Assessment of the combined inputs of antimicrobials from top soil improvers and irrigation waters on green leafy vegetable fields

Figures & data

Figure 1. Inventoried animal farms and slaughterhouses pressures on the water sampling points. Data kindly provided by Regione Emilia Romagna Veterinary Office.

Table 1. Applied extraction and clean-up protocols to irrigation water and top soil improvers samples.

		Top soil improvers
Step	Irrigation water^a	Protocol A^a^,b	Protocol B^c
Test portion	20 mL	0.5 g	1.5 g
Extraction – 1	10 µL Na2EDTA 0.1 M pH = 3	15 mL McIlvane buffer pH = 7 containing 0.1 M of Na2EDTA/MeOH 40/60 v/v	7 mL ACN/H2O 80/20 (v/v) containing HCOOH 0.5% and 390 mg di Na2EDTA x 2H2O
Extraction – 2		5 mL of Na2EDTA 0.1 M and 10 mL McIlvane buffer pH = 3 / MeOH 40/60 v/v	3 mL of ACN/H2O 80:20 v/v, containing 1 % of NH4OH
Extraction – 3		5 mL Na2EDTA 0.1 M and 10 mL of ammonium chloride/ammonia pH = 10 / MeOH 40/60 v/v
Extraction – 4		Evaporation of organic phase, dilution and pH adjustment to 3
SPE clean-up	OASIS HLB/Strata X-C	OASIS HLB/Strata X-C	NH2
SPE conditioning – 1	6 mL MeOH	6 mL MeOH	6 mL ACN
SPE conditioning – 2	6 mL CH3COOH 0.1%	6 mL CH3COOH 0.1%
Sample loading	Discard	Discard	Collect
Wash	6 mL of water	6 mL of water
Elution – 1	3 mL ACN/NH4OH 30% 70/30 v/v (Strata X-C)	3 mL ACN/NH4OH 30% 70/30 v/v (Strata X-C)	1.5 mL of ACN
Elution – 2	3 mL MeOH/NH4OH 30% 70/30 v/v (Strata X-C)	3 mL MeOH/NH4OH 30% 70/30 v/v (Strata X-C)
Elution – 3	6 mL MeOH	6 mL MeOH
Evaporation	40 °C under nitrogen stream	40 °C under nitrogen stream	40 °C under nitrogen stream
Final solvent	200 µL ammonium acetate 0.2M	1.5 mL of ammonium acetate 0.2M	1.5 mL ammonium acetate 0.2M / ACN 70/30 v/v
Centrifugation	12000 rpm for 5 min.	12000 rpm for 5 min.	12000 rpm for 5 min.

^aSargenti et al. (2020).

^bProtocol applied to ‘sludge’, ‘mixed compost’, ‘sludge with lime’, ‘bovine manure and bone meals’, ‘mixed animal manure and green compost’, ‘mixed compost and minerals’, ‘poultry litter’; ^cProtocol applied to ‘digestate’, ‘bovine manure’ and ‘pig slurry’

Table 2. Determined antimicrobials (ng g^-1 wet weight) in top soil improvers, according to the different origin.

		Sulfonamides								Tetracyclines			Fluoroquinolones
ID	Category	GUA	ANI	DIA	MER	MET	MMT	DMX	TRM	TET^a	OXY^b	DOX	NOR	CPR	FLU	ENR	MRB	TYL	LIN
1	Mixed compost and minerals	<LOQ	1832	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
4	Mixed compost and minerals	<LOQ	1710	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
2	Bovine manure and bone meals	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	27	57	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	111	<LOQ	<LOQ	<LOQ
3	Mixed animal manure and green compost	<LOQ	<LOQ	<LOQ	<LOQ	29	<LOQ	51	178	<LOQ	<LOQ	95	<LOQ	<LOQ	<LOQ	95	<LOQ	<LOQ	<LOQ
7	Bovine manure from organic farm	37	<LOQ	41	<LOQ	33	<LOQ	80	274	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	179	<LOQ	<LOQ	<LOQ
8	Pig slurry	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	190
10	Pig slurry	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	76	54	<LOQ	<LOQ	<LOQ	<LOQ	14	<LOQ	959
26	Bovine manure	<LOQ	<LOQ	36	14	14	20	<LOQ	<LOQ	<LOQ	58	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
30	Poultry litter	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	4487	<LOQ
9	Digestate	<LOQ	<LOQ	12	<LOQ	11	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
24	Digestate	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	13	<LOQ	<LOQ
16	Sludge from cWWTP	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	202	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
18	Sludge from cWWTP	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	30	<LOQ	63	<LOQ	353	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
20	Sludge from cWWTP	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	34	29	885	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
21	Sludge from cWWTP	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	41	36	632	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
22	Sludge with lime	<LOQ	<LOQ	58	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	81	<LOQ	470	<LOQ	406	27	<LOQ	<LOQ	<LOQ	<LOQ
23	Sludge with lime	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	572	58	<LOQ	<LOQ	<LOQ	<LOQ
27	Sludge with lime	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	307	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ

Abbreviations: GUA = Sulfaguanidine; ANI = Sulfanilamide; DIA = Sulfadiazine; MER = Sulfamerazine; MET = Sulfamethazine; MMT = sulfamomnometoxine; DMX = Sulfadimetoxine; TRM = Trimethoprim; TET = Tetracycline; OXY = Oxytetracycline; DOX = Doxicycline; NOR = Norfloxacin; CPR = Cipofloxacin; FLU = Flumequine; ENR = Enrofloxacin; MRB = Marbofloxacin; TYL = Tylosin A; LIN = Lincomycine; ^aSum of tetracycline and its epimer; ^bSum of oxytetracycline and its epimer. Samples with all the analytes < LOQ are not reported

Table 3. Determined antimicrobials (ng L⁻¹) in the 11 samples of irrigation water, according to the different origin.

	Sampling date	ID	GUA	DIA	PYR	ANI	MTX	TRM	CPR	SPR	DOX	TIA	FLU
Category	dd/mm/yy
Reused waters	23/05/2022	1297	22	<LOQ	26	<LOQ	136	50	12	30	61	11	10
River freshwater	23/05/2022	1298	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Irrigation channel	23/05/2022	1299	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Irrigation channel	23/05/2022	1301	14	<LOQ	13	<LOQ	77	18	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Reused waters	20/06/2022	1551	25	11	22	11	181	35	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Irrigation channel	20/06/2022	1552	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
River freshwater	20/06/2022	1553	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Irrigation channel	20/06/2022	1554	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Reused waters	30/08/2022	2211	28	<LOQ	17	<LOQ	221	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Irrigation channel	30/08/2022	2212	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Irrigation channel	30/08/2022	2213	16	<LOQ	<LOQ	<LOQ	15	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ

Abbreviations: GUA = Sulfaguanidine; DIA = Sulfadiazine; PYR = Sulfapyridine; ANI Sulfanilamide; MTX = Sulfametoxazole; TRM = Trimethoprim; CPR = Cipofloxacin; SPR = Spiramicyn; DOX = Doxicycline; TIA = Tiamuline; FLU = Flumequine

Figure 2. ‘Extracted ion chromatograms referred to the analysis of the fluoroquinolone ciprofloxacin in top soil improvers and irrigation waters’. From top to bottom: analytical standard, cWWTP biosolid sample (Table 2, sample No. 20), and irrigation water sample from river (Table 3, ID #1298).

Table 4. Computed inputs (mg) and expected concentration (ng g⁻¹) of antimicrobials from selected biosolids and irrigation waters in soils intended for lettuce production, according to the considered stancard agriculture practices.

			Sulfonamides								Tetracyclines		Macrolides			Fluoroquinolones
Inputs			GUA	ANI	DIA	MET	DMX	MTX	PYR	TRP	DOX	OXY*	TYL	SPR	LYN	ENR	NOR	CPR	MRB	TIA
TSI	ID	tons
manure	7	0.30	11.1	NC	12.3	9.9	24	NC	NC	82.2	NC	NC	NC	NC	NC	53.7	NC	NC	NC	NC
Mixed Compost	1	0.15	NC	275	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC
Sludge compost	15	0.15	NC	NC	NC	NC	NC	NC	NC	NC	2.04	NC	NC	NC	NC	NC	1.74	53.1	NC	NC
Poultry Litter	30	0.02	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	89.7	NC	NC	NC	NC	NC	NC	NC
Pig Slurry	10	0.02	NC	NC	NC	NC	NC	NC	NC	NC	1.08	1.52	NC	NC	19.2	NC	NC	NC	0.28	NC
Waters		tons
Reused (N = 3)		350	8.75	1.30	1.30	NC	NC	62.7	7.70	9.80	7.00	NC	NC	3.50	NC	NC	NC	1.40	NC	1.30
Rivers (N = 2)		350	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC
Channels (N = 6)		350	1.75	NC	0.63	NC	NC	5.25	0.77	1.05	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC
Worst case
Top soil + water		mg	12.9	276	12.3	9.90	24.0	62.7	7.70	82.2	2.04	1.52	89.6	3.50	19.8	53.7	1.74	53.1	0.28	1.30
Dilution factor	1542	tons
expected concentration		ng g^−1	0.008	0.179	0.008	0.006	0.016	0.042	0.005	0.053	0.001	0.001	0.058	0.002	0.012	0.035	0.001	0.034	0.001	0.001

Sludge compost values computed as 40% of the recorded contamination in the biosolid from cWWTP.

Abbreviations: GUA = Sulfaguanidine; ANI = Sulfanilamide; DIA = Sulfadiazine; MET = Sulfamethazine; DMX = Sulfadimetoxine; MTX = Sulfametoxazole; PYR = Sulfapyridine; TRP = Trimethoprim; DOX = Doxicycline; OXY = Oxytetracycline; TYL = Tylosin A; LIN = Lincomycine; ENR = Enrofloxacin; NOR = Norfloxacin; CPR = Ciprofloxacin; MRB = Marbofloxacin; SPR = Spiramycin; TIA = Tiamulin; * and its epimer. NC = Not Computable.

Table 5. Hazard Index for antimicrobial resistance (HI_AMR), accounting for worst case (wc) concentration (ppb) found in top soil improvers (TSI), irrigation water (IW) and for modelled inputs in soil.

	PNEC AMR	TSI wc	IW wc	Soil wc	HI AMR	HI AMR	HI AMR
Antimicrobials	(ng g^−1; ng mL^−1)				TSI	IW	Soil
Ciprofloxacin	0.064	885	0.012	0.034	13828	0.19	0.53
Enrofloxacin	0.064	179	nc	0.035	2797	nc	0.55
Ciprofloxacin + Enrofloxacin	0.064	nc	nc	0.069	nc	nc	1.08
Norofloxacin	0.500	470	nc	0.001	940	nc	<0.01
Doxicycline	2.000	470	0.061	0.001	940	0.03	<0.01
Oxytetracycline	0.500	76	nc	0.001	235	nc	<0.01
Tetracycline	1.000	81	nc	0.010	81	nc	0.01
Sulfamethoxazole	16.00	nc	0.221	0.042	nc	0.01	<0.01
Trimethoprim	0.500	274	0.050	0.053	548	0.1	0.11
Sulfamethoxazole + trimethoprim	0.500	nc	0.216	0.095	nc	0.43	0.19
Tylosin A	4.000	4487	nc	0.058	1122	nc	0.01
Lincomycin	2.000	959	nc	0.012	480	nc	<0.01

Predicted no effect concentration for antimicrobial resistance (PNEC_AMR) derived from Bengtsson-Palme and Larsson (2016). nc = not computable.

Bengtsson-Palme J, Larsson DJ. 2016. Concentrations of antibiotics predicted to select for resistant bacteria: proposed limits for environmental regulation. Environ Int. 86:140–149. doi:10.1016/j.envint.2015.10.015.

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