2 (↓) T-2 (↓) T-2 (↓) - > - < T-1(↓) > >

Planorbidae sp. > > - > > - > T-2 (↓) T-2 (↓) T-2 (↓) - > - > T-2 (↓) >

Gerridae sp. > > - > T-2 (↓) - T-2 (↓) < T-1 (↓) T-1 (↓) T-1 (↓) - T-2 (↓) - T-1 (↓) T-1 (↓) >

Dyscidae sp. > > - > T-1 (↓) - T-1 (↓) < T-1 (↓) T-1 (↓) T-1 (↓) - > - T-2 (↓) T-1 (↓) >

Notonecdae sp. > > - > T-2 (↓) - T-2 (↓) T-1 (↓) T-2 (↓) T-2 (↓) - T-2 (↓) - T-2 (↓) > >

P. minussima >> - > > - T-2 (↓) T-2 (↓) T-1 (↓) T-2 (↓) - T-2 (↓) - T-1 (↓) > >

Corixidae sp. > > - > > - T-1 (↓) < T-1 (↓) T-1 (↓) T-1 (↓) - T-2 (↓) - T-2 (↓) T-2 (↓) >

Chironomidae sp. > > - > > - T-1 (↓) T-1 (↓) T-2 (↓) T-1 (↓) - T-1 (↓) - T-2 (↓) > >

Culicidae sp. > > - > T-2 (↓) - T-1 (↓) T-1 (↓) T-1 (↓) T-1 (↓) - T-1 (↓) - T-1 (↓) T-2 (↓) T-1 (↓)

C. horaria >> - T-1 (↓)

C. dipterum >> - T-1 (↓)

Organic maer decomposion

% weight loss - - > - - > - T-2 (↓) T-1 (↓) > > > > - - - aNote that the concentration of T-1 = 0.02 μg/L (based on the analytical results obtained for T-2 and T-3), T-2 = 0.124 μg/L (TWA), T-3 = 2.09 μg/L (TWA).

9 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167 macroinvertebrate community exposed to a single dose of 3.2 μg/L was 3.2.4. Physicochemical parameters not observed after 27 weeks. In contrast, in a mesocosm study by Rico Water quality variables showed no consistent effects (significant on et al. (2018) a shorter recovery period (56 days) was reported for the at least two consecutive samplings), nor a consistent increasing or de- macroinvertebrate community following a single application of ≤250 creasing trend in time, except for temperature (Table 4; Fig. SI3). For μg/L of the insecticide (Table SI4). This difference in results may be ex- more information see supplementary information section A. plained partly due to differences in species composition as our cosm contained 82% insects (9 taxa) and 18% molluscs (2 taxa), while the 3.2.5. Organic matter decomposition pond cosms established by Rico et al. (2018) had 69% insects (9 taxa), The mean (and range) values of organic matter decomposition rate 15% molluscs (2 taxa) and 8% each from Arachnida (1 taxon) and Crus- (% loss/day) were 0.63 (0.48–0.95), 0.60 (0.47–0.92), 0.59 (0.48–0.89) tacea (1 taxon). The differences in exposure scenario between our study and 0.58 (0.48–0.91) for the control, T-1, T-2 and T-3 treatment levels, (chronic, i.e. multiple applications) and Rico et al. (2018) (acute,i.e.sin- respectively (see Table SI6). The Williams test showed that decomposi- gle application) can be also one of the factors for the difference in recov- tion rate was significantly decreased in the highest treatment (2.09 μg/L ery pattern (Table SI4). In addition to life cycle characteristics, aerial (TWA)) on day 28 and 42 (Table 4; Fig. SI5), indicating direct or indirect recolonization of the treated cosms from an external source for mobile effects on microbial activity. Although statistically a significant differ- species (e.g., insects) is an important route for the quick recovery of spe- ence was observed for organic matter decomposition rate in the highest cies (Beketov et al., 2008; Kattwinkel et al., 2012; Gergs et al., 2016). treatment (0.58% loss/day) compared to control (0.63% loss/day), the However, as our experiment was carried out during the rainy season, effect was small and may not have an ecological significance. A single we hypothesize that heavy rain inhibited aerial recolonization from species toxicity test performed in central Europe, Slovenia (Tišler nearby water bodies (Lake, 2000). et al., 2009) evaluated the toxic effect of imidacloprid (both analytical grade and Confidor SL 200 formulation) to bacteria (Vibrio fischeri) 3.2.3. Periphyton and phytoplankton and reported a 30 min IC20 of 11,200 μg/L with luminescence inhibition Chlorophyll-a was measured to approximate the effect of as the endpoint. In contrast to our result, many studies (Kreutzweiser imidacloprid on phytoplankton and periphyton communities. The et al., 2007; Pestana et al., 2009; Sumon et al., 2018) reported no signif- mean values of chlorophyll-a were 608 ± 170 μg/L, 577 ± 164 icant effect of imidacloprid on organic matter decomposition rate and μg/L, 632 ± 175 μg/L and 654 ± 184 μg/L for phytoplankton, and microbial activity. Recovery of the functional endpoint was observed 2.3 ± 2.9 μg/cm2,2.8±3.4μg/cm2,3.3±3.1μg/cm2 and 3.0 ± 2.6 on day 35 after the last application. μg/cm2 for periphyton in the control, T-1, T-2 and T-3 treatments, re- spectively (Fig. SI4). Our result showed that the concentration levels 3.2.6. Hypothesised overall effect chain of chlorophyll-a for both phytoplankton and periphyton were signif- The effects of imidacloprid on structural and functional endpoints as icantly higher in the two highest imidacloprid test concentrations observed in the highest treatment are summarized and depicted in (NOECs = 0.02 μg/L) compared to the control and lowest treatment Fig. 3. Significant direct effects of imidacloprid were measured on the (Table 4; Fig. SI4). The effects remained until 86 days and 84 days for abundance of several invertebrate taxa in the cosms with the highest phytoplankton and periphyton, respectively (Table 4). This can be two treatment concentration levels (0.124 and 2.09 μg/L, TWA). The ef- explained by an indirect effect of imidacloprid on algae, due to a fect observed on rotifers requires further sensitivity testing to evaluate loss of grazing pressure as a result of the decrease in invertebrates whether the observed effects should be classified as direct or indirect ef- (see section 3.2.7). fect of the imidacloprid. The largest responses to the imidacloprid

Fig. 3. The effect chain diagram showing the hypothesised structural and functional effects of imidacloprid on structural and functional characteristics of mesocosm ecosystem established in tropical climatic condition in Ethiopia. The symbol (↓), (↓?) and (+) represent: decrease due to direct effect, decrease but it is unclear whether it is due to direct or indirect effects and increase, respectively.

10 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167 exposure were observed for the macroinvertebrate species C. dipterum, using single species acute toxicity tests and outdoor mesocosms. The C. horaria and Culicidae sp. and the zooplankton taxa Afrocyclops sp., concentrations used in our cosm studies are within the concentration Brachionus sp., Filinia sp. and nauplii. The study showed an increase of range (0.04–7.2 μg/L) of imidacloprid previously reported for African chlorophyll-a concentrations of periphyton and phytoplankton in the aquatic ecosystems (Jansen and Harmsen, 2011; Curchod et al., 2020). T-2 and T-3 treatments, likely an indirect effect (Fleeger et al., 2003) Our results suggest that imidacloprid can result in significant adverse ef- resulting from the decrease in abundance of grazers (e.g., C. dipterum, fects on invertebrates and, possible, small effects on organic matter de- Afrocyclops sp. and Brachionus sp.) and scrapers (e.g., Physidae sp. and composition in aquatic ecosystems exposed to ≥0.02 μg/L. The study Planorbidae sp.) resulting from the direct effect of the insecticide. further showed that a long-term alteration of the structure of macroin- vertebrate community, especially concerning mayflies and mosquitos 3.3. Sensitivity differences between tropical and temperate aquatic species can occur in water bodies where imidacloprid reached ≥0.124 μg/L. Fur- ther to previous studies under sub-tropical (Sumon et al., 2018) and In general, we observed lower effect threshold values for Mediterranean (Rico et al., 2018; Sumon et al., 2018) conditions, our re- imidacloprid in the tropical macroinvertebrate and zooplankton com- sults highlight elevated sensitivity of tropical aquatic species to munities compared to the results from temperate climatic conditions. imidacloprid relative to their temperate counterparts. Similar findings were reported by Sumon et al. (2018) for sub-tropical communities in Bangladesh and by Rico et al. (2018) for aquatic species CRediT authorship contribution statement from the Mediterranean climatic region. These differences in sensitivity can be a result of multiple factors such as variations in temperature and Lemessa B. Merga: Conceptualization, Investigation, Project admin- generation status (e.g. continuously reproducing or in winter hiberna- istration, Writing – original draft, Formal analysis. Paul J. Van den tion state) of the test organisms. The temperature of the cosms in this Brink: Conceptualization, Supervision, Writing – review & editing, study was only slightly higher compared to many temperate cosm stud- Formal analysis, Project administration. ies. We acknowledge that there can be geographically related other fac- tors that may explain the differences in results observed between our Declaration of competing interest study and studies from temperate regions. This section discusses some of the factors reported in the literature including temperature and gen- The authors declare that they have no known competing financial eration status of the tested organisms. interests or personal relationships that could have appeared to influ- The difference in temperature between tropical and temperate re- ence the work reported in this paper. gions can be one of the factors contributing to the differences as temper- ature increases the rate of uptake and circulation of the chemicals in the Acknowledgments test organisms (Castillo et al., 1997; Kwok et al., 2007; Camp and Buchwalter, 2016). For example, Van den Brink et al. (2016) reported The study was financially supported by Netherlands fellowship that the mortality and immobility of C. dipterum acutely (96-h) exposed programmes, NUFFIC/ PhD studies, grant NFP - PhD.16/0019, reference to imidacloprid was increased by a factor of 4.2 and 1.7, respectively number WIMEK2015 02. Special thanks to Ambo University, Ethiopia when the temperature of the test system was increased from 10 °C to for providing laboratory facilities during the experiments. This study 18 °C (at 10 °C LC50 = 154 μg/L, EC50 = 31 μg/L; at 18 °C LC50 = 37 was co-funded by the GETREAL (Incorporating spatial and seasonal var- μg/L, EC50 = 18 μg/L). Camp and Buchwalter (2016) also reported that in- iability in community sensitivity into chemical risk assessment) project, creasing the test temperature from 15 °C to 24 °C decreased the time-to- which was funded by the European Chemical Industry Council long- effect (immobility endpoint) of imidacloprid by about a factor of 3 for the range research initiative (CEFIC-LRi project ECO 50). We declare that mayfly, Isonychia bicolor. Furthermore, Macaulay et al. (2020) demon- there are no conflicts of interest. strated synergistic effects of temperature-imidacloprid interactions on survivorship, mobility and moulting frequency of the freshwater mayflies Appendix A. Supplementary data Deleatidium sp. and Coloburiscus humeralis.Theauthors(Macaulay et al., 2020) reported that increasing the test water temperature from 9 °C to Supplementary data to this article can be found online at https://doi. 15 °C reduced the mobility of the mayfly C. humeralis by 100% when ex- org/10.1016/j.scitotenv.2021.147167. posed for 96 h to 12.5 μg/L of imidacloprid. Moreover, Van den Brink et al. (2016) reported the higher sensitivity of the reproducing summer References generations of freshwater arthropods to imidacloprid compared to the overwintering generations. As most tropical aquatic arthropods repro- Alexander, A.C., Heard, K.S., Culp, J.M., 2008. Emergent body size of mayfly survivors. Freshw. Biol. 53, 171–180. duce continuously (Brittain, 1982; Sumon et al., 2018), this may contrib- Anderson, J.C., Dubetz, C., Palace, V.P., 2015. Neonicotinoids in the Canadian aquatic envi- ute to the relatively high vulnerability of aquatic populations in the ronment: a literature review on current use products with a focus on fate, exposure, tropics, although the mechanistic pathway remains unknown. and biological effects. Sci. Total Environ. 505, 409–422. The altitude of 2102 m at the location where the organisms were col- Bartlett, A.J., Hedges, A.M., Intini, K.D., Brown, L.R., Maisonneuve, F.J., Robinson, S.A., Gillis, P.L., de Solla, S.R., 2019. Acute and chronic toxicity of neonicotinoid and butenolide lected could explain why our toxicity test results were in between those insecticides to the freshwater amphipod, Hyalella azteca. Ecotoxicol. Environ. Saf. collected in temperate and tropical regions, making the climate cooler 175, 215–223. than other tropical regions. 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11 L.B. Merga and P.J. Van den Brink Science of the Total Environment 784 (2021) 147167

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