Supporting Information/Supplementary Material

Supporting Information/Supplementary Material

A multidisciplinary assessment of river surface water quality in areas heavily influenced by human activities.

Nikolina Charalampous1, Aggeliki Kindou2, Dimitris Vlastos2,Vasiliki Tsarpali1, Maria Antonopoulou2, Ioannis Konstantinou2 and Stefanos Dailianis1*

1Department of Biology, Section of Animal Biology, School of Natural Sciences, University of Patras, 26500, Patras, Greece.

2Department of Environmental and Natural Resources Management, University of Patras, 30100, Agrinio, Greece.

* Corresponding author

Tel.: +32610-969213

Fax: +32610-969213

E-mail:

Section of Animal Biology

Department of Biology

Faculty of Sciences, University of Patras

26 500 PATRAS

GREECE

Table of contents

SM 2.3.3 Determination of pesticides.

SM 2.4.1Surface water sample toxicity in microalgae Chlorococcum sp. and Scenedesmus rubescens.

SM Table 1. Characteristics (limits of detection (LODs) and quantification (LOQs), total uncertainty) of the methods followed for the determination of total Cr, Cr(VI), Pg, Cd, Ni, Hg and Zn in surface water samples collected from 3 sampling stations (S1, S2 and S3) along Asopos river.

SM Table 2. Pesticide concentrations (μg L-1) in water samples from S1, S2 and S3 stations of Asopos river.

SM Table 3. Cell number (x104) and growth rate (μ values expressed as day-1 in the parenthesis) of Scenedesmus rubescens (A), and24h IC values (including lower and upper bound confidence intervals within parenthesis), as obtained by Probit analysis (p<0.05) (B),after exposure to surface water samples from S1, S2 and S3 along Asopos River. The results are mean  SDs (N=3, each measurement was performed in duplicate). Values with asterisks in each column differ significantly from the respective control in each case (Mann-Whitney U test, p<0.05). Values in each row that share the same letter differ significantly in each case (Friedman test, p<0.05).

SM Table 4. Cell number (x104) and growth rate (μ values expressed as day-1 in the parenthesis) of Chlorococcum sp.,(A), and24h IC values (including lower and upper bound confidence intervals within parenthesis), as obtained by Probit analysis (p<0.05) (B),after exposure to surface water samples from S1, S2 and S3 along Asopos River. The results are mean  SDs (N=3, each measurement was performed in duplicate). Values with asterisks in each column differ significantly from the respective control in each case (Mann-Whitney U test, p<0.05). Values in each row that share the same letter differ significantly in each case (Friedman test, p<0.05).

SM Table 5. Determination of (A) 24h LC values in Thamnocephalus platyurus and Brachionus calyciflorus and (B) hazard classification of water samples collected from S1, S2 and S3 along Asopos River. 24h LC values (expressed as % v/v) are mean ± SD from 4 independent measurements.

SM 2.3.3 Determinationofpesticides.

LCseparationswereperformedinaAccelaThermoUPLCAccelapump (ThermoFisherScientificInc, USA) usinga 100 mm × 2.1 mmHypersilGold 1.9 μmcolumn (ThermoScientific). A sample volume of 10 μL was injected with a Thermo Accelaautosampler. The mobile phase was composed of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) at a constant flow of 0.5 mL min-1. The gradient was programmed to increase the amount of B from an initial 20% to 100% in 5 min, returning to the initial conditions (20% B) in 0.1 min (from 5 to 5.1 min). Mass spectrometry was performed on a Thermo LC-MS/MS TSQ Quantum Access Max (Thermo Scientific) fitted with an ESI interface and controlled by Thermo TSQ Tune master software (version 2.1.0.1030). Typical interface conditions were optimised for maximum intensity of the precursor ions as follows: nebulizer and desolvation (drying gas) N2 flows were set at 650 and 150 L h-1, respectively, source block and desolvation temperatures were 100 and 350oC, respectively. The ESI polarity ionisation mode was set individually for each target compound, the switch time between positive and negative mode was 20 ms. For the MS–MS fragmentation argon was used as collision gas at a pressure of 3.7×10−3mBar. TraceFinder software was used for data acquisition under selective reaction monitoring (SRM) mode. Selection and tuning of SRM transitions were performed individually for each analyte. For optimising the mass spectrometer, direct infusion of a 100 μg L-1 standard solution of each analyte was used.

GC-MS analysis was performed using a QP-2010 Shimadzu (Kyoto, Japan) instrument equipped with a SPB-5ms (30 m x 0.25 mm x 0.25 μm) capillary column was used at the following chromatographic conditions: Helium was used as carrier gas at 87.5 kPa and 1.4 mL/min flow rate. The injection temperature was 230 oC. The column was programmed from 70 oC to 180 oC at 30 oC/min (first step), from 180 oC to 230 oC (second step) at 1.8 oC/min and from 230oC to 300oC (25 min) (final step). The interface was kept at 300 oC.The quadrupole mass spectrometer was operated in electron impact (EI) ionization mode at 70 eV and monitored ions from m/z 80 to 450. Selected Ion Monitoring (SIM) mode was used for the determination of selected pesticides. Two ions of m/z >200 or three ions of m/z >100 were used as the minimum requirements for confirmation according to the quality control procedures established by the EU. The base peak was used for quantification, and the other ions were used for confirmation purposes. Groupings in ion sets were defined on the basis of the retention times of the analyzed pesticides to increase the sensitivity of the MS analysis. The injection volume was 1 μL for all standards and samples in splitless mode.For both LC-MS/MS and GC MS analyses limits of detection ranged between 3-15 ng L-1. Linearity over 5 to 500 ng L-1 was always better than r2=0.9916 and the mean recoveries for six replicates ranged between 73% to 112%.

SM 2.4.1Surface water sample toxicity in microalgae Chlorococcum sp. and Scenedesmus rubescens.

Equation 1 for estimating growth rate:

μn: algal growth rate (day-1) after n days (24, 48, 72 or 96h)

X0 = number of cells/ml at time 0 (t0)

Xn= number of cells/ml at tn

t0 = time of first measurement after beginning of test

tn = time of nth measurement after beginning of test

Equation 2 for estimating inhibition rate:

%I: percent inhibition in average specific growth rate

μc: mean value for average specific growth rate (μ) in the control group

μn: average specific growth rate for the treatment replicate.

SM Table 1. Characteristics (limits of detection (LODs) and quantification (LOQs), total uncertainty) of the methods followed for the determination of total Cr, Cr(VI), Pg, Cd, Ni, Hg and Zn in surface water samples collected from 3 sampling stations (S1, S2 and S3) along Asopos river.

Metal ion / Method of analysis / LODs
(μg L-1) / LOQs
(μg L-1) / Total uncertainty
(%)
Total Cr / In house based in APHA 3113A and B by AAS / 1.5 / 5 / 14.2
Cr(VI) / APHA: 21th EDITION 2005 / 3 / 10 / 25.0
Pb / In house based in 3111A and B by AAS / 0.5 / 1.5 / 16.7
Cd / In house based in APHA 3111A and B by AAS / 0.03 / 0.1 / 40.0
Ni / In house based in APHA 3113A and B by AAS / 0.3 / 1 / 28.1
Hg / In house based in APHA 3112B by AAS/Hydride / 0.1 / 0.3 / 16.7
Zn / In house based in APHA 3113A and B by AAS / 30 / 100 / 37.0

SM Table 2. Pesticide concentrations (μg L-1) in water samples from S1, S2 and S3 stations of Asopos river.

Pesticide / Method for analysis / Sampling stations
S1 / S2 / S3

Abamectin

aLC

/ c<0.01 / <0.01 / <0.01

Acetamiprid

LC

/ <0.01 / <0.01 / <0.01

Acetochlor

LC

/ <0.01 / <0.01 / <0.01

Aclonifen

bGC

/ <0.01 / <0.01 / <0.01
Acrinathrin /

GC

/ <0.05 / <0.05 / <0.05
Amitraz /

LC

/ <0.01 / <0.01 / <0.01
Azinphos-methyl /

LC

/ <0.01 / <0.01 / <0.01
Azoxystrobin /

LC

/ <0.01 / <0.01 / <0.01
Bifethrin /

LC

/ <0.01 / <0.01 / <0.01
Bitertanol /

LC

/ <0.01 / <0.01 / <0.01
Boscalid /

LC

/ <0.01 / d0.03 / d0.03
Bromuconazole /

/ <0.01 / <0.01 / <0.01
Bupirimate /

/ <0.01 / <0.01 / <0.01
Buprofezin /

/ <0.01 / <0.01 / <0.01
Captan /

/ <0.01 / <0.01 / <0.01
Carbaryl /

/ <0.01 / <0.01 / <0.01
Carbendazim /

/ <0.01 / <0.01 / <0.01
Chlorantraniliprole /

/ <0.01 / <0.01 / <0.01
Chlorbromuron /

/ <0.01 / <0.01 / <0.01
Chlormephos /

/ <0.01 / <0.01 / <0.01
Chlorothalonil /

/ <0.01 / <0.01 / <0.01
Chlorpyriphos /

/ <0.01 / <0.01 / <0.01
Chlorpyriphos-methyl /

/ <0.01 / <0.01 / <0.01
Clofentezin /

/ <0.01 / <0.01 / <0.01
Clothianidin /

/ <0.01 / <0.01 / <0.01
Cycloxydim /

/ <0.01 / <0.01 / <0.01
Cyfluthrin /

/ <0.03 / <0.03 / <0.03
Cyhexatin (Sum of Azocyclotin and Cyhexatin) /

/ <0.05 / <0.05 / <0.05
Cypermethrin (Sum) /

/ <0.05 / <0.05 / <0.05
Cyproconazole /

/ <0.01 / <0.01 / <0.01
Cyprodinil /

/ <0.01 / <0.01 / <0.01
Cyromazin /

/ <0.01 / <0.01 / <0.01
Deltamethrin /

/ <0.01 / <0.01 / <0.01
Diazinon /

/ <0.01 / <0.01 / <0.01
Dichlorvos /

/ <0.01 / <0.01 / <0.01
Dicofol /

/ <0.01 / <0.01 / <0.01
Diethofencarb /

/ <0.01 / <0.01 / <0.01
Forchlorfenuron / LC / <0.01 / <0.01 / <0.01
Formetanate / LC / <0.01 / <0.01 / <0.01
Hexaconazole / LC / <0.01 / <0.01 / <0.01
Hexythiazox / LC / <0.01 / <0.01 / <0.01
Imazalil / LC / <0.01 / <0.01 / <0.01
Imidacloprid / LC / <0.01 / <0.01 / <0.01
Indoxacarb / LC / <0.01 / <0.01 / <0.01
Iprodione / GC / <0.01 / <0.01 / <0.01
Iprovalicarb / LC / <0.01 / <0.01 / <0.01
Isoprothiolane / LC / <0.01 / <0.01 / <0.01
Kresoxim-methyl / LC / <0.01 / <0.01 / <0.01
Linuron / LC / <0.01 / <0.01 / <0.01
Lufenuron /

/ <0.01 / <0.01 / <0.01
Malathion /

/ d 0.04 / d 0.04 / d 0.01
Mandipropamid /

/ <0.01 / <0.01 / <0.01
Mecarbam /

/ <0.01 / <0.01 / <0.01
Mepanipyrim / LC / <0.01 / <0.01 / <0.01
Mercaprodimethur / LC / <0.01 / <0.01 / <0.01
Metaflumizone / LC / <0.01 / <0.01 / <0.01
Metalaxyl /

/ <0.01 / <0.01 / <0.01
Methamidophos /

/ <0.05 / <0.05 / <0.05
Methidathion /

/ <0.01 / <0.01 / <0.01
Methomyl (Methomyl and Thiodicarb Sum) /

/ <0.01 / <0.01 / <0.01
Methoxyfenozide /

/ <0.01 / <0.01 / <0.01
Metobromuron /

/ <0.01 / <0.01 / <0.01
Myclobutanil /

/ <0.01 / <0.01 / d 0.01
Ofurace / LC / <0.01 / <0.01 / <0.01
Omethoate /

/ <0.01 / <0.01 / <0.01
Oxadiazon /

/ <0.01 / <0.01 / <0.01
Oxamyl /

/ <0.01 / <0.01 / <0.01
Oxyfluorfen /

/ <0.01 / <0.01 / <0.01
Parathion-methyl /

/ <0.01 / <0.01 / <0.01
Penconazole / LC / <0.01 / <0.01 / <0.01
Pencycuron / LC / <0.01 / <0.01 / <0.01
Pendimethanil /

/ <0.01 / <0.01 / <0.01
Phosalone /

/ <0.01 / <0.01 / <0.01
Phosmet /

/ <0.01 / <0.01 / <0.01
Pirimicarb /

/ <0.01 / <0.01 / <0.01
Pirimiphos-methyl /

/ <0.01 / <0.01 / <0.01
Prochloraz /

/ <0.01 / <0.01 / <0.01
Procymidon /

/ <0.01 / <0.01 / <0.01
Difenoconazole /

/ <0.01 / <0.01 / <0.01
Diflubenzuron /

/ <0.01 / <0.01 / <0.01
Dimethoate / LC / <0.01 / <0.01 / <0.01
Dimethomorph / LC / <0.01 / <0.01 / <0.01
Diphenylamine / LC / <0.01 / <0.01 / <0.01
Dodine / LC / <0.01 / <0.01 / <0.01
Endosulfan (Sum) / GC / <0.01 / <0.01 / <0.01
Esfevalerate / GC / <0.01 / <0.01 / <0.01
Ethiofencarb / LC / <0.01 / <0.01 / <0.01
Ethion / LC / <0.01 / <0.01 / <0.01
Ethiprole / LC / <0.01 / <0.01 / <0.01
Ethoprophos / LC / <0.01 / <0.01 / <0.01
Etofenprox / LC / <0.01 / <0.01 / <0.01
Etoxazole / GC / <0.02 / <0.02 / <0.02
Fenamidone / LC / <0.01 / <0.01 / <0.01
Fenamiphos / LC / <0.01 / <0.01 / <0.01
Fenarimol / LC / <0.01 / <0.01 / <0.01
Fenazaquin / LC / <0.01 / <0.01 / <0.01
Fenbuconazole / LC / <0.01 / <0.01 / <0.01
Fenbutatinoxide / LC / <0.05 / <0.05 / <0.05
Fenhexamid / LC / <0.01 / <0.01 / <0.01
Fenitrothion / GC / <0.01 / <0.01 / <0.01
Fenoxycarb / LC / <0.01 / <0.01 / <0.01
Fenpropimorph / LC / <0.01 / <0.01 / <0.01
Fenpyroximate / LC / <0.01 / <0.01 / <0.01
Fenthion / LC / <0.01 / <0.01 / <0.01
Fenvalerate / GC / <0.01 / <0.01 / <0.01
Fluabendiamide / LC / <0.01 / <0.01 / <0.01
Fluapicolide / LC / <0.01 / <0.01 / <0.01
Fluazifop-Butyl / LC / <0.01 / <0.01 / <0.01
Fluazinam / LC / <0.01 / <0.01 / <0.01
Flucythrinate / LC / <0.01 / <0.01 / <0.01
Fludioxonil / LC / <0.01 / <0.01 / <0.01
Flufenoxuron / LC / <0.01 / <0.01 / <0.01
Fluquinconazole / LC / <0.01 / <0.01 / <0.01
Flusilasole / LC / <0.01 / <0.01 / <0.01
Flutriafol / LC / <0.01 / <0.01 / <0.01
Folpet / GC / <0.02 / <0.02 / <0.02
Propamocarb /

/ <0.01 / <0.01 / <0.01
Propaquizafop / LC / <0.01 / d 0.01 / d 0.01
Propargite /

/ <0.01 / <0.01 / <0.01
Propyzamide / LC / <0.01 / <0.01 / <0.01
Prosulfocarb / LC / <0.01 / <0.01 / <0.01
Pymetrozine /

/ <0.01 / <0.01 / <0.01
Pyraclostrobin /

/ <0.01 / <0.01 / <0.01
Pyridaben /

/ <0.01 / <0.01 / <0.01
Pyrimethanil /

/ <0.01 / <0.01 / <0.01
Pyriproxyfen /

/ <0.01 / <0.01 / <0.01
Quinoxyfen / LC / <0.01 / <0.01 / <0.01
Quizalofop-ethyl /

/ <0.01 / <0.01 / <0.01
Rimsulfuron / LC / <0.01 / <0.01 / <0.01
Rotenone /

/ <0.01 / <0.01 / <0.01
SpinosadA+D /

/ <0.01 / <0.01 / <0.01
Spirodiclofen /

/ <0.01 / <0.01 / <0.01
Spiromesifen / LC / <0.01 / <0.01 / <0.01
Spiroxamine / LC / <0.01 / <0.01 / <0.01
Tebuconazole /

/ <0.01 / <0.01 / d 0.02
Tebufenozide /

/ <0.01 / <0.01 / <0.01
Tebufenpyrad /

/ <0.01 / <0.01 / <0.01
Teflubenzuron /

/ 0.01 / 0.01 / 0.01
Temephos /

/ <0.01 / <0.01 / <0.01
Tetraconazole /

/ <0.01 / <0.01 / <0.01
Thiabendazole /

/ <0.01 / <0.01 / <0.01
Thiacloprid /

/ <0.01 / <0.01 / <0.01
Thiamethoxam (Thiamethoxam and Clothianidin Sum) /

/ <0.01 / <0.01 / <0.01
Thiophanate-methyl /

/ <0.01 / <0.01 / <0.01
Tolcofos-methyl /

/ <0.01 / <0.01 / <0.01
Thiabendazole / LC / <0.01 / <0.01 / <0.01
Triadimefon /

/ <0.01 / <0.01 / <0.01
Triadimenol /

/ <0.01 / <0.01 / <0.01
Triclopyr /

/ <0.01 / <0.01 / <0.01
Trifloxystrobin /

/ <0.01 / <0.01 / <0.01
Triflumuron /

/ <0.01 / <0.01 / <0.01
Zoxamide / LC / <0.01 / <0.01 / <0.01
α-Cypermethrin /

/ <0.005 / d 0.006 / d 0.006
λ-Cyhalothrin /

/ <0.02 / <0.02 / <0.02
τ-Fluvalinate /

/ <0.01 / <0.01 / <0.01
Values in all cases are expressed as μg/L-1. Values in italics exceed the detection limits.

a determination by LC-MS/MS; b determination by GC-MS; cValues with < represent quantification limits; d Values in italics exceed the detection limits.

SM Table 3. Cell number (x104) and growth rate (μvalues expressed as day-1 in the parenthesis) of Scenedesmus rubescens (A), and24h IC values (including lower and upper bound confidence intervals within parenthesis), as obtained by Probit analysis (p<0.05)(B),after exposure to surface water samples from S1, S2 and S3 along Asopos River. The results are mean  SDs (N=3, each measurement was performed in duplicate). Values with asterisks in each column differ significantly from the respective control in each case (Mann-Whitney U test, p<0.05). Values in each row that share the same letter differ significantly in each case (Friedman test, p<0.05).

A / Incubation time (h)
S1 / 24 / 48 / 72 / 96
control / 15.132.45
(2.710.16) / 29.630.43
(1.690.01) / 40.152.06
(1.230.02) / 45.270.72
(0.950.00)
6.25 / 13.082.45
(2.560.20)abc / 24.250.58
(1.590.01)ade / 36.881.94
(1.200.02)bdf / 40.130.92
(0.920.01) *cef
12.5 / 10.920.72
(2.390.06) *abc / 17.881.01
(1.440.03) *ade / 31.752.87
(1.150.03) *bdf / 36.781.35
(0.900.01) *cef
25 / 10.001.15
(2.300.12) *abc / 15.754.04
(1.370.13) * ad / 26.171.59
(1.090.02) * bf / 32.251.77
(0.870.01) *cdf
50 / 8.750.00
(2.170.00) *abc / 11.424.19
(1.200.17) * ad / 22.002.60
(1.030.04) * be / 29.391.51
(0.840.01) *cde
100 / 5.250.35
(1.660.07) *abc / 10.500.00
(1.180.00) *ade / 19.081.44
(0.980.02) *bdf / 27.391.77
(0.830.02) *cef
S2 / 24 / 48 / 72 / 96
control / 14.000.59
(2.640.04) / 26.332.09
(1.630.04) / 37.061.88
(1.200.02) / 44.500.58
(0.950.00)
6.25 / 11.350.52
(2.430.05) *abc / 22.041.00
(1.550.02) ade / 33.331.28
(1.170.01) bdf / 39.631.65
(0.920.01)cef
12.5 / 9.331.28
(2.230.13) *abc / 19.880.72
(1.490.02) *ade / 31.672.89
(1.150.03) bdf / 36.831.88
(0.900.01) *cef
25 / 7.921.15
(2.060.14) *abc / 16.582.77
(1.400.08) *ade / 31.752.05
(1.150.02) bdf / 36.000.71
(0.900.00) *cef
50 / 6.880.43
(1.930.06) *abc / 13.282.00
(1.290.08) * ad / 29.671.91
(1.130.02) * be / 33.961.46
(0.880.01) *cde
100 / 5.170.35
(1.640.07) *abc / 10.390.35
(1.170.02) *ade / 20.131.36
(1.000.02) *bdf / 25.332.52
(0.810.02) *cef
S3 / 24 / 48 / 72 / 96
control / 13.920.48
(2.630.03) / 25.982.54
(1.630.05) / 38.251.71
(1.210.02) / 43.440.66
(0.940.00)
6.25 / 11.380.53
(2.430.05) *abc / 21.630.72
(1.540.02)ade / 32.500.43
(1.160.00)bdf / 37.860.47
(0.910.00) *cef
12.5 / 9.360.63
(2.240.07) *abc / 18.231.49
(1.450.04) *ade / 30.901.79
(1.140.02) *bdf / 36.691.07
(0.900.01) *cef
25 / 8.250.87
(2.110.11) *abc / 17.350.74
(1.430.02) *ade / 29.711.48
(1.130.02) *bdf / 34.690.75
(0.890.01) *cef
50 / 6.381.01
(1.840.16) *abc / 15.000.29
(1.350.01) *ade / 28.671.91
(1.120.02) *bdf / 33.851.51
(0.880.01) *cef
100 / 4.380.43
(1.470.10) *abc / 8.581.09
(1.070.06) * ad / 15.291.11
(0.910.03) * be / 23.232.79
(0.780.03) *cde
B / 24h IC10 / 24h IC20 / 24h IC50
S1 / 10.7 (5.7-15.7) / 29.2 (21.1-39.7) / >100
S2 / 6.9 (2.6-11.4) / 23.2 (15.1-32.9) / >100
S3 / 9 (4.8-13.2) / 23.3(16.7-30.6) / >100

SM Table 4. Cell number (x104) and growth rate (μvalues expressed as day-1 in the parenthesis) of Chlorococcum sp.,(A), and24h IC values (including lower and upper bound confidence intervals within parenthesis), as obtained by Probit analysis (p<0.05)(B),after exposure to surface water samples from S1, S2 and S3 along Asopos River. The results are mean  SDs (N=3, each measurement was performed in duplicate). Values with asterisks in each column differ significantly from the respective control in each case (Mann-Whitney U test, p<0.05). Values in each row that share the same letter differ significantly in each case (Friedman test, p<0.05).

A / Incubation time (h)
S1 / 24 / 48 / 72 / 96
control / 10.171.73
(2.310.17) / 20.671.54
(1.510.04) / 32.136.78
(1.150.07) / 42.381.59
(0.940.01)
6.25 / 7.750.87
(2.040.11)abc / 14.721.68
(1.340.06)*ade / 22.421.01
(1.040.02)*bdf / 30.672.02
(0.860.02)*cef
12.5 / 7.000.58
(1.940.08)*abc / 13.500.58
(1.300.02)*ade / 19.920.14
(1.000.00)*bdf / 28.130.14
(0.830.00)*cef
25 / 5.500.00
(1.700.00)*abc / 11.670.58
(1.230.03)*ade / 18.390.63
(0.970.01)*bdf / 27.810.82
(0.830.01)*cef
50 / 3.500.29
(1.250.08)*abc / 7.880.43
(1.030.03)*ade / 16.501.30
(0.930.03)*bdf / 26.130.72
(0.820.01)*cef
100 / 2.330.00
(0.850.00)* a / 7.130.72
(0.980.05)*ab / 14.501.73
(0.890.04)* c / 22.250.29
(0.780.00)*bc
S2 / 24 / 48 / 72 / 96
control / 10.921.72
(2.380.16) / 18.191.93
(1.450.05) / 42.001.15
(1.250.01) / 55.902.53
(1.010.01)
6.25 / 8.130.14
(2.090.02)abc / 14.500.50
(1.340.02)*ade / 33.133.71
(1.170.04)*bdf / 41.891.39
(0.930.01)*cef
12.5 / 7.750.00
(2.050.00)*abc / 12.280.82
(1.250.03)*ade / 26.780.38
(1.100.00)*bdf / 39.111.92
(0.920.01)*cef
25 / 4.670.38
(1.540.08)*abc / 11.640.85
(1.230.04)*ade / 26.830.76
(1.100.01)*bdf / 33.850.95
(0.880.01)*cef
50 / 3.520.49
(1.250.13)*ab / 9.830.58
(1.140.03)* c / 24.281.84
(1.060.03)* ad / 31.000.71
(0.860.01)*bcf
100 / 2.480.65
(0.880.28)* / 7.310.96
(0.990.07)* a / 21.481.23
(1.020.02)* b / 27.670.58
(0.830.01)*ab
S3 / 24 / 48 / 72 / 96
control / 7.130.14
(1.960.02) / 24.000.38
(1.590.01) / 37.507.51
(1.200.07) / 49.960.34
(0.980.00)
6.25 / 6.630.14
(1.890.02)*abc / 21.882.17
(1.540.05)ade / 30.750.29
(1.140.00)bdf / 38.880.43
(0.920.00)*cef
12.5 / 5.580.58
(1.720.10)*abc / 16.001.15
(1.390.04)*ade / 26.170.29
(1.090.00)*bdf / 33.171.15
(0.880.01)*cef
25 / 5.390.24
(1.680.04)*abc / 16.170.19
(1.390.01)*ade / 24.221.54
(1.060.02)*bdf / 28.331.44
(0.840.01)*cef
50 / 4.560.10
(1.520.02)*abc / 15.500.00
(1.370.00)*ade / 25.000.43
(1.070.01)*bdf / 29.582.41
(0.850.02)*cef
100 / 3.680.03
(1.300.01)*ab / 13.784.23
(1.300.14)*cd / 21.890.96
(1.030.01)* ace / 27.942.21
(0.830.02)*bde
B / 24h IC10 / 24h IC20 / 24h IC50
S1 / 6.2 (3.7-8.9) / 13.7 (9.8-17.6) / 61.6 (48.4-84.9)
S2 / 5.4 (3.1-7.9) / 12.1 (8.4-15.7) / 55.3 (43.8-75)
S3 / 13.8 (7.6-19.8) / 38.2 (27.7-54.2) / >100

SM Table 5. Determination of (A) 24h LC values in Thamnocephalus platyurus and Brachionus calyciflorus and (B) hazard classification of water samples collected from S1, S2 and S3 along Asopos River. 24h LC values (expressed as % v/v) are mean ± SD from 4 independent measurements.

A / T. platyurus / B. calyciflorus
24h LC10 / 24h LC20 / 24h LC50 / 24h LC10 / 24h LC20 / 24h LC50
S1 / 22.11
(nd) / 25.04
(nd) / 31.76
(nd) / 16.25
(2.72-28.55) / 27.86
(10.1-47.85) / 78.12
(45.76-348.6)
S2 / 13.45
(nd) / 15.82
(nd) / 21.58
(nd) / 5.31
(0.75-10.8) / 11.7
(3.51-19.72) / 53
(32.8-130.27
S3 / 11.61
(0.12-22.72) / 16.57
(0.74-30.76) / 32.76
(12.16-100.47) / 5.25
(0.07-12.77) / 13.26
(1.51-26.45) / 82.71
(40.90-1252.3)
LC values (including lower and upper bound confidence intervals within parenthesis) were obtained by Probit analysis (p<0.05. N= 6).
nd: not detectable
B / T.platyurus / B. calyciflorus
24h LC50 / TU / HC / 24h LC50 / TU / HC
S1 / 31.76 / 3.15 / III / acute toxicity / 78.1 / 1.28 / III / acute toxicity
S2 / 21.58 / 4.63 / III / acute toxicity / 53 / 1.89 / III / acute toxicity
S3 / 32.76 / 3.05 / III / acute toxicity / 82.7 / 1.21 / III / acute toxicity
The characterization was based on the conversion of the obtained LC50 values to toxic units (TU) that are proportional to toxicity. Hazard classification (HC) of each sample was performed as follows: HC I, TU<0.4 no acute toxicity; HC II, 0.4<TU<1, slight acute toxicity; HC III, 1<TU<10, acute toxicity; HC IV, 10<TU, high acute toxicity; HC V, TU>100, very high acute toxicity (Sprague and Ramsay, 1965).