Supporting Information Variation in the toxicity of sediment-associated substituted phenylamine antioxidants to an epibenthic (Hyalella azteca) and endobenthic (Tubifex tubifex) invertebrate Prosser, R.S. 1,3* , Bartlett, A.J. 1 , Milani, D. 2 , Holman, E.A.M. 1 , Ikert, H. 1 , Schissler, D. 1 , Toito, J. 1 , Parrott, J.L. 1 , Gillis, P.L. 1 , and Balakrishnan, V.K. 1 1 Environment and Climate Change Canada, Aquatic Contaminants Research Division, Burlington, Ontario, Canada 2 Environment and Climate Change Canada, Watershed Hydrology and Ecology Research Division, Burlington, Ontario, Canada * Corresponding Author Current affiliation: 3 University of Guelph, School of Environmental Sciences, Guelph, Ontario, Canada Page 1 of 39
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Supporting Information
Variation in the toxicity of sediment-associated substituted phenylamine antioxidants to an epibenthic (Hyalella azteca) and endobenthic (Tubifex tubifex) invertebrate
Prosser, R.S. 1,3*, Bartlett, A.J. 1, Milani, D. 2, Holman, E.A.M. 1, Ikert, H. 1, Schissler, D. 1, Toito, J. 1, Parrott, J.L. 1, Gillis, P.L. 1, and Balakrishnan, V.K.1
1 Environment and Climate Change Canada, Aquatic Contaminants Research Division, Burlington, Ontario, Canada2 Environment and Climate Change Canada, Watershed Hydrology and Ecology Research Division, Burlington, Ontario, Canada
* Corresponding Author
Current affiliation:3 University of Guelph, School of Environmental Sciences, Guelph, Ontario, Canada
Corresponding Author:Ryan Prosser Ph.D.University of GuelphSchool of Environmental SciencesGuelph, Ontario, [email protected]@canada.ca
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Table S1 Physicochemical properties of sediments collected from Long Point Marsh and Long Point Bay in Lake Erie located in Ontario, Canada (n = 4). Physicochemical properties were determined using standard methods developed by ECCC’s National Laboratory for Environmental Testing (NLET).
Long Point Marsh Long Point Bay
Property Units Mean Standard deviation Mean Standard
Table S2 Physicochemical properties of mixed sediment used in toxicity testing (n = 6). Physicochemical properties were determined using standard methods developed by ECCC’s NLET.
Table S3 Concentration of selected inorganic and organic contaminants in mixed sediment used in toxicity testing (n = 4). Analyses were conducted using standard methods developed by ECCC’s NLET.
Table S4 Physicochemical properties of culture water (dechlorinated Burlington, ON, tap water) used in toxicity testing (n = 4). Physicochemical properties were determined using standard methods developed by ECCC’s NLET.
Table S5 Concentration of selected inorganic and organic contaminants in culture water used in toxicity testing (n = 4). Analyses were conducted using standard methods developed by ECCC’s NLET.
Contaminant Mean concentration (µg/L) Standard deviation
Table S6 Mean, standard deviation, maximum, and minimum of water quality parameters in replicate test vessels across treatments at the initiation and conclusions of tests exposing juvenile Hyalella azteca to SPAs for 96 h.
Experiment pH Dissolved oxygen (mg/L)
Conductivity (µS/cm)
Ammonia (mg/L)
DPA t = 0 d Mean 8.26 8.41 360 <0.01Standard Deviation 0.03 0.27 3Max. 8.29 8.58 363 <0.01Min. 8.21 7.82 357 <0.01
t = 28 d Mean 8.26 9.40 374 0.004Standard Deviation 0.04 0.19 5 0.1Max. 8.31 9.56 380 <0.01Min. 8.24 9.08 364 0.025
PNA t = 0 d Mean 8.24 8.57 357 <0.01Standard Deviation 0.03 0.24 6Max. 8.26 8.93 370 <0.01Min. 8.19 8.31 351 <0.01
t = 28 d Mean 8.29 8.01 357 0.02Standard Deviation 0.19 0.36 8 0.02Max. 8.72 8.33 371 0.025Min. 8.17 7.26 352 <0.01
DPPDA t = 0 d Mean 8.33 9.25 357 <0.01Standard Deviation 0.03 0.48 17Max. 8.38 9.37 394 <0.01Min. 8.27 8.52 348 <0.01
t = 28 d Mean 8.28 7.72 347 0.004Standard Deviation 0.05 0.33 10 0.1Max. 8.32 7.99 377 0.025Min. 8.18 7.01 358 <0.01
MBA t = 0 d Mean 8.29 7.98 350 <0.01Standard Deviation 0.05 0.62 3Max. 8.33 8.45 353 <0.01Min. 8.20 6.94 349 <0.01
t = 28 d Mean 8.06 8.18 375 0.014Standard Deviation 0.05 0.63 10 0.02Max. 8.13 8.94 391 0.05Min. 7.98 6.89 365 <0.01
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Table S7 Mean, standard deviation, maximum, and minimum of water quality parameters in replicate test vessels across treatments at the initiation and conclusions of tests exposing juvenile Hyalella azteca to sediment spiked with SPAs.
Experiment pH Dissolved oxygen (mg/L)
Conductivity (µS/cm)
Ammonia (mg/L)
DPA t = 0 d Mean 8.39 7.94 453 0.01Standard Deviation 0.03 0.20 15 0.02Max. 8.41 8.22 478 0.05Min. 8.32 7.67 436 <0.01
t = 28 d Mean 8.41 8.03 514 <0.01Standard Deviation 0.06 0.20 20Max. 8.53 8.29 539 <0.01Min. 8.37 7.66 483 <0.01
PNA t = 0 d Mean 8.44 8.74 516 <0.01Standard Deviation 0.10 0.12 54Max. 8.67 8.91 586 <0.01Min. 8.38 8.53 449 <0.01
t = 28 d Mean 8.34 8.11 531 <0.01Standard Deviation 0.04 0.11 28Max. 8.40 8.27 565 <0.01Min. 8.30 7.91 496 <0.01
DPPDA t = 0 d Mean 8.37 7.78 420 0.06Standard Deviation 0.07 0.38 35 0.05Max. 8.44 8.20 484 0.1Min. 8.30 7.22 383 <0.01
t = 28 d Mean 8.47 8.19 470 0.07Standard Deviation 0.03 0.11 50 0.15Max. 8.51 8.33 571 0.4Min. 8.43 8.05 430 <0.01
MBA t = 0 d Mean 8.46 8.24 471 <0.01Standard Deviation 0.04 0.18 35Max. 8.49 8.44 545 <0.01Min. 8.37 8.03 437 <0.01
t = 28 d Mean 8.36 7.86 485 <0.01Standard Deviation 0.08 0.24 27Max. 8.50 8.20 524 <0.01Min. 8.27 7.47 445 <0.01
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Table S8 Mean, standard deviation, maximum, and minimum of water quality parameters in replicate test vessels across treatments at the initiation and conclusions of tests exposing Tubifex tubifex to sediment spiked with SPAs.
Experiment pH Dissolved oxygen (mg/L)
Conductivity (µS/cm)
Ammonia (mg/L)
DPA t = 0 d Mean 8.41 7.74 446 0.01Standard Deviation 0.06 0.29 8 0.04Max. 8.47 8.26 460 0.1Min. 8.32 7.44 439 <0.01
t = 28 d Mean 8.38 7.66 476 <0.01Standard Deviation 0.06 0.13 23Max. 8.44 7.79 509 <0.01Min. 8.28 7.39 449 <0.01
PNA t = 0 d Mean 8.36 7.89 489 <0.01Standard Deviation 0.03 0.11 40Max. 8.41 8.06 535 <0.01Min. 8.34 7.73 438 <0.01
t = 28 d Mean 8.44 7.96 496 <0.01Standard Deviation 0.06 0.14 33Max. 8.51 8.13 529 <0.01Min. 8.34 7.74 452 <0.01
DPPDA t = 0 d Mean 8.37 7.62 438 0.004Standard Deviation 0.06 0.09 33 0.01Max. 8.43 7.71 475 0.025Min. 8.28 7.47 398 <0.01
t = 28 d Mean 8.52 7.77 482 0.03Standard Deviation 0.04 0.08 72 0.05Max. 8.58 7.90 630 0.1Min. 8.48 7.69 428 <0.01
MBA t = 0 d Mean 8.41 7.88 497 <0.01Standard Deviation 0.03 0.13 32Max. 8.45 8.09 533 <0.01Min. 8.34 7.67 443 <0.01
t = 28 d Mean 8.59 8.50 514 <0.01Standard Deviation 0.04 0.12 40Max. 8.62 8.66 567 <0.01Min. 8.54 8.26 462 <0.01
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Table S9 Mortality for each replicate in experiments exposing Hyalella azteca to water spiked with SPAs for 96 h.
Nominal concentration
of DPA in water(μg/L)
Mortality(/10)
Nominal concentration
of PNA in water(μg/L)
Mortality(/10)
Nominal concentration of DPPDA in
water(μg/L)
Mortality(/15)
Nominal concentration
of MBA in water(μg/L)
Mortality(/10)
Control 0 Control 0 Control 1 Control 1Control 1 Control 2 Control 0 Control 0Control 1 Control 1 Control 0 Control 1Solvent 0 Solvent 2 Solvent 0 Solvent 1Solvent 0 Solvent 0 Solvent 1 Solvent 0Solvent 1 Solvent 1 Solvent 1 Solvent 1
Table S10 Mortality, growth (g dry weight (dw)/ amphipod), and production of biomass (g dw / test vessel) for each replicate in experiments exposing Hyalella azteca to sediment spiked with SPAs for 28 days.
Nominal concentration
of DPA in sediment(μg/g dw)
Mortality(/15)
Growth Biomass production
Nominal concentration
of PNA in sediment(μg/g dw)
Mortality(/15)
Growth Biomassproduction
Nominal concentration of DPPDA in
sediment(μg/g dw)
Mortality(/15)
Growth Biomassproduction
Nominal concentration of
MBA in sediment(μg/g dw)
Mortality(/15)
Growth Biomassproduction
Control 1 0.000386 0.000360 Control 0 0.00055 0.00036 Control 0 0.000313 0.000313 Control 1 0.000657 0.000613Control 1 0.000536 0.000500 Control 0 0.00043 0.00055 Control 0 0.000307 0.000307 Control 1 0.000529 0.000493Control 0 0.000473 0.000473 Control 0 0.00162 0.00043 Control 2 0.000723 0.000627 Control 0 0.000540 0.000540Control 0 0.000456 0.000456 Control 0 0.00034 0.00141 Control 0 0.000627 0.000627 Control 1 0.000636 0.000593Control 0 0.000333 0.000333 Control 0 0.00030 0.00034 Control 0 0.000587 0.000587 Control 4 0.000818 0.000600Control 0 0.000453 0.000453 Control 0 0.00040 0.00040 Control 0 0.000393 0.000393 Control 0 0.000467 0.000467Solvent 2 0.000569 0.000493 Solvent 0 0.00051 0.00051 Solvent 0 0.000820 0.000820 Solvent 0 0.000453 0.000453Solvent 1 0.000471 0.000440 Solvent 0 0.00030 0.00030 Solvent 0 0.000327 0.000327 Solvent 2 0.000746 0.000647Solvent 0 0.000593 0.000593 Solvent 2 0.00081 0.00070 Solvent 2 0.000769 0.000667 Solvent 0 0.000513 0.000513Solvent 0 0.000733 0.000733 Solvent 0 0.00037 0.00035 Solvent 0 0.000767 0.000767 Solvent 2 0.000562 0.000487Solvent 0 0.000520 0.000520 Solvent 1 0.00044 0.00041 Solvent 1 0.000629 0.000587 Solvent 1 0.000664 0.000620Solvent 3 0.000425 0.000340 Solvent 0 0.00025 0.00023 Solvent 0 0.000673 0.000673 Solvent 0 0.000633 0.000633
Table S11 Adult mortality, number of juvenile worms >500 µm, number of juvenile worms <500 µm, number of full cocoons, number of empty cocoons, total number of cocoons and total number of juveniles for each replicate in experiments exposing Tubifex tubifex to sediment spiked with SPAs for 28 days.
Figure S1 Images of mature Tubifex tubifex used in the current study along with juvenile worm >500 µm in size (A; x10 magnification) and full and empty cocoons (B; x20 magnification).
A
B
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Juvenile worm >500 µm
Mature worm
Cocoon
Analysis of toxicity dataRed – data file nameBlue – variable name (top of column)Green – new function file namePurple – R output
R coding for determination of LCx and ECx values in drc
The scale of the data will determine whether a lower or upper limit is set on the model.
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Table S12 Mean concentrations of SPAs in exposure solutions used in 96-h water-only tests with Hyalella azteca and percent differences in measured concentration from Day 0 to 4. The number in the brackets is the standard deviation of triplicate samples. Percent differences between nominal concentrations in water and measured concentrations in water at day 0 and percent differences between measured concentrations in water from day 0 to 4.
Mean concentration (µg/L)
SPA Nominal concentration (µg/L) Day 0 Day 4 Percent difference –
Table S13 Mean concentrations of SPAs in sediment and overlying water of test vessels at the initiation and conclusion of the 28-day sediment exposures with Hyalella azteca. The number in brackets is the standard deviation of triplicate samples. Percent differences in nominal concentration in sediment and measured concentrations in sediment on day 0, percent differences between measured concentrations in sediment at day 0 and 28, and percent differences between measured concentrations in overlying water on day 0 and 28.
Mean concentration – sediment (µg/g dw) Mean concentration – overlying water (µg/L)
Table S14 Mean concentrations of SPAs in overlying water of test vessels at the initiation and conclusion of 28-day sediment exposures with Tubifex tubifex. The number in brackets is the standard deviation of triplicate samples. Percent differences in nominal concentrations in sediment and measured concentrations in sediment on day 0, percent differences between measured concentrations in sediment at day 0 and 28, and percent differences between measured concentrations in overlying water on day 0 and 28.
Mean concentration – sediment (g/g dw) Mean concentration – overlying water (µg/L)
Table S15 Measured concentrations of substituted phenylamine antioxidants (SPA) at the conclusion of the test in water causing 10, 25, or 50% mortality in Hyalella azteca in 96-h water-only tests and associated 95% confidence intervals. The number in brackets is the standard error for each LC.
SPA LC10 (µg/L) 95% CI LC25 (µg/L) 95% CI LC50 (µg/L) 95% CI
Table S16 Measured concentrations of substituted phenylamine antioxidants (SPA) in overlying water and sediment at the conclusion of the test causing 10, 25, and 50% mortality or reduction in growth or production of biomass and associated 95% confidence intervals for Hyalella azteca in 28-day sediment tests. The number in brackets is the standard error for each LC/EC.
SPA Matrix Endpoint LC/EC10 95% CI LC/EC25 95% CI LC/EC50 95% CI
: 95% confidence interval included zero: Unable to calculate LC/ECs because of concentrations of PNA in overlying water were < MDL in all treatments except the highest concentration tested and the effect at the highest concentration tested was > 50%: LC/ECs could not be calculated and LC/EC50s were greater than the concentration of MBA in the overlying water and sediment in the treatment with the highest concentration of MBA due to 50% mortality or 50% reduction in production of biomass not being observed in this treatment.NA: drc package not able to estimate standard error or 95% confidence interval due to relatively high variability in the data
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Table S16 Measured concentrations of substituted phenylamine antioxidants (SPA) in sediment at the conclusion of the test causing 10, 25, and 50% mortality in adult Tubifex tubifex and 10, 25, and 50% reduction in production of juvenile worms >500 µm, juvenile worms >500 µm, full cocoons, empty cocoons, total cocoons or total juveniles and associated 95% confidence interval for 28-day sediment tests. The number in brackets is the standard error for each EC.