Occurrence and Risk Assessment of Veterinary Antimicrobials in Commercial Organic Fertilizers on Chinese Markets

Abstract

Commercial organic fertilizers (COFs), as an alternative to chemical fertilizers, have been widely promoted and applied to agricultural soils to improve soil fertility and develop green agriculture. However, the residues of veterinary antimicrobials in COFs could be transferred to agricultural soils and pose ecological risk that should not be ignored. This study quantified the occurrence of fifty-seven veterinary antimicrobials, covering five classes (i.e., twenty-three sulfonamides, nineteen quinolones, seven macrolides, six tetracyclines, and two lincosamides) in ninety-three COFs collected from five provinces in China. Twenty-two veterinary antimicrobials, including eleven quinolones, six sulfonamides, four macrolides, and one lincosamide, were detected in the COFs with total contents up to 3870 ng/g. The contents of individual antimicrobials ranged from 0.66 to 3310 ng/g, and their detection frequencies were between 2% and 49%. The composition and contents of antimicrobials in the COFs varied significantly, depending on their raw materials, production processes, and source regions. Seven antimicrobials, including ciprofloxacin, enrofloxacin, and tilmicosin, could pose low to medium potential ecological risk to soil organisms in the amended soils. The wide occurrence of antimicrobials in COFs and their potential ecological risk indicate the urgent need to establish regulatory limits of antimicrobial residues in COFs to control and prevent antimicrobial pollution in agricultural soils brought by their amendment.

1. Introduction

With the intensification of livestock farming, antimicrobials have been widely used for disease treatment and prevention, as well as growth promotion on animals. The total consumption of veterinary antimicrobials used in livestock farms was approximately 84,240 tons in China in 2013 [1]. The majority of these antimicrobials are not degraded in animal bodies and are mostly excreted with urine and feces [2]. When livestock manure containing antimicrobial residues is composted and processed into commercial organic fertilizers (COFs), the antimicrobials are not fully degraded and may even become concentrated due to the partial breakdown of organic matter [3]. Consequently, COFs frequently contain elevated levels of antimicrobials.

Studies have reported high levels of quinolones, sulfonamides, and tetracyclines in COFs, with contents and composition varying significantly with the raw materials and source regions. Analysis of 143 livestock manure samples covering chicken manure, cow manure, and pig manure in China revealed that the residue levels of most antimicrobials (e.g., sulfonamides and quinolones) were significantly different among different provinces and animal species [4]. For example, the levels of enrofloxacin detected in chicken manure (up to 1420 mg/kg) were two orders of magnitude higher than those detected in pig manure (up to 33.3 mg/kg) and cow manure (up to 46.7 mg/kg) [4]. The composition and contents of antimicrobials also differed greatly among the COFs collected in China, Malaysia, and Austria [5,6]. Despite the fact that COFs often contain high levels of antimicrobials, regulations on their contents in COFs are lacking worldwide. Although some countries have begun to develop relevant standards and regulations regarding antimicrobial residues, most of them focus on meat and dairy products [7].

The Chinese government has implemented a series of policies to encourage the use of organic fertilizers instead of chemical fertilizers to improve soil fertility and promote sustainable agricultural practices. As a result, the production and application of COFs to agricultural soils have reached 18.3 and 9.47 million tons in China, respectively [8]. Despite the benefits on soil quality and crop yields, the application of COFs could input substantial amounts of antimicrobials into farmland soils. The antimicrobials transferred into agricultural soils and their subsequent uptake by crops pose potential risk to ecosystems and human health.

While antimicrobial pollution has become a major environmental and agricultural concern in China, the residues of antimicrobials in COFs have not received significant attention yet. This study investigated the levels of antimicrobial residues in 93 COFs collected from five provinces in China. The composition and contents of antimicrobials in the COFs made from various raw materials and by different processes were compared, and their potential ecological risk on soil organisms was assessed. These findings demonstrate the urgent need to improve antimicrobial degradation in composting and reduce veterinary antimicrobial use in animal husbandry to significantly lower the contents of antimicrobial residues in COFs.

2. Materials and Methods

2.1. Target Analytes

A total of fifty-seven veterinary antimicrobials, covering five classes (twenty-three sulfonamides, nineteen quinolones, seven macrolides, six tetracyclines, and two lincosamides), were selected as the target analytes due to their widely veterinary use and high detection frequencies and contents in livestock manure worldwide. Mixed standards of the target analytes were purchased from Alta Scientific (Tianjin, China). Further details on these target analytes are shown in

Table A1. Instrumental details for the analysis of the target antimicrobials in this study.

Analyte	Precursor Ion (m/z)	Quantitative Ion (m/z)	Qualitive Ion (m/z)	Retention Time (min)	Collision Energy (V)	Entrance Voltage (V)	Lens Voltage (V)	MDL (ng/L)	MQL (ng/L)
Chlortetracycline	479	444	462	9.18	−28/−20	30/30	−122/−92	24	80
Cinoxacin	263	217	245	9.11	−29/−21	16/19	−52/−48	13	43
Ciprofloxacin	332	314	288	8.45	−31/−24	1/0	−68/−56	14	47
Clindamycin	425	126.3	377	9.54	−40/−25	25/36	−76/−84	13	45
Danofloxacin	358	340	82	8.55	−33/−58	0/4	−76/−80	8	26
Demeclocycline	465	448	430	8.88	−23/−27	18/22	−100/−116	75	249
Difluoxacin	400	382	356	8.71	−32/−27	17/33	−76/−68	11	36
Doxycycline	445	428	154	9.47	−24/−40	30/30	−80/−82	34	112
Enoxacin	321	303	234	8.12	−27/−29	22/0	−60/−64	8	28
Enrofloxacin	360	316	342	8.52	−27/−31	22/35	−60/−64	9	31
Erythromycin	734	158	576	9.8	−38/−24	29/30	−128/−168	58	193
Fleroxacin	370	326	269	7.81	−25/−36	28/13	−72/−92	21	71
Flumequine	262	244	202	9.71	−26/−44	10/6	−44/−72	3	10
Isochlortetracycline	479	461.9	444	8.71	−27/−29	16/6	−92/−124	40	134
Josamycin	828	174.2	109.4	9.92	−45/−58	36/47	−144/−124	22	73
Leucomycin	702	174.2	558.2	9.14	−40/−32	40/36	−112/−128	55	184
Lincomycin	407	126	359	7.81	−38/−24	25/30	−80/−84	1	3
Lomefloxacin	352	265	308	8.64	−32/−23	28/18	−60/−56	17	55
Marbofloxacin	363	72	319	7.67	−31/−24	31/24	−64/−76	15	50
Nalidixic acid	233	215	187	9.63	−19/−34	6/6	−48/−68	6	20
Norfloxacin	320	302	276	8.26	−31/−24	2/2	−60/−52	14	46
Ofloxacin	362	318	261	8.1	−27/−37	28/35	−68/−72	13	43
Oleandomycin	688	158.3	544.4	9.59	−38/−23	16/16	−116/−96	28	94
Orbifloxacin	396	352	295	8.69	−23/−30	0/16	−76/−96	6	22
Oxolinic acid	461	426	443	8.52	−24/−17	20/20	−76/−72	4	15
Oxytetracycline	262	244	202	9.26	−24/−42	13/7	−64/−108	23	76
Pefloxacin	334	316	290	8.12	−29/−24	0/0	−64/−56	9	31
Pipemidic acid	304	217	189	7.39	−30/−44	4/21	−72/−68	19	64
Sarafloxacin	386	299	368	8.81	−39/−33	16/19	−88/−72	15	51
Sparfloxacin	393	292	349	8.97	−30/−27	8/15	−88/−80	18	59
Spiramycin	844	174	109	9.82	−45/−56	42/52	−168/−144	32	107
Sulfabenzamide	277	156	92	8.9	−16/−47	14/6	−40/−52	4	12
Sulfacetamide	215	156.1	108	1.63	−15/−30	9/7	−36/−44	8	26
Sulfachlorpyridazine	284.9	156	108.1	8.29	−20/−37	18/15	−56/−56	11	36
Sulfadiazine	250.9	156.1	108.2	4.76	−21/−36	15/16	−52/−56	15	50
Sulfadimethoxine	311	156	108.1	8.71	−26/−38	14/19	−60/−68	7	23
Sulfadoxine	311	156	108.1	9.16	−27/−38	14/15	−60/−64	12	40
Sulfaguanidine	215	156	92.1	3.5	−18/−34	13/10	−36–32	17	56
Sulfisomidine	279	124	185.9	7.79	−26/−22	12/15	−48/−56	6	18
Sulfamerazine	264.9	155.9	172.2	6.6	−23/−22	14/24	−52/−52	6	19
Sulfameter	280.9	156	108	8.55	−23/−38	20/19	−64/−64	23	76
Sulfamethazine	279	186	156	5.23	−24/−25	24/22	−52/−60	8	26
Sulfamethiazole	271	156.1	108.2	7.62	−19/−34	19/19	−52/−56	21	71
Sulfamethoxazole	254	155.9	108.1	8.47	−21/−35	18/20	−48/−48	26	87
Sulfamethoxypyridazine	281	155.9	92	7.43	−22/−42	18/17	−56/−52	15	50
Sulfamonomethoxine	280.9	156	108	8	−23/−38	24/24	−56/−64	14	48
Sulfamoxole	268	92	155.9	8.73	−46/−20	25/14	−56/−44	7	22
Sulfanitran	336	134.2	197.8	9.54	−38/−29	22/26	−80/−48	7	25
Sulfaphenazole	315	156	222	9.02	−28/−26	29/31	−72/−68	6	21
Sulfapyridine	249.9	156	184.1	6.13	−23/−23	23/26	−52/−48	10	34
Sulfaquinoxaline	301	156	92	9.26	−25/−56	28/28	−48/−68	6	22
Sulfathiazole	255.9	156.1	108	5.63	−20/−34	22/24	−52/−48	20	67
Sulfisoxazole	268	156.1	107.9	7.6	−19/−35	17/21	−48/−56	7	23
Tetracycline	445	410	427	8.38	−25/−17	20/27	−88/−66	21	71
Tilmicosin	869	174	132	9.35	−60/−67	30/51	−280/−196	3	11
Trimethoprim	291	230	261	7.84	−31/−33	34/33	−52/−56	13	42
Tylosin	917	773.2	174.2	9.68	−40/−51	44/39	−164/−172	11	35

.

2.2. Sample Collection and Pretreatment

A total of 93 COFs were collected from twelve cities in Hebei Province, Shandong Province, Hubei Province, Jiangsu Province, and the Inner Mongolia Autonomous Region. While 47 out of 93 COFs were primarily made from poultry manure, pig manure, cattle manure, or sheep manure, the other samples contained mixed feedstocks and bio-organic ingredients, with detailed information on the raw materials unknown (

Table A2. Description of the 93 COF samples collected in this study.

Raw Material	Sample Number	Main Treatment and Processes
Poultry manure	20	Mix, ferment, crush
Sheep manure	17	Mix, ferment, crush
Cattle manure	8	Mix, ferment, crush
Pig manure	2	Mix, ferment, crush
Bio-organic materials (mushroom residues, bean dreg, Chinese herb residues, etc.)	14	Ferment, mix, crush, pellet
Mixed feedstocks	32	Ferment, mix, crush, pellet
Total	93	Mix, ferment, crush, pellet

).

Pretreatment and extraction of the target antimicrobials were conducted following the procedures developed in our previous work [9]. Briefly, 0.5 g of freeze-dried and sieved COF samples were spiked with the surrogates then added with 30 mL of EDTA–McIlvaine buffer/acetonitrile/methanol/acetone (5:2:2:1 by vol.), vortexed for 30 s, and then ultrasonicated for 10 min. After centrifugation, the supernatants were diluted to 250 mL with ultrapure water. The solid phase extraction process involved the sequential use of a strong anion exchange cartridge (SAX, 500 mg, 6 mL) and a hydrophile–lipophile balance cartridge (HLB, 500 mg, 6 mL; CNW, Anpel Laboratory Technologies, Shanghai, China). The final reconstitution solutions were filtered with 0.22 μm polytetrafluorethylene (PTFE) syringe filters and then transferred into sample vials. Twenty nanograms of enrofloxacin-d5 was added into each sample as an internal standard. All samples were stored at −20 °C until analysis, typically within a week.

2.3. Instrumental Analysis and Quality Assurance and Quality Control (QA/QC)

The target antimicrobials were analyzed on a Qsight LX50 ultra-high pressure liquid chromatograph coupled with a QSight 210 triple quadrupole mass spectrometer (UPLC-MS/MS, PerkinElmer, Waltham, MA, USA) using the analytical method developed in our previous work [9]. Compound separation was performed on an XBridge BEH C18 column (2.1 × 150 mm; 2.7 μm) at 40 °C. The analytes were separated by gradient elution using ultrapure water with 0.1% formic acid (mobile phase A) and LC-MS grade acetonitrile (mobile phase B). The flow rate was 0.35 mL/min, and the sample injection volume was 5 μL. The electrospray ionization (ESI) source was operated under positive mode, and the source temperature was set at 300 °C. The temperature of the desolvation gas was set at 250 °C. The capillary voltage was at 5500 V. The limits of detection (LODs) for individual antimicrobials ranged from 1 to 75 ng/L during the instrumental analysis, while their method detection limits (MDLs) in the COF samples ranged from 0.002 to 0.206 ng/g, which is sufficient for quantitative analysis. Further details on compound-specific operating parameters and instrumental detection limits are also shown in Table A1.

Laboratory blanks were routinely analyzed, and no antimicrobial was detected. The recoveries were evaluated by adding 50 μL of 1 mg/L mixture of deuterated chemicals (i.e., ciprofloxacin-d8, erythromycin-d3, sulfadiazine-d4, sulfamethoxazole-d4, and tetracycline-d6), and the matrix effect was quantified by spiking 20 μL of 1 mg/L enrofloxacin-d5 as an internal standard into each sample prior to instrumental analysis. The recoveries were generally >70%, whereas the matrix factors (i.e., the ratios of the analyte peaks in the presence and absence of matrix component) were between 1.05 and 1.09. The effectiveness of pretreatment procedures and the accuracy and robustness of the instrumental analysis method have been comprehensively validated in our previous work [9]. Figure 1 shows selected ion monitoring chromatograms of ofloxacin and sulfamethoxazole in a mixed standard (20 µg/L) and the extract of a representative COF sample. The excellent consistency in peak shape and retention time, as well as the low noises, demonstrate the effectiveness of sample pretreatment and the sensitivity of instrumental detection.

2.4. Risk Assessment

For risk assessment of these antimicrobials in the COFs on agricultural soils, the contents of antimicrobials transferred from COF to soil were estimated as follows:

$$C_{soil}={\displaystyle \frac{C_{COF}\times M}{A_s\times H_s\times {\rho }_s}}$$

(1)

where C_soil is the estimated content of individual antimicrobials transferred from COF to soil, C_COF is the measured content of individual antimicrobials in the COF sample, M is the annual applied amount of COFs (6000 kg), A_s is the field area where manure was applied (0.067 ha), H_s is the depth of the ploughed soil layer (20 cm), and ρ_s is the soil density (1500 kg/m³) [10]. It is worth mentioning that the annual applied amount of COFs was estimated based on the inquiry from the local farmers, which is also at a comparable level with that reported in the literature [10]. Other processes that may occur during the migration of antimicrobials from COF to soil, such as runoff, plant uptake, and degradation, were not considered for a conservative assessment. The potential ecological risk of the individual antimicrobial on soil was evaluated by calculating the risk quotient (RQ):

$$RQ={\displaystyle \frac{C_{soil}}{PNE{C}_{soil}}}$$

(2)

where PNEC_soil is the predicted no-effect concentration of individual antimicrobial to the sensitive species (ng/g). The PNEC_soil values of the target antimicrobials were calculated as follows:

$$PNE{C}_{soil}={\displaystyle \frac{TOX}{AF}}$$

(3)

where TOX is the toxicity data collected from the ECOTOX database, including median lethal concentration (LC₅₀), median effect concentration (EC₅₀), lowest observed effect concentration/lowest observed effect level (LOEC/LOEL), and no observed effect concentration/no observed effect level (NOEC/NOEL) for soil organisms. The assessment factor (AF) for LC₅₀, EC₅₀, LOEC/LOEL, and NOEC/NOEL were 1000, 1000, 10, and 10, respectively. The lowest value was adopted for a conservative assessment when multiple toxicity data were available for the same species. The lowest toxicity data within the same class were used when the toxicity data for a given analyte was not available.

Mixture toxicity models, such as concentration addition (CA) and independent action (IA), have been used to predict the toxicity of chemical mixtures. The CA model is commonly applied to chemicals that share the same mode of action, while the IA model is suitable for chemicals with different modes of action. For this study, the co-occurrence of different antimicrobials often exhibits synergistic or additive effects. We chose to evaluate the ecological risk on an individual basis to avoid bias and obtain a robust assessment. The ecological risk of individual antimicrobials was evaluated based on the commonly used ranking criteria: RQ ≥ 1, high risk; 0.1 ≤ RQ < 1, medium risk; 0.01 ≤ RQ < 0.1, low risk; RQ < 0.01, no risk.

3. Results and Discussion

3.1. Levels of Antimicrobials in the COFs

Among the ninety-three COF samples analyzed, a total of twenty-two veterinary antimicrobials were detected, including eleven quinolones, six sulfonamides, four macrolides, and one lincosamide (Figure 2 and

Table A3. Summary of the contents of antimicrobial residues in the 93 COF samples.

Antimicrobial	Maximum (ng/g)	Median (ng/g)	Minimum (ng/g)	Mean (ng/g)	Standard Deviation (ng/g)	Detection Frequency (%)
Quinolones
Ciprofloxacin	621	32.0	4.84	75.7	144	17
Enrofloxacin	417	47.9	3.60	106	119	20
Fleroxacin	14.4	9.32	5.07	9.58	3.32	5
Flumequine	32.5	7.65	4.25	13.0	11.4	4
Lomefloxacin	119	24.1	9.18	45.2	39.4	11
Marbofloxacin	15.1	6.37	2.24	6.55	4.13	8
Nalidixic acid	50.0	10.1	1.45	13.1	10.3	30
Norfloxacin	83.9	68.0	38.1	66.9	14.4	8
Ofloxacin	146	23.6	2.71	37.4	36.2	49
Oxolinic acid	178	79.0	43.1	100	57.2	3
Sparfloxacin	13.2	6.04	3.81	7.27	3.54	4
Sulfonamides
Sulfadiazine	24.5	6.27	0.99	8.58	8.39	5
Sulfamethoxazole	21.8	11.4	0.95	11.4	10.4	2
Sulfaphenazole	24.5	4.23	1.94	6.61	6.06	14
Sulfapyridine	128	1.98	0.76	33.1	54.6	4
Sulfaquinoxaline	17.3	2.23	0.66	3.95	4.04	17
Sulfamoxole	76.2	12.8	2.14	22.4	25.9	6
Macrolides
Erythromycin	163	6.59	3.54	19.9	41.5	14
Leucomycin	260	58.7	14.1	87.9	79.7	20
Tilmicosin	3310	42.2	11.8	458	1080	9
Tylosin	485	55.9	37.3	193	207	3
Lincosamides
Lincomycin	5.47	3.12	0.67	3.09	1.96	3

). The total contents of antimicrobials ranged between 0.95 and 3870 ng/g, with a median content of 53.8 ng/g and a detection frequency of 86% (Figure 2 and Table A3). No tetracyclines were detected in the COFs. Previous studies have also reported the absence of tetracyclines in livestock manure, likely due to their relatively fast degradation in the processing of livestock manure [6,11,12]. It was reported that chlortetracycline in cattle manure was transformed into several degradation products, including iso-chlortetracycline, 4-epi-chlortetracycline, and anhydro-chlortetracycline, during composting [11]. In addition, no antimicrobials were detected in thirteen COFs, which were mostly made from bio-organic materials, such as mushroom residues and Chinese herbal residues.

The contents of individual antimicrobials in the COF samples ranged from 0.66 to 3310 ng/g (Figure 2 and Table A3). Ofloxacin was the most abundant quinolone, with a detection frequency of 49% and a median content of 23.6 ng/g, while ciprofloxacin was detected at content up to 621 ng/g. Ciprofloxacin is both a commonly used antimicrobial and a main metabolite from enrofloxacin, which is one of the most widely consumed veterinary antimicrobials in China [1]. The overall detection frequencies of sulfonamides, macrolides, and lincosamides were generally low (i.e., ≤20%). Sulfaquinoxaline was the most frequently detected sulfonamide, with a detection frequency of 17% and a median content of 2.23 ng/g. Sulfaquinoxaline is a veterinary antimicrobial that is widely administered on cattle and sheep to treat coccidiosis, and it has been detected in livestock manure at levels up to 6100 ng/g [13]. Among the macrolides, leucomycin was the most abundant compound (detection frequency: 20%), whereas tilmicosin had the highest maximum content (3310 ng/g). Lincomycin was the only lincosamide detected at a rather low detection frequency (3%) and low contents (median: 3.12 ng/g).

Veterinary antimicrobials, such as ciprofloxacin, fleroxacin, sulfamethoxazole, and tylosin, were among the most commonly monitored antimicrobials in livestock manure and organic fertilizers (

Table 1. Comparison of levels of antimicrobial residues in the COF samples of this study with those reported for animal manure and COF in the literature (unit: ng/g).

	Ciprofloxacin	Enrofloxacin	Fleroxacin	Marbofloxacin	Norfloxacin	Ofloxacin	Sulfadiazine	Sulfamethoxazole	Lincomycin	Tilmicosin	Tylosin	Reference
COFs (n = 93)	75.7 ± 144	106 ± 119	9.58 ± 3.32	6.55 ± 4.13	66.9 ± 14.4	37.4 ± 36.2	8.58 ± 8.39	11.4 ± 10.4	3.09 ± 1.96	458 ± 1080	193 ± 207	This study
Manure (n = 20)	300–3000	NA	NA	NA	NA	NA	2300–5200	4500–18,700	NA	NA	NA	[14]
Pig manure (n = 61)	2010 ± 8350	2090 ± 8050	2230 ± 1440	NA	2090 ± 1450	NA	210 ± 400	510 ± 310	NA	NA	NA	[4]
Chicken manure (n = 54)	3780 ± 11,800	4650 ± 366,000	3110 ± 24,200	NA	4680 ± 54,810	NA	150 ± 900	780 ± 1470	NA	NA	NA	[4]
Cow manure (n = 28)	3440 ± 10,700	6790 ± 11,400	2220	NA	1840 ± 1080	NA	ND	ND	NA	NA	NA	[4]
Manure (n = 10)	NA	112–26,900	NA	NA	30.7–1890	NA	<MQL−5770	NA	NA	<MQL−85.2	99.5–13,700	[15]
Manure (n = 17)	1310	1250	NA	NA	692	NA	3.9	7.5	NA	NA	NA	[16]
Manure (n = 32)	808 ± 2310	NA	NA	NA	NA	NA	NA	18.9 ± 20.1	NA	NA	ND−37.1	[17]
Compost (n = 20)	86.5	309	NA	NA	31.9	42.5	3.3	19.7	NA	NA	NA	[21]
Cow manure	28.1	27.3	NA	NA	13.8	NA	NA	ND	NA	NA	ND	[18]
Pig manure	1.0	16.2	NA	NA	1.8	NA	NA	0.3	NA	NA	ND	[18]
Chicken manure	3.4	8.2	NA	NA	2.2	NA	NA	2.4	NA	NA	0.2	[18]
Pig manure (n = 34)	1.52	0.534	NA	NA	NA	NA	NA	<MDL	NA	NA	NA	[19]
Cattle manure (n = 21)	<MQL−70.4	ND−65.1	ND−1.2	ND−12.1	<MQL−74.6	<MQL−71.2	NA	NA	NA	NA	NA	[20]
Pig manure	40–70	50–70	ND-60	NA	ND−60	40	ND	ND	ND	310–7890	NA	[22]
Cow manure	NA	NA	NA	NA	NA	NA	ND	ND	NA	NA	NA	[13]
Chicken manure	NA	NA	NA	NA	NA	NA	8.3–18.8	ND	NA	NA	NA	[13]
Pig manure	NA	NA	NA	NA	NA	NA	7.1–37.1	ND	NA	NA	NA	[13]
Duck manure	NA	NA	NA	NA	NA	NA	ND	ND	NA	NA	NA	[13]
COF (n = 6)	NA	NA	NA	NA	NA	NA	NA	608	NA	NA	NA	[23]
Cow manure (n = 23)	5.2	3	NA	NA	0.12	NA	4.34	4.63	NA	NA	28.64	[24]
Cattle manure (n = 5)	3380	22,100	NA	NA	266	NA	4030	ND	NA	NA	ND	[24]
Pig manure (n = 23)	363	105	NA	NA	8.02	NA	21.7	0.5	NA	NA	166	[24]

Notes: NA—not available; ND—not detected.

). The levels of ciprofloxacin, enrofloxacin, fleroxacin, norfloxacin, sulfadiazine, and sulfamethoxazole quantified in this study were generally 1–2 orders of magnitude lower compared to those measured in the livestock manure (Table 1) [4,14,15,16,17]. However, compared to more recent studies [13,18,19,20,21,22], the levels of antimicrobials in this study were comparable or higher than those reported in the literature (Table 1). For example, ciprofloxacin and enrofloxacin at levels up to 70.4 and 65.1 ng/g were reported in cattle manure [20], whereas their mean contents in the COF samples of this study were 75.7 and 106 ng/g, respectively. Qian and co-workers reported high levels of sulfamethoxazole (mean: 608 ng/g) in the organic fertilizers [23], but generally low levels of sulfonamides were often reported in pig manure, cow manure, and duck manure (Table 1) [13,22,24]. Although several studies have investigated the occurrence of a few common antimicrobials in livestock manure and organic fertilizers, other antimicrobials have yet to receive adequate attention.

3.2. Antimicrobial Residues in COFs Made from Different Raw Materials

The antimicrobials in COFs originate from the various types of raw materials used, such as cattle manure, sheep manure, poultry manure, pig manure, and other bio-organic materials. Overall, the COFs made from bio-organic materials, such as mushroom residues and bean dregs, exhibited the lowest levels of antimicrobial residues, whereas the antimicrobial contents in the COFs made from livestock manure generally decreased in the order of cattle manure > sheep manure > poultry manure > pig manure (Figure 3a). Quinolones were the dominant class of antimicrobials in the COFs made from sheep manure, poultry manure, cattle manure, and other bio-organic materials, whereas only macrolides were detected in the COFs processed from pig manure (Figure 3b).

Besides animal manure, a range of organic wastes, such as mushroom residues, furfural residues, and Chinese herbal residues, are also utilized to produce COFs. These materials often contain low levels of antimicrobial residues (or no antimicrobial residues) compared to livestock manure. No antimicrobials were detected in the COF samples made from mushroom residues, furfural residues, and Chinese herbal residues in this study. Therefore, the choice of raw materials for COFs significantly impacts the composition and contents of antimicrobial residues. Using alternative organic materials, such as agricultural waste or biogas residues, can reduce the levels of antimicrobial residues in the COFs and the associated ecological risk.

3.3. Antimicrobial Residues in COFs Made by Different Processes

Minerals (e.g., lime and clay) and salts (e.g., ferrous sulfate and calcium/magnesium superphosphate) are often added in the composting of manure or in the granulation of COFs. As a result, the contents in COFs in different shapes, which are made by different processes, could vary significantly. The total contents of antimicrobials in the powdered COFs (mean: 114 ng/g) were higher than those in the granular COFs (mean: 51.4 ng/g; Figure 4a). The composition also varied between the powdered and granular COFs. Macrolides dominated in powdered COFs, whereas quinolones were the dominant class in granular COFs (Figure 4b). Powdered COFs, mainly derived from livestock manure or other bio-organic materials, generally contained higher levels of antimicrobials compared to granular COFs. This is likely due to the fact that powdered COFs are less processed and retain higher levels of antimicrobials from the raw materials, whereas granular COFs often undergo more controlled production processes and contain additives, such as clay and turfy soil, which dilute the levels of antimicrobials in the final product. Composting, a commonly used method for producing granular COFs, can reduce the residues of antimicrobials, such as tetracyclines and sulfonamides, by over 60% during the process [25]. Nevertheless, the effectiveness of composting depends on many factors, such as temperature, duration, and microbial activity. Interestingly, the pellet COFs exhibited even higher levels of antimicrobials compared to the powdered and granular COFs. This is attributed to the fact that their main raw materials were livestock manure (e.g., sheep manure), which often contains high levels of antimicrobial residues.

The shape of COFs may impact the release rates and transfer of antimicrobials in agricultural soils. It is expected that antimicrobials and nutrients would be released from the granular COFs more slowly compared to the powdered COFs, which is beneficial for long-term soil conditioning and sustained plant growth. While the production processes may result in COF products with different levels of antimicrobial residues, it is important to reduce and even eliminate their contents in the raw materials used in COF production.

3.4. Antimicrobial Residues in COFs Produced from Different Regions

Antimicrobial residues in COFs vary widely across source regions, with hotspots in the areas characterized by intensive agricultural activities and high livestock density. A comprehensive study analyzed the antimicrobial resistance genes (ARGs) in agricultural soils from seventeen countries and reported that Central North America, Eastern Europe, Western Asia, and Northeast China were the hotspots for antimicrobial resistance burden because of dense population and intensive agricultural activities, such as crop farming, livestock husbandry, and manure application [26]. Regions with intensive livestock farming and frequent use of antimicrobials in livestock husbandry tend to have higher levels of antimicrobial residues in COFs. Seventeen antimicrobials were detected in the manure-based COFs collected in East China, with total contents exceeding 15 mg/kg, which highlights the substantial enrichment of antimicrobial residues in the COFs produced in this region [27].

The COF samples in this study were collected from twelve cities in Hebei Province, Shandong Province, Jiangsu Province, Hubei Province, and the Inner Mongolia Autonomous Region, which are among the major agricultural provinces in China. For example, Shandong Province has a total agricultural output, including crop farming, animal husbandry, fishery, and forestry, of CNY 1.28 trillion in 2024, ranking first in the country [28]. COFs from Nantong, Jiangsu Province, exhibited the highest levels of antimicrobial residues (mean: 451 ng/g), followed by those from Tianjin (mean: 302 ng/g), Cangzhou (mean: 174 ng/g), Ji’ning (mean: 163 ng/g), and Shijiazhuang (mean: 159 ng/g; Figure 5a). Nantong is a major livestock and poultry breeding region and ranks fourth in the total number of livestock and poultry farmed in Jiangsu Province. The contents of antimicrobial residues in the COFs collected from Ji’ning and Ji’nan, Shandong Province, were relatively high, which is also consistent with the fact that they are both important livestock and poultry breeding bases in Shandong Province. On the contrary, the levels of antimicrobial residues in the COFs collected from Bayannur, Inner Mongolia Autonomous Region, were much lower compared to the others. Bayannur is renowned for the production of sheep and cows by free-range farming, which uses much less antimicrobials compared to the factory farming of pigs and poultry. The is also a significant difference in the composition of antimicrobials in the COF samples produced from different regions (Figure 5b). For example, macrolides were the dominant class of antimicrobials in the COFs from Shijiazhuang, Handan, and Ji’ning, whereas quinolones were the most abundant antimicrobial class in those from Huanggang and Nantong. These results are consistent with the findings of a previous study, which reports that fluoroquinolones were used more heavily in animal farming in Southeast China compared to North China [17].

3.5. Ecological Risk of Antimicrobials in COFs on Agricultural Soils

The antimicrobial residues in COFs would be transferred into agricultural soils with their amendment and subsequently pose a potential ecological risk to soil organisms. Based on the estimated RQ values, only tilmicosin could pose low to medium ecological risk, while six antimicrobials, including ciprofloxacin, enrofloxacin, oxolinic acid, erythromycin, leucomycin, and tylosin, would pose low ecological risk (Figure 6). Among these seven antimicrobials, the potential ecological risk posed by individual compounds decreases in the order of tilmicosin > tylosin > ciprofloxacin > enrofloxacin > leucomycin > erythromycin > oxolinic acid. The other antimicrobials in the COF samples would pose no ecological risk to soil organisms. It is worth noting that only the risk from individual compounds is assessed here, while the co-occurrence of multiple antimicrobials could result in additive, synergistic, or antagonistic effects and, thus, have a much more complex toxic effect on soil organisms. For instance, ciprofloxacin and norfloxacin can exhibit strong synergistic effects, whereas erythromycin and tetracycline are known to present antagonistic effects on aquatic organisms [29,30]. Detailed knowledge of the mode of action of these antimicrobials is needed for more accurate prediction of their potential ecological risk.

The application of COFs is a major source of antimicrobials in agricultural soils [31]. The above results demonstrate that the continuous application of COFs could result in the accumulation of antimicrobials and pose a potential ecological risk to soil organisms. In addition, COFs could function as crucial reservoirs of ARGs and pathogens, along with a wide range of contaminants [32]. The antimicrobials inputted by long-term amendment of COFs could significantly reduce the bacterial diversity in agricultural soils and change the dominant bacterial species in the microbial community [33]. Moreover, the long-term application of COFs could greatly increase the abundance and diversity of ARGs in agricultural soils and accelerate the spread of ARGs from soil to plants, eventually threatening human health [25,34]. Therefore, it is critical to lower the residues of antimicrobials in COFs to control and prevent antimicrobial pollution in soil from the source. There is an urgent need to establish regulatory standards for the levels of antimicrobial residues in COFs, as no such regulations exist at the moment [35]. The composting and other production processes of COFs should be optimized to enhance the removal efficiency of antimicrobials in animal manure. The removal of antimicrobials during common aerobic composting and anaerobic digestion varies largely. Several sulfonamides, such as sulfadiazine, sulfadimethoxine, and sulfamethoxazole, could be almost completely removed during anaerobic digestion, whereas other sulfonamides, including sulfamethoxypyridazine, sulfamethazine, and sulfathiazole, are quite recalcitrant [36]. It was reported that aerobic composting could effectively remove tetracyclines, quinolones, and sulfonamides [37,38]. However, the degradation efficiency of antimicrobials could be significantly affected by their initial levels. For example, the degradation of ciprofloxacin in swine manure during composting was inhibited when its initial content exceeded 10 mg/kg [39]. To reduce the level of antimicrobial residues in animal manure, it is necessary to curb the prevalent overuse and misuse of antimicrobials in animal husbandry in China [40,41,42].

4. Conclusions

The occurrence of twenty-two veterinary antimicrobials, including eleven quinolones, six sulfonamides, four macrolides, and one lincosamide, were quantified in ninety-three COF samples collected from five provinces in China. While the total contents of antimicrobials in these COF samples ranged between 0.95 and 3870 ng/g, the levels of individual antimicrobials were up to 3310 ng/g. The composition and contents of antimicrobials in the COFs varied largely with different raw materials, production processes, and source regions. Seven antimicrobials could pose low to medium ecological risk to soil organisms, and their risk decreased in the order of tilmicosin > tylosin > ciprofloxacin > enrofloxacin > leucomycin > erythromycin > oxolinic acid. Although the majority of antimicrobials could pose only very low or no ecological risk, the actual risk to soil organisms in the amended soils from exposure to multiple antimicrobials introduced by COFs is difficult to predict. While COF application has been widely promoted as an alternative to chemical fertilization to improve soil fertility and develop green agriculture, the negative impact associated with the antimicrobial residues deserves significant attention. The wide occurrence of antimicrobials in COFs and their potential ecological risk indicate the urgent need to lower the antimicrobial residues in COFs to control and prevent antimicrobial pollution in agricultural soils.