Assessing Emerging and Priority Micropollutants in Sewage Sludge: Environmental Insights and Analytical Approaches

Diana Rede1,2, Ivan Teixeira1, Cristina Delerue-Matos1, and Virgínia Cruz Fernandes1,*

1REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida, 431, 4249-015 Porto, Portugal

2Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169– 007 Porto, Portugal.

*Corresponding author: vir@isep.ipp.pt

S.1 Materials and Methods

S.1.1 Physical, Chemical, and Elemental Characterization of Sewage Sludge

The physicochemical characterization of the samples was carried out according to standardized methods. All parameters were analyzed in duplicate, and blanks were also prepared. For instance, the pH in water (pHw) was determined using SW-846 Test Method 9045D: Soil and Waste pH from EPA (USEPA, 1986). The measurements were performed at room temperature in a 1:2 (m/V) SS/water suspension using an electronic pH meter (micropH 2002 Crison, Spain) after 20-minute of contact time. Dry matter (DM) and organic matter (OM) content were assessed according to EN 12457-4 (CEN, 2002) and the Laboratory Guide for Conducting Soil Tests and Plant Analysis (J.B.Jones, 2001), respectively. DM was determined by weighing the difference, using a forced-draft oven (J.P. Selecta S.A 2000200), where the samples were placed overnight at 105 °C. OM was estimated using the loss-on-ignition methodology. The previous dried sampleswere placed into a muffle furnace (Nabertherm - B 180, Germany) at 450 °C for 4 hours. Total carbon (TOC) was determined using a Shimadzu TOC-Vscn E200V analyzer with SSM-5000A Solid Sample Module (Shimadzu Corporation, Kyoto, Japan).

For the determination of mineral and metal content, samples were digested using HNO3, in open vessels covered with a watch glass and heated conventional (APHA, 1992). After cooling, the volume of the digested samples was adjusted to 10 mL using deionized water. The concentrations of Co, Mo, Hg, Cd, Ni, Pb, Cr, Cu, Zn, Li, Be, V, As, Se, Rb, Sr, Rh, Sb, Cs, Ba, Ir, Tl, Mg, Ca, Mn, and Fe were determined using Ion Coupled Plasma Mass Spectrometry (ICP-MS) performed with a Thermo Fisher Scientific (Bremen, Germany) iCAP™ Q instrument, which was equipped with a MicroMist™ nebulizer (Glass Expansion, Port Melbourne Vic, Australia). The instrument also included a Peltier-cooled baffled cyclonic spray chamber, a standard quartz torch, and a two-cone interface design with Ni sample and skimmer cones., High-purity argon (99.9997%; Praxair, Portugal) was used as the carrier gas. Prior to use, all the labware was properly soaked in10% HNO3 and rinsed with deionized water.

S.1.2 Chromatographic Analysis

PAHs were quantified using an LC system (Shimadzu Corporation, Kyoto, Japan), equipped with an LC-20AD pump, DGU-20AS degasser, and photodiode array SPD-M20A, as well as fluorescence RF-10AXL detectors. The LC system was operated using LC Solution Shimadzu software. Chromatographic separation was performed at 20 ± 1 °C by injecting 15.0 μL of sample extracts onto a NUCLEOSIL® 100-5 C18 PAH column (150 x 4.0 mm and 5 μm particle size) from Macherey–Nagel (Duren, Germany), with a flow rate of 0.8 mL min-1. The chromatographic conditions consisted of a mixture of 50% ACN and 50% ultrapure water. Then, the elution gradient was increased to 100% can over a period of 15 minutes, and these conditions were maintained for an additional 13 minutes (Oliveira et al., 2015).

OPPs and OPEs were determined using a Shimadzu GC-FPD operated by the Shimadzu GC Solution software. The injector and detector temperatures were maintained at 250 and 290 °C, respectively. 1.0 µL of the sample solution was injected in splitless mode on a capillary column TRB-5 (30 m in length, 0.32 mm in inner diameter (i.d.), with a 0.25 µm film thickness, manufactured by Teknokroma, Barcelona, Spain). The GC oven temperature was programmed as follows: 100 °C for 1 min, ramped at 20 °C min-1 to 150 °C, held for 1 min, ramped at 2 °C min-1 to 180 °C, held for 2 min, and finally ramped at 20 °C min-1 to 290 °C, and held at the final temperature for 5 min. Helium (purity 99.99% from Linde Sogás) was used at a pressure of 83.6 KPa, linear velocity of 25.4 cm s-1, and flow rate of 1 mL min-1.

The analysis of PBDEs, PCBs, OCPs, and PYRs was performed in splitless mode with a 2 μL injection volume using a GC-ECD operated by GC Solution Shimadzu software. A capillary column, Zebron-5MS (30 m × 0.25 mm × 0.25 μm), from Phenomenex Spain, was used. The oven temperature was initially set at 40 °C (held for 1 min), then increased to 120 °C at a rate of 15 °C min-1 per minute and held for 1 min. After the temperature was increased to 200 °C at a rate of 10 °C min-1 (held for 1 min), it was further increased to 290 °C at a rate of 7 °C min-1 and maintained for 10 min. The injector and detector temperatures were set at 250 °C and 300 °C, respectively. The flow rate of the helium carrier gas (Nippon Gases, Portugal) was set at 1.3 mL min-1 with a linear velocity of 32.7 cm s-1, and the nitrogen make-up gas (Nippon Gases, Portugal) at a rate of 30 mL min-1.

GC-MS analyses were performed using a Thermo Fisher Scientific instrument (Waltham, USA) coupled with an ion trap mass detector (Thermo Polaris). The analytes were separated on a Zebron-5ms capillary column (30 m × 250 μm i.d., 0.25 μm thickness, Phenomenex, Spain). The oven temperature was initially maintained at 40 °C for 1 minute, and then the temperature was increased to 290 °C as follows: 120 °C at a rate of 15 °C per minute, held for 1 minute; 150 °C (10 °C perminute), held for 1 minute; 180 °C (10 °C per minute), held for 1 minute; 200 °C (10 °C per minute), held for 1 minute; and 290 °C (10 °C per minute), held for 10 minutes. The helium carrier gas (purity ≥ 99.999%) was maintained at a constant flow rate of 1.0 mL/min. The temperatures of the inlet, ion source, and transfer line were maintained at 250 °C. The injection volume of 1 μL entered the GC-MS system in splitless mode. The mass spectrometer was operated with electron impact (eI) ionization (-70 eV) with a filament current of 200 μA in the SIM and tandem MS modes. The retention time, precursor, and fragment ions are listed in Table S1. At least three fragment ions were monitored for each compound. To maximize the detector signal, the most abundant and characteristic ion in the spectrum was selected for confirmation.

Table S1: SIM program used to confirm the OPPs, OPEs, PCBs, PBDEs, OCPs, and PYRs detected in the SS samples.

Compound	m/z	Retention time (min)	Dwell time (ms)
TiBP	99,155, 211, 265	13.16	99
Chlorpyrifos-methyl	109, 125, 197, 286	19.07	286
Malathion	93, 99, 125, 158, 173	20.01	173
α-endosulfan	195, 241, 265, 277	21.82	195
PCB118	145, 184, 219, 254, 256, 326	23.28	326
BDE 28	168, 223, 247, 406	23.42	406
TPhP	77, 169, 215, 233, 326	24.66	215
TEHP	57, 99, 113, 211, 323	24.91	99
BDE 99	404, 564, 566	27.78	404
Cypermethrin	91, 127, 152, 163	28.11	163
Deltamethrin	77, 93, 172, 209, 253	30.13	253
BDE 183	561, 564, 723	34.17	561

S.2 Results

S.2.1 Physical, Chemical, and Elemental Characterization of Sewage Sludge Samples

Table S2: Physicochemical properties, heavy metals and other trace elements content of SS samples (n=2) (mean ± SD) (ND = Not detected).

Parameter	Sewage Sludge Samples
A	B	C	D	E	F	G
pH	8.42	6.08	7.98	6.61	6.08	6.93	7.38
DM (%)	24.1	15.5	17.0	14.4	13.4	20.7	19.8
OM (% dw)	68.7	85.1	74.1	85.8	81.5	85.2	73.5
Primary nutrients
TOC (%)	36.8	40.5	32.8	27.3	16.8	44.3	35.2
Secondary nutrients
Ca (mg kg-1dw)	12,473	2,857	10,541	10,162	7,579	12,265	13,016
Mg (mg kg-1dw)	1,619	1,129	2,743-	2,927	1,799	2,242	2,779
Micro nutrients
Co (mg kg-1dw)	2.35	1.26	60.2	2.31	1.57	1.56	2.84
Mo (mg kg-1dw)	3.23	2.14	4.93	3.75	2.88	3.06	4.00
Mn (mg kg-1dw)	105	43.6	643	75.2	108	71.6	127
Fe (mg kg-1dw)	3,957	1,412	8,856	2,867	3,041	2,247	6,994
Heavy metals
Hg (mg kg-1dw)	0.060	0.050	0.110	0.170	0.190	0.050	0.210
Cd (mg kg-1dw)	0.730	0.290	7.89	0.790	0.660	0.810	1.10
Ni (mg kg-1dw)	59.5	16.5	110	29.7	14.2	11.0	20.1
Pb (mg kg-1dw)	27.8	8.51	38.3	22.8	14.9	20.6	32.9
Cr (mg kg-1dw)	125	12.3	32.2	27.7	13.9	12.7	25.2
Cu (mg kg-1dw)	172	115	246	181	140	179	269
Zn (mg kg-1dw)	906	238	2187	634	453	689	935
Other trace elements
Li (µg/g)	7.80	1.44	7.01	3.55	8.37	3.67	16.9
Be (mg kg-1dw)	0.450	0.190	2.62	0.180	0.550	0.330	1.34
V (mg kg-1dw)	6.71	1.52	9.62	5.56	4.42	5.32	8.69
As (mg kg-1dw)	5.07	2.22	13.3	6.50	8.24	5.59	8.62
Se (mg kg-1dw)	3.17	2.89	6.80	2.62	2.54	2.53	3.80
Rb (mg kg-1dw)	8.67	3.72	8.95	6.63	1.38	5.42	19.8
Sr (mg kg-1dw)	66.7	14.4	67.8	106.4	26.2	42.7	37.7
Rh (mg kg-1dw)	ND	ND	ND	ND	ND	ND	0.860
Sb (mg kg-1dw)	0.120	0.050	0.250	0.150	0.060	0.560	0.060
Cs (mg kg-1dw)	2.06	0.850	2.61	1.99	5.22	3.33	4.45
Ba (mg kg-1dw)	232	40.8	303	99.3	71.1	110	154
Ir (mg kg-1dw)	ND	ND	0.190	ND	ND	1.33	1.34
Tl (mg kg-1dw)	0.110	0.0500	0.310	0.0800	0.140	0.150	0.210

S.2.2 Levels of Organic Pollutants in Sewage Sludge Samples

Table S3: Concentrations (μg g-1 dw) of OPPs, OPEs, PBDEs, PCBs, OCPs, PYRs, and PAHS in sewage sludge collected from municipal wastewater treatment plants located in the north of Portugal (number of replicates = 2; ND: not detected; <LOQ: below method quantification limit).

Target analyte	SS samples
A	B	C	D	E	F	G
OPPs	Dimethoate	ND	ND	ND	ND	ND	ND	ND
Chlorpyrifos methyl	ND	ND	<LOQ	ND	ND	ND	ND
Parathion methyl	ND	ND	ND	ND	ND	ND	ND
Malathion	ND	ND	ND	<LOQ	ND	ND	ND
Chlorpyrifos	ND	ND	ND	ND	ND	ND	ND
Chlorfenvinphos	ND	ND	ND	ND	ND	ND	ND
∑6 OPP
OPEs	TiBP	ND	ND	ND	ND	ND	26.8±0.013	ND
TnBP	ND	ND	ND	ND	ND	ND	ND
TCEP	ND	ND	ND	ND	ND	ND	ND
TPhP	ND	ND	ND	<LOQ	2.05±0.012	ND	ND
TBEP	ND	1.06±0.19	ND	ND	ND	ND	ND
TEHP	ND	ND	ND	ND	ND	1.53±0.023	ND
.	TCP	ND	ND	ND	ND	ND	ND	ND
∑7 OPE	1.06±0.19	2.05±0.012	28.3±0.068
PCBs	PCB 28	ND	ND	ND	ND	ND	ND	ND
PCB 118	0.302±0.056	ND	ND	ND	ND	ND	ND
PCB 153	ND	ND	ND	ND	ND	ND	ND
PCB 180	ND	ND	ND	ND	ND	ND	ND
∑4 PCB	0.302±0.056
PBDEs	BDE 28	0.128±0.027	<LOQ	ND	ND	ND	ND	ND
BDE 47	ND	ND	ND	ND	ND	ND	ND
BDE 100	ND	ND	ND	ND	ND	ND	ND
BDE 99	ND	ND	0.054±0.012	ND	ND	<LOQ	ND
BDE 154	ND	ND	ND	ND	ND	ND	ND
BDE 153	ND	ND	ND	ND	ND	ND	ND
BDE 183	<LOQ	ND	ND	ND	ND	ND	ND
∑7 BDE	0.128±0.027	0.054±0.012
OCPs	o-HCH	ND	ND	ND	ND	ND	ND	ND
α-Endosulfan	0.110±0.007	0.170±0.005	0.490±0.084	0.571±0.037	0.366±0.010	0.282±0.019	0.286±0.022
p,p´-DDE	ND	ND	ND	ND	ND	ND	ND
p,p´-DDD	ND	ND	ND	ND	ND	ND	ND
Dieldrin	ND	ND	ND	ND	ND	ND	ND
∑5 OCP	0.110±0.007	0.170±0.005	0.490±0.084	0.571±0.037	0.366±0.010	0.282±0.019	0.286±0.022
PYRs	Bifenthrin	ND	ND	ND	ND	ND	ND	ND
λ-Cyhalothrin	ND	ND	ND	ND	ND	ND	ND
Cypermethrin	0.0870±0.012	ND	0.0573±0.011	ND	ND	<LOQ	ND
Fenvalerate	ND	ND	ND	ND	ND	ND	ND
Deltamethrin	0.254±0.0560	ND	ND	<LOQ	ND	ND	ND
∑5 PYR	0.341±0.068	0.0573±0.011
PAHs	Fluoranthene	ND	ND	0.435±0.0042	ND	ND	ND	0.401±0.0143
Benzo[b+j]fluoranthen	ND	ND	ND	ND	ND	ND	ND
Benzo[a]pyrene	ND	ND	0.0442±0.0002	ND	0.0418±0.00003	ND	ND
Dibenzo[a,l]pyrene	ND	ND	ND	ND	ND	ND	ND
Dibenz[a,h]anthracene	ND	ND	<LOQ	ND	ND	ND	<LOQ
Benzo[ghi]perylene	ND	ND	ND	ND	ND	ND	ND
Indeno[1,2,3‐cd]pyrene	ND	ND	ND	ND	ND	ND	ND
∑9 PAH	0.479±0.0044	0.0418±0.00003	0.401±0.0143
Total pollutant load per SS sample (μg g-1 dw)	0.881	1.26	2.25	0.571	2.46	28.6	0.687
Total POP load per SS sample (μg g-1 dw)	0.540	0.170	1.02	0.571	0.408	0.282	0.687
Number of pollutants detected per SS sample	6	3	7	4	3	5	2

Fig. 1S Chromatographic data for OPPs and OPEs obtained by GC-FPD. a) Chromatogram of a matrix-matched standard solution; b) chromatogram of a reagent-only standard solution; and c) chromatogram of sample F

Fig. 2S Chromatographic information of TPhP in sample E: a) chromatogram of sample E obtained by GC-FPD, b) chromatogram of sample E obtained by GC-MS, and c) mass spectrum of TPhP in sample E

Fig. 3S Chromatographic data for PCBs PBDEs, OCPs, and PYRs obtained using GC-ECD. a) Chromatogram of a matrix-matched standard solution, b) chromatogram of a reagent-only standard solution, and c) chromatogram of sample A

Fig. 4S Chromatographic data for PAHs obtained using LC-PDA-FLD. a) Chromatogram of a reagent-only standard solution, b) chromatogram of a matrix-matched standard solution, and c) chromatogram of sample C

References

APHA, 1992. SM 3050B, in: Andrew D. Eaton, Rodger B. Baird, E.W.R. (Ed.), Standard Methods for the Examination of Water and Wastewater.

CEN, 2002. EN 12457-4, in: Characterization of Waste -Leaching - Compliance Test for Leaching of Granular Waste Materias and Sludges.

J.B.Jones, 2001. Laboratory Guide for conducting soil tests and plant analysis, Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis. CRC Press LLC.

USEPA, 1986. EPA 9045D, in: Test Methods for Evaluating Solid Waste. Springfield.