Occurrence, elimination, and risk of anticoagulant rodenticides in wastewater and sludge

Silvia Lacorte, Cristian Gómez- Canela

Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26, 08034 Barcelona Rats and super-rats Neverending story

1967 Coumachlor 1 tn rodenticides /city per campaign “It will be the LAST ONE”

Rodenticides

Biocides: use regulated according to EU.

Used mainly as bait formulations. First generation: multiple feedings, less persistent in tissues, commensal and outdoor use. Second generation: single feeding (more toxic), more persistent in tissue, commensal use only.

Toxic: vitamin K antagonists that cause mortality by blocking an animal’s ability to produce several key blood clotting factors. High oral, dermal and inhalation toxicity. Origin and fate of rodenticides Study site: Catalonia (7.5 M inhabitants)

1693 km of sewage corridor 13 fluvial tanks (70.000 m3) 130,000,000 € / 8 YEARS 32,000 km2

378,742 kg/y AI 2,077,000 € Objectives

1. To develop an analytical method to determine most widely used rodenticides in wastewater and sludge. 2. To monitor the presence of rodenticides within 9 WWTP receiving urban and agricultural waters. 3. To evaluate the risk of rodenticides using Daphnia magna as aquatic toxicological model. 4. To study the accumulation of rodenticides in sludge. Compounds studied

Coumachlor* Pindone C19H15ClO4 C14H14O3

Dicoumarol Warfarin C19H12O6 C19H16O4

Coumatetralyl Ferulenol FGARs C19H16O3 C24H30O3

Acenocoumarol Chlorophacinone • Solubility C19H15NO6 C23H15ClO3 0.001-128 mg/L

• pKa 3.4-6.6 Flocoumafen Bromadiolone C H F O C H BrO 33 25 3 40 30 23 4 • Log P 1.92-8.5

Brodifacoum Fluindione C H BrO 31 23 3 C15H9FO2 SGARs

Difenacoum Fenindione C31H24O3 C15H10O2 1. Analytical methodology Extraction Oasis HLB®, ENV+, Centrifugation Strata AW, Isolute C18 at 10 min at 4000 rpm to remove particulate matter

Evaporation

Ultrasonic Reconstitution: extraction 500 µL

1:1 MeOH:H2O HPLC (5mM NH COOH) Clean up 4

Mass spectral characterization (FIA) SGARs . 4 - hydroxicoumarin derivatives.

+ + [C10 H6 O 3 ] [C 14H 10] Bromadiolone_10ppm_4 Brodifacoum_10ppm_4 Bromadiolone_10ppm_4 7 (0.291) 4: Daughters of 509ES+ Brodifacoum_10ppm_4 8 (0.368) 178 1: Daughters of 523ES+ 173 5.22e5 9.56e5 100 Bromadiolone 100 m/z = 251 Brodifacoum m/z = 173 + [C16 H11 O 3 ] 251

m/z = 178

m/z = 335

% % [335 -Br] + + [C 20H 15Br] 256 335

[ M+H ] + + [M- H 2 O] 523/525 509/511

0 m/z 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

+ + Difenacoum4 [C 20H 17] [C 24 H 19 O 3 ] Difenacoum4 7 (0.342) 4: Daughters of 445ES+ Flocoumafen_10ppm_4 257 2.85e6 Flocoumafen_10ppm_4 3 (0.248) 355 9: Daughters of 543ES+ 100 100 Flocoumafen 3.25e6 Difenacoum m/z = 257

m/z = 179 m/z = 159

m/z = 355

+ [C 14H 11]

179 % %

+ [C 8 H 6 F 3 ] + [ M+H ] + 159 [ M+H ] 543 445

0 m/z 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 RC_6_1ppmR RC_6_1ppmR Sm (Mn, 5x2) MRM of 28 Channels ES+ 8.76 100 8.76 223 > 176 (Phenindione)+223 > 205 (Phenindione) 5.80e5 % 0  Zorbax Eclipse 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 Column: RC_6_1ppmR Sm (Mn, 5x2) MRM of 28 Channels ES+ 9.65 100 9.65 241 > 165 (Fluindione)+241 > 194 (Fluindione) 1.49e5 % 0 XDB-C18 (150x2.1 mm, 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR Sm (Mn, 5x2) MRM of 28 Channels ES+ 11.00 100 354 > 163 (Acenocoumarol)+354 > 296 (Acenocoumarol) 11.00 9.59e5 % 0 5 µm) 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR Sm (Mn, 5x2) MRM of 28 Channels ES+ 11.10 100 293 > 91 (Coumatetraryl)+293 > 175 (Coumatetraryl) 11.10 3.90e6 % 0  Gradient: MeOH:HPLC 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR Sm (Mn, 5x2) MRM of 28 Channels ES+ 11.39 RC_6_1ppmR100 Sm (Mn, 5x2) MRM of231 28 >Channels 147 (Pindone) ES+ 11.36 11.38 100 231 > 57 (Pindone)+231 > 147 (Pindone)1.41e3 % 1.04e4 0 % water (5 mM NH COOH) 00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 4 RC_6_1ppmR0.00 Sm1.00 (Mn, 5x2)2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 MRM17.00 of 28 Channels18.00 ES+ RC_6_1ppmR 11.38 MRM of 23128 Channels > 57 (Pindone) ES+ 100 11.59 100 309 > 121 (Warfarin)+309 > 163 (Warfarin)9.02e3

% 11.59 2.34e6 -1 0 %  Flow: 0.3 mL min 00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR0.00 Sm1.00 (Mn, 5x2)2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 MRM17.00 of 28 Channels18.00 ES+ RC_6_1ppmR 11.59 MRM of309 28 > Channels 163 (Warfarin) ES+ 100 12.53 100 337 > 121 (Dicoumarol)+337 > 163 (Dicoumarol)1.16e6

% 12.53 3.30e5 %  0 Acquisition: MRM 00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR Sm (Mn, 5x2) MRM of 28 Channels ES+ RC_6_1ppmR MRM of 28 Channels ES+ 11.59 12.60 309 > 121 (Warfarin) 100 343 > 163 (Coumachlor)+343 > 285 (Coumachlor) 12.60 3.38e53.82e5 % % 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0010.00 11.0011.00 12.0012.00 13.0013.00 14.0014.00 15.0015.00 16.0016.00 17.0017.00 18.0018.00 RC_6_1ppmR Sm (Mn, 5x2) MRMMRM ofof 2828 ChannelsChannels ES+ES+ 12.53 13.59 337 > 163 (Dicoumarol) 100 375 > 77 (Chlorophacinone)+375 > 263 (Chlorophacinone) 13.59 4.58e41.53e5 % % 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0010.00 11.0011.00 12.0012.00 13.0013.00 14.0014.00 15.0015.00 16.0016.00 17.0017.00 18.0018.00 RC_6_1ppmR Sm (Mn, 5x2) MRMMRM ofof 2828 ChannelsChannels ES+ES+ 12.53 337 > 121 (Dicoumarol) 100 509 > 173 (bromodiolone)+509 > 251 (bromodiolone) 14.32 3.56e54.65e4 % % 0 Time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0010.00 11.0011.00 12.0012.00 13.0013.00 14.0014.00 15.0015.00 16.0016.00 17.0017.00 18.0018.00 RC_6_1ppmR MRM of 28 Channels ES+ 100 445 > 179 (Diphenacoum)+445 > 257 (Diphenacoum) 14.71 2.85e6 % 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR MRM of 28 Channels ES+ 100 543 > 159 (Flocoumafen)+543 > 355 (Flocoumafen) 14.97 3.09e6 % 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR MRM of 28 Channels ES+ 100 523 > 178 (brodifacoum)+523 > 335 (brodifacoum) 15.30 1.25e6 % 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 RC_6_1ppmR MRM of 28 Channels ES+ 100 367 > 107 (Ferulenol)+367 > 175 (Ferulenol) 15.41 6.21e6 % 0 Time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 Method performance

% R (SPE) % R (SPE) % R (LLE) IDL Repetitivity Reproduc. MDL Compounds ±RSD, n=3 ±RSD, n=3 ±RSD, n=3 (ng) (0.25 ng µL-1) (0.25 ng µL-1) (ng L-1) Milli-Q WW WW Fenindione 0.01 6.3 8.7 60±3* 58±4* n.r. 13* Fluindione 0.02 1.6 12.2 53±8* 48±6* n.r. 11* Acenocoumarol 0.01 2.0 12.0 71±3 92±2 105±15 0.34 Coumatetraryl 0.14 5.6 8.3 101±5 99±5 80±8 4.9 Pindone 3.88 20.2 21.3 93±8 109±15 n.r. 9.7 Warfarine 0.01 0.7 8 89±4 103±3 91±9 1.6 220 Dicoumarol 0.17 1.2 6.9 71±6 141±12 76±6 14 200 Chlorophacinone 0.13 9.3 4.6 89±6 71±1 85±5 20 180 y = 0.6357x + 32.314 Bromadiolone 0.05160 3.9 14.0 102±3 134±20 107±12 5.1

R² = 0.8116 Difenacoum ) 0.02140 1 16.1 75±2 118±21 138±6 0.65 1 - Flocoumafen 0.04120 1.9 11.9 79±4 95±10 113±16 0.55

Brodifacoum 0.06100 2.5 12.5 81±6 99±9 99±20 12 80 Ferulenol 0.03 5.3 16.3 102±12 112±25 34±3 16 60 LLE (ng L 40 20 0 0 20 40 60 80 100 120 140 160 180 SPE (ng L-1) 2. Monitoring

9 WWTP from Catalonia

Influent Biological Effluent reactor

24h composite sampling

3000000 y = 5.4258x - 23643 R² = 0.98 2500000

2000000 /day)

3 1500000

1000000 inflow (m

500000

0 0 100000 200000 300000 400000 500000 600000 inhab-eq % compounds detected

Warfarine 15 Coumatetralyl 1 6 Chlorophacinone 7 Ferulenol 1 Acenocoumarol Flocoumafen 10 47 Brodifacoum 6 Bromadiolone 1 Difenacoum Rodenticides in wastewater

400

350 % removal: 31-100%

300

250 ) 1 -

200 Agricultural Barcelona No traces in effluents [∑ARs]L (ng 150

100

50

0 Inf Bio Eff Inf Bio Eff Inf Bio Eff Inf Bio Eff Inf Bio Eff Inf Bio Eff Inf Bio Eff Inf Bio Eff Inf Bio Eff WWTP1 WWTP2 WWTP3 WWTP4 WWTP5 WWTP6 WWTP7 WWTP8 WWTP9 N= 3 (days sampled) Water influent vs concentration Discharge of rodenticides to receiving waters

WWTP 2

WWTP 1 WWTP 8 WWTP 5 WWTP 9 WWTP 3

WWTP 4 WWTP 7

WWTP 6

0 100 -2500 >2500 mg / day- 3. Aquatic risk

OECD 202 Test Acute Immobilisation Test and Reproduction

Bromadiolone Clorophacinone Test (48 h) Warfarin

LC50= 0.56 (0.01) LC50= 49.68 (4.42) LC50= 170.19(3.45) 100

50 % Dead %

0

0.01 0.1 1 10 100 Concentration (mg/L) All compounds detected at concentration <<< EC50 Fish toxicity

mg/kg bw Warfarin 730 Coumatretralyl 48 Dicoumarol 7.5 Bromadiolone 1.4 Chlorophacinone 0.35 Flocumafen 0.15 Potential risk Difenacoum 0.064 for fish

Bennet, S.M., 2012. PiedPiper Northern Limited. http://www.the-piedpiper.co.uk/. 4. Accumulation in sludge

100 %R 80 60 40 20 0 Warfarin Ferulenol Fluindione Dicoumarol Coumachlor Phenindione Brodifacoum Flocoumafen Diphenacoum Bromodiolone Chlorophacino… Coumatretralyl Acenocoumarol

ng/g dw Warfarin 0.6 -112 Coumachlor 3 - 97 Ferulenol 1.4 - 2.9 Brodifacoum 3 - 1700 More rats than human beings! Conclusions

• AG rodenticides are an emerging family of contaminants for which little information is available regarding the specific sources, levels in different types of waters and impact on the aquatic environment. • AG rodenticides pose mammals and birds at high risk, specifically affecting protected species.

Needs: • Environmental stability in water and soil • Accumulation in agricultural soils • Impact on wildlife inhabiting in agricultural areas

Thank you very much for your attention