SUPPLEMENTAL DATA for:

“Chronic effects of an environmentally-relevant, short-term neonicotinoid insecticide pulse on four aquatic invertebrates”

Melanie Raby, Xiaoming Zhao, Chunyan Hao, David G. Poirier, Paul K. Sibley

Science of the Total Environment, 2018

Contents:Table S1. Summary of culture methods

S1. Culturing of Navicula sp. diatoms for Neocloeon triangulifer feeding

Table S2. Physicochemical properties of dechlorinated water

S2. Analytical methods for neonicotinoids

S3. Descriptions of literature-derived neonicotinoid monitoring studies

Figure S1. Plotted results of Ontario Ministry of the Environment and Climate Change (MOECC) neonicotinoid stream monitoring program for 2015

Table S3. Survival of Hyalella azteca after 24-h pulse of imidacloprid, and 28 and 42 d of recovery

Table S4. Hyalella azteca mean growth and reproduction after 24-h pulse of imidacloprid, and 28 and 42 d of recovery in reference conditions

Table S5. Chironomus dilutus mean % survival and % immobilization after 24-h pulse of imidacloprid

Table S6. Chironomus dilutus emergence and reproduction after 24-h pulse of imidacloprid followed by recovery in reference conditions

Table S7. Chironomus dilutus mean % survival and % immobilization after 24-h pulse of thiamethoxam

Table S8. Chironomus dilutus emergence and reproduction after 24-h pulse of thiamethoxam followed by recovery in reference conditions

Figure S2. Chironomus dilutus emergence, days to emergence, reproduction, and adult lifespan for after 24-h pulse of imidacloprid followed by post-treatment conditions until mortality or emergence

Table S9. Wet weight of Hexagenia spp. nymphs at test initiation, prior to pulse exposure

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Table S10. Survival and wet weight of Hexagenia spp. after a 24-h pulse exposure to imidacloprid or thiamethoxam and 21-d recovery in reference conditions

Table S11. Survival and days to emergence of Neocloeon triangulifer after 24-h pulse exposure to imidacloprid or thiamethoxam and recovery in reference conditions

Table S12. Sediment analysis for Long Point reference sediment used in Hexagenia spp. test

Table S13. Results of tests for statistical significance

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Table S1. Summary of culture methodsNeocloeon triangulifer

Hyalella azteca Chironomus dilutus Hexagenia spp.

Reference (Weaver et al., 2015) (Ontario Ministry of the Environment and Climate Change Aquatic Toxicology Unit, 2016b)

(Ontario Ministry of the Environment and Climate Change Aquatic Toxicology Unit, 2016c)

(Ontario Ministry of the Environment and Climate Change Aquatic Toxicology Unit, 2016d)

Temperature (°C)

25 ± 2 23 ± 2 23 ± 2 23 ± 2

Diurnal cycle 16:8 light: dark cycle

Light intensity (lux)

100 – 500 lux 500 – 2500 lux 5000 – 1000 lux 100 – 1000 lux

Culture vessel and media

0 - ~10 d: 1 L flat-bottom glass dish with ~700 mL dechlorinated water

~10 d – emergence: 10 L tank with 3 L dechlorinated water

~2 L polyethylene containers, filled with culture water

Breeding tanks: 20 L glass aquaria, half full with sand and dechlorinated water

Rearing trays: white phenolic trays with several cm of dechlorinated water

10 L aquaria, with 1 L sediment and ~8 L dechlorinated water

Substrate None 2x5 cm pieces of cotton gauze

Breeding tanks: 1.6 L silica sand

Rearing trays: none

Long Point sediment

Aeration None None Breeding tanks: Constant gentle aeration

Rearing trays: none

Constant gentle aeration

Feeding Diatom (Navicula sp.) biofilms grown on frosted slides, added as needed a

~20 mg ground Nutrafin daily

Slurry made of 3:2 cereal grass: ground Nutrafin in dechlorinated water; feeding as needed

Slurry as per C. dilutus feeding; 5-10 mL 1-2x weekly, based on a ramped feeding regime

Water change frequency

Half refreshed 1-2x weekly

Half refreshed 1-2x monthly

Partially refreshed as needed Tanks topped up daily

Additional details

None None Egg cases collected from breeding tanks daily; hatched egg cases used to start rearing trays; breeding tank started with ~10-12 d old larvae

Eggs obtained from J. Ciborowski (University of Windsor) and stored at 7 ± 2°C for up to 12 months

a See section S1 for details on Navicula sp. culturing

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S1. Culturing of Navicula sp. diatoms for Neocloeon triangulifer feeding

Navicula sp. culturing is based on methods in Soucek & Dickinson (2015). Navicula sp.

were grown in Kent® Proculture F/2 algal medium supplemented with sodium metasilicate. Free

diatom stocks were grown under sterile conditions in vented tissue culture flasks (Falcon™) in

Kent media at a concentration of 0.23 mL Kent solution A, 0.23 Kent solution B, and 26.5 mg

sodium metasilicate per 500 mL autoclaved dechlorinated water. Flasks were seeded with 50 mL

of concentrated, previously grown Navicula sp. stock. Flasks were incubated at approximately

20°C under continuous, indirect fluorescent light (300-500 lux) with no agitation except

occasional shaking to observe growth. New flasks were created monthly. Free diatoms were

concentrated by allowing diatoms to settle, then carefully pouring off approximately 80% of the

overlying media.

Navicula sp. biofilms were grown on frosted slides (Fisher Scientific, cat. No. 12 544

5CY). 20 slides were laid, frosted side up, in the bottom of a rectangular autoclavable container.

2 L of dechlorinated water was added, and the container was covered in foil and autoclaved

(121°C, 30 min). Kent media was added at a concentration of 1.44 mL Kent solution A, 1.44

Kent solution B, and 170 mg sodium metasilicate per 2 L dechlorinated water. Containers were

seeded with approximately 100 mL of concentrated Navicula sp., and incubated at 20°C with

continuous, indirect fluorescent light (500-700 lux) with gentle aeration. Slides were ready for

feeding in approximately two weeks.

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Table S2. Physicochemical properties of dechlorinated water (n = 2 or 3 for all properties)Property Unit Mean Std Dev RangeChloride mg/L 29.03 3.62 24.90 – 36.80Sulphate mg/L 28.33 1.17 26.70 – 29.90Conductivity µS/cm 332.5 18.3 313 - 361pH 8.01 0.15 7.72 – 8.21Alkalinity mg/L CaCO3 77.70 3.88 69.90 – 81.80NH4+/NH3 mg/L 0.05 0.07 0.01 – 0.27NO3-/NO2- mg/L 0.58 0.15 0.27 – 0.72Phosphorus/phosphate mg/L 0.74 0.13 0.58 – 0.93Dissolved organic carbon mg/L 1.19 0.49 0.10 – 1.70Dissolved inorganic carbon mg/L 21.36 0.98 19.60 – 23.30Silicon/reactive silicate mg/L 0.56 0.07 0.46 – 0.66Bromide µg/L 4.65 3.08 1.21 – 10.30Magnesium mg/L 8.43 0.38 7.82 – 8.96Potassium mg/L 1.53 0.08 1.42 – 1.64Sodium mg/L 15.10 1.64 13.60 – 18.10Copper µg/L 0.90 0.45 0.26 – 1.80Hardness mg/L 123 4.94 112 - 128

S2. Analytical methods for neonicotinoids

Analysis was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS)

on a Shimadzu Prominence HPLC system and Sciex 5500QTrap mass spectrometer following

MOECC routine method E3544 “The determination of neonicotinoids in water by liquid

chromatography-tandem mass spectrometry analysis” (available upon request from

laboratoryservicesbranch@ontario.ca). LC-MS/MS analysis was conducted using an electrospray

ionization source (ESI) in positive ionization and Multiple Reaction Monitoring (MRM) scan

mode. Identification and quantification of the target analytes are based on retention times (RTs)

and the existence of the MRM transitions by running a sample against the prepared standards

under the same instrumental conditions and comparing obtained responses of samples to those of

the standards. Target compounds in aqueous environmental matrices were measured without

sample concentration by direct aqueous injection (injection volume of 90 µL), and where results

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exceeded the calibration range of an analyte (500-2000 ng/L for different analytes), samples

were diluted as needed with HPLC Grade water. Method detection limits were 0.010 µg L-1 for

both imidacloprid and thiamethoxam.

S3. Descriptions of literature-derived neonicotinoid monitoring studies

The MOECC study sampled five sites from April 10 – November 11 2015, using grab

and wet weather-triggered sampling methods (MOECC, 2016). Data and associated metadata is

available at: https://www.ontario.ca/data/stream-neonicotinoid-monitoring-study.

Struger et al. (2017) sampled 17 sites (nine agricultural; one urban; one reference) using

grab sampling methods. Data available in Supplemental Data,

10.1016/j.chemosphere.2016.11.036).

6B

A

Figure S1. (A) Plotted results of Ontario Ministry of the Environment and Climate Change (MOECC) neonicotinoid stream monitoring program for 2015 for the three most frequently detected neonicotinoids: clothianidin, imidacloprid, and thiamethoxam. (B) Empirical cumulative distribution functions (ECDFs) of grab samples only for imidacloprid and thiamethoxam. ECDFs of grab samples only were constructed using Kaplan-Meier methods, with the R package ‘NADA’ (v. 1.6-1) in R v. 3.3.2 (R Core Team, 2016).

Table S3. Survival of Hyalella azteca after 24-h pulse of imidacloprid, and 28 and 42 d of recovery

Mean % survivalImidacloprid concentration(µg L-1)

After 24 h pulse d 28 d 42Mean SD N Mean SD N Mean SD N

0 99.1 3.0 11 93.6 10.3 11 91.3 11.3 82.3 99.1 3.0 11 98.2 4.0 11 98.8 3.5 84.7 100.0 0.0 11 97.3 6.5 11 93.8 7.4 88.9 100.0 0.0 11 99.1 3.0 11 98.8 3.5 8100 93.6 6.7 11 86.4 10.3 11 82.5 10.4 8

Table S4. Hyalella azteca mean growth and reproduction after 24-h pulse of imidacloprid, and 28 and 42 d of recovery in reference conditions

Imidacloprid concentration(µg L-1)

d 28 d 42mg dw/individual mg dw/individual juveniles/femaleMean SD N Mean SD N Mean SD N

0 0.80 0.06 3 1.13 0.17 8 16.1 5.3 82.3 0.69 0.16 3 1.28 0.13 8 17.3 3.5 84.7 0.80 0.08 3 0.94 0.24 8 16.0 6.7 88.9 0.84 0.16 3 1.05 0.17 8 16.3 6.6 8100 0.52 0.06 3 1.11 0.36 8 11.6 9.1 8

Table S5. Chironomus dilutus mean % survival and % immobilization after 24-h pulse of imidacloprid

Imidacloprid concentration(µg L-1)

After 24 h pulse (t = 24 h) After 24 h recovery (t = 48 h)% survival % immobilization

N

% survival % immobilization

NMean

SD Mean SD Mean SD Mean SD

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95% quantile0.025 µg L-1 95% quantile

0.34 µg L-1

0 98.8 3.4 0 0 16 98.3 3.9 0 0 122.3 98.8 3.4 5.0 10.3 16 99.2 2.9 0 0 124.6 91.9 6.6 15.0 21.9 16 88.3 7.2 3.3 6.5 128.8 92.1 4.3 36.4 25.0 14 86.7 7.1 4.4 5.3 9

Table S6. Chironomus dilutus emergence and reproduction after 24-h pulse of imidacloprid followed by recovery in reference conditionsImidacloprid concentration(µg L-1)

Percent emergence Days to emergence Eggs produced Adult lifespan

NMean SD Mean SD Mean SD Mean SD

0 54.2 18.3 39.0 3.6 995 266 3.6 1.0 162.3 66.7 12.8 41.1 4.4 903 333 3.3 0.6 164.6 57.6 14.0 36.8 5.0 946 440 3.5 1.0 168.8 55.6 9.6 41.5 4.3 999 327 3.2 0.7 16

Table S7. Chironomus dilutus mean % survival and % immobilization after 24-h pulse of thiamethoxam

Thiamethoxam concentration(µg L-1)

After 24 h pulse (t = 24 h) After 24 h recovery (t = 48 h)% survival % immobilization

N

% survival % immobilization

NMean SD Mean SD Mea

nSD Mean SD

0 100 0 0 0 16 100 0 0 0 162 99.4 2.5 0 0 16 99.4 2.5 0 0 164.1 98.8 5.0 0 0 16 98.8 5.0 0 0 167.8 100 0 0 0 16 99.4 2.5 0 0 1612.3 100 0 0.63 2.5 16 100 0 0 0 16

Table S8. Chironomus dilutus emergence and reproduction after 24-h pulse of thiamethoxam followed by recovery in reference conditionsThiamethoxam concentration(µg L-1)

Percent emergence Days to emergence Eggs produced Adult lifespan

NMean SD Mean SD Mean SD Mean SD

0 55.6 11.4 37.3 3.6 813 217 2.7 0.9 162.0 62.5 10.4 39.0 2.7 830 388 3.2 0.7 164.1 61.1 13.5 35.0 2.1 951 364 2.5 0.6 167.8 64.6 12.9 38.2 3.2 955 167 3.1 0.5 1612.3 59.7 13.7 38.6 2.9 789 231 2.2 0.9 16

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Figure S2. Responses of Chironomus dilutus after 24-h pulse of imidacloprid (A, C, E, G) and thiamethoxam (B, D, F, H), followed by post-treatment conditions until growth measurement (A, B), or mortality or emergence (remainder of plots): (C, D) mean emergence to adult; (E, F) mean time (in days) to emergence; (G, H) mean number of eggs produced per egg case; (I, J) mean

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adult lifespan (in days). Results are presented as mean of n=3 replicates for growth (A, B), or n=16 replicates (remainder of plots) with standard deviations for each measured concentration of pulse of neonicotinoid. Negative control is represented by “C.” No significant differences (p > 0.05) between treatments and the control were detected as determined by a Kruskal-Wallis with post-hoc Dunn’s method test or one-way ANOVA, with post-hoc Tukey test. See Supplemental Data, Table S13 for results of tests for statistical significance.

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Table S9. Wet weight of Hexagenia spp. nymphs at test initiation, prior to pulse exposureHexagenia spp. wet weight (mg)

Individual (replicate) Imidacloprid Thiamethoxam1 7.11 8.882 6.95 10.583 4.29 9.244 6.72 2.805 2.76 6.546 7.92 5.447 4.60 6.448 5.39 8.929 2.27 5.22

10 2.60 9.3411 6.67 14.2612 9.49 6.6513 6.60 3.7414 6.47 6.1815 3.53 15.1216 3.56 7.9117 5.60 6.5218 9.89 2.8419 3.23 3.0420 4.94 3.61

Mean 5.53 7.16SD 2.21 3.47

Min 2.27 2.80Max 9.89 15.12

Table S10. Survival and wet weight of Hexagenia spp. after a 24-h pulse exposure to imidacloprid or thiamethoxam and 21-d recovery in reference conditions

Imidacloprid Thiamethoxam

Concentration(µg L-1)

Percent survival

Individual wet weight (mg)

NConcentration(µg L-1)

Percent survival

Individual wet weight (mg)

NMean SD Mean SD Mean SD Mean SD0 97.5 0.046 26.490 3.369 8 0 97.5 0.046 22.012 1.926 82.3 98.8 0.035 24.908 2.761 8 1.9 93.8 0.141 21.579 1.815 84.5 96.3 0.052 26.286 4.513 8 4 97.5 0.046 19.668 1.595 89 95.0 0.076 27.314 4.089 8 7.8 93.8 0.052 20.916 3.291 8

Table S11. Survival and days to emergence of Neocloeon triangulifer after 24-h pulse exposure to imidacloprid or thiamethoxam and recovery in reference conditions

Imidacloprid ThiamethoxamConcentration(µg L-1)

Percent emergence Days to emergenceN

Concentration(µg L-1)

Percent emergence Days to emergenceNMean SD Mean SD Mean SD Mean SD

0 90.0 21.1 25.7 0.327 10

0 85.0 24.2 22.3 2.070 10

2.3 85.0 24.2 26.1 0.556 10

2 85.0 24.2 21.2 0.345 10

4.7 90.0 21.1 26.3 0.378 10

4.1 95.0 15.8 21.7 0.773 10

8.9 95.0 15.8 26.8 0.825 10

8.4 95.0 15.8 21.8 0.898 10

Table S12. Sediment analysis for Long Point reference sediment used in Hexagenia spp. testParameter Unit (dw, unless stated) Value Method detection limitTotal Organic Carbon mg/g 65 N/A% Total Organic Carbon mg/g 6.5 N/ASulphate; water soluble µg/g 529. 6.187

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Parameter Unit (dw, unless stated) Value Method detection limitSolids; total, loss on ign mg/g 110 N/AMoisture % 58. N/A% < 2.63 µm, > 0.10 µm, sum %V 8.1 N/A% < 62 µm, > 2.63 µm, sum %V 67.0 N/A% < 1000 µm, > 62 µm, sum %V 24.9 N/ANaphthalene ng/g 15. 52-methylnaphthalene ng/g 6.0 51-methylnaphthalene ng/g < MDL 5Acenaphthylene ng/g 7.0 5Acenaphthene ng/g < MDL 5Fluorene ng/g 16. 5Phenanthrene ng/g 35. 5Anthracene ng/g 7.0 5Fluoranthene ng/g 35. 5Pyrene ng/g 24. 5Benz(a)anthracene ng/g 8.0 5Chrysene ng/g 12. 5Benzo(b)fluoranthene ng/g 18. 5Benzo(k)fluoranthene ng/g 7.0 5Benzo(e)pyrene ng/g 10. 5Benzo(a)pyrene ng/g 9.0 5Perylene ng/g 27. 5Indeno(1,2,3-c,d)pyrene ng/g 11. 5Dibenz(a,h)anthracene ng/g < MDL 5Benzo(g,h,i)perylene ng/g 12 5Aluminum µg/g 7360 50Arsenic µg/g < MDL 5Barium µg/g 49.2 0.5Beryllium µg/g 0.390 0.025Boron µg/g 6.39 0.25Calcium µg/g 63400 50Cadmium µg/g 0.2 0.1Chromium µg/g 11.3 0.2Cobalt µg/g 5.2 0.1Copper µg/g 13.6 0.2Iron µg/g 12900 50Lead µg/g 9.8 0.5Magnesium µg/g 11800 50Manganese µg/g 435. 1Molybdenum µg/g < MDL 0.5Nickel µg/g 11.5 0.1Potassium µg/g 960. 50Phosphorus µg/g 700. 50Silver µg/g < MDL 0.2Sodium µg/g 190. 50Strontium µg/g 86.5 0.5Sulphur µg/g 2830 50Tin µg/g 4.6 2.5Titanium µg/g 213. 0.5Vanadium µg/g 17.7 0.25Zinc µg/g 43.2 0.25

< MDL = below method detection limit

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Table S13. Results of tests for statistical significance Imidacloprid Thiamethoxam

Species Endpoint Statistics StatisticsHyalella azteca 24 h survival Normality test failed (p < 0.050)

Kruskal-Wallis, H = 18.309, df = 4, p = 0.001Post-hoc tests (Tukey, Dunn, SNK, Dunnett’s) all showed no pairwise comparison differences.

N/A

28 d growth Normality test passed (p = 0.094)Equal variance test passed (p = 0.779)One-way ANOVA, F = 3.871, p = 0.038Post-hoc (Dunnett’s): 0/100 (p <0.044)Power with alpha of 0.050 = 0.559

N/A

42 d growth Normality test passed (p = 0.196)Equal variance test passed (p = 0.226)One-way ANOVA, F = 2.270, p = 0.081Power with alpha of 0.050 = 0.347; below desired power of 0.800, so interpret negative results cautiously.

N/A

28 d survival Normality test failed (p < 0.050)Kruskal-Wallis, H = 17.562, df = 4, p = 0.002Post-hoc (Dunn’s): No statistically significant differences compared to control.

N/A

42 d survival Normality test failed (p = 0.026)Equal variance test failed (p = 0.017)Kruskal-Wallis, H = 15.135, df = 4, p = 0.004Post-hoc (Dunnett’s): No statistically significant differences compared to control.

N/A

42 d reproduction Normality test passed (p = 0.116)Equal variance test failed (p < 0.050)Kruskal-Wallis, H = 8.405, df = 4, p = 0.078

N/A

Chironomus dilutus 24 h survival Normality test failed (p < 0.050)Kruskal-Wallis, H = 23.345, df = 3, p < 0.001Post-hoc (Dunn’s): 0/4.6 (p < 0.05); 0/8.8 (p < 0.05)

Normality test failed (p < 0.050)Kruskal-Wallis, H = 3.039, df = 4, p = 0.551

24 h immobilization Normality test failed (p < 0.050)Kruskal-Wallis, H = 28.436, df = 3, p < 0.001Post-hoc (Dunn’s): 0/4.6 (p < 0.05); 0/8.8 (p < 0.05)

Normality test failed (p < 0.050)Kruskal-Wallis, H = 4.000, df = 4, p = 0.406

48 h survival Normality test failed (p < 0.050)Kruskal-Wallis, H = 24.680, df = 3, p < 0.001Post-hoc (Dunn’s): 0/4.6 (p < 0.05); 0/8.8 (p < 0.05)

Normality test failed (p < 0.050)Kruskal-Wallis, H = 2.053, df = 4, p = 0.726

48 h immobilization Normality test failed (p < 0.050)Kruskal-Wallis, H = 10.428, df = 3, p = 0.015Post-hoc (Dunn’s): No statistically significant differences compared to control.

Normality test failed (p < 0.050)Kruskal-Wallis, H = 0.000, df = 4, p = 1.000

14 d growth Normality test passed (p = 0.311)Equal variance test passed (p = 0.277)One-way ANOVA, F = 1.116, p = 0.381Power with alpha of 0.050 = 0.065; below desired power of 0.800, so interpret negative results cautiously.

Normality test passed (p = 0.545)Equal variance test passed (p = 0.516)One-way ANOVA, F = 1.486, p = 0.256Power with alpha of 0.050 = 0.133; below desired power of 0.800, so interpret negative results cautiously.

Percent emergence Normality test failed (p < 0.050)Kruskal-Wallis, H = 5.898, df = 3, p = 0.117

Normality test passed (p = 0.214)Equal variance test passed (p = 0.976)One-way ANOVA, F = 0.892, df = 4, p = 0.475Power with alpha of 0.050 = 0.049; below desired power of 0.800, so interpret negative results cautiously.

Number eggs produced

Normality test passed (p = 0.142)Equal variance test passed (p = 0.770)One-way ANOVA, F = 0.145, p = 0.932

Normality test passed (p = 0.493)Equal variance test passed (p = 0.377)One-way ANOVA, F = 0.605, df = 4, p = 0.661

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Power with alpha of 0.050 = 0.049; below desired power of 0.800, so interpret negative results cautiously.

Power with alpha of 0.050 = 0.050; below desired power of 0.800, so interpret negative results cautiously.

Adult lifespan Normality test failed (p < 0.050)Kruskal-Wallis, H = 2.597, df = 3, p = 0.458

Normality test passed (p = 0.539)Equal variance test passed (p = 0.522)One-way ANOVA, F = 3.562, df = 4, p = 0.012Post-hoc (Dunnett’s): No statistically significant differences compared to control.

Days to emergence Normality test passed (p = 0.297)Equal variance test passed (p = 0.917)One-way ANOVA, F = 3.000, p = 0.041Post-hoc (Dunnett’s): No statistically significant differences compared to control.

Normality test passed (p = 0.701)Equal variance test passed (p = 0.569)One-way ANOVA, F = 3.428, df = 4, p = 0.014Post-hoc (Dunnett’s): No statistically significant differences compared to control.

Hexagenia spp. 24 h survival Normality test failed (p < 0.050)Kruskal-Wallis, H = 3.920, df = 3, p = 0.270

Normality test failed (p < 0.050)Kruskal-Wallis, H = 3.000, df = 3, p = 0.392

21 d survival Normality test failed (p < 0.050)Kruskal-Wallis, H = 1.807, df = 3, p = 0.613

Normality test failed (p < 0.050)Kruskal-Wallis, H = 3.133, df = 3, p = 0.372

21 d wet weight Normality test passed (p = 0.085)Equal variance test passed (p = 0.876)One-way ANOVA, F = 0.569, p = 0.640

Normality test failed (p < 0.050)Kruskal-Wallis, H = 5.139, df = 3, p = 0.162

Neocloeon triangulifer

24 h immobilization Normality test failed (p < 0.050)Kruskal-Wallis, H = 79.000, df = 3, p < 0.001Post-hoc (Dunn’s): 0/8.9 (p < 0.05)

No immobilization in any treatment

Percent emergence Normality test failed (p < 0.050)Kruskal-Wallis, H = 1.219, df = 3, p = 0.749

Normality test failed (p < 0.050)Kruskal-Wallis, H = 2.438, df = 3, p = 0.487

Days to emergence Normality test passed (p = 0.067)Equal variance test passed (p = 0.857)One-way ANOVA, F = 6.405, df = 3, p = 0.001Post-hoc (Dunnett’s): 0/8.9 (p < 0.001)Power of test with alpha of 0.050 = 0.917.

Normality test failed (p < 0.050)Kruskal-Wallis, H = 7.264, df = 3, p = 0.064

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