Antibiotic Residues and Resistance in Three Wastewater Treatment Plants in Romania
Abstract
:1. Introduction
2. Results
2.1. Occurrence and Seasonal Variation of Antibiotics in Wastewaters
2.2. Antibiotic Removal Efficiency from Wastewater Treatment Plants
- CIWW—Influent antibiotic concentration
- CEWW—Effluent antibiotic concentration
2.3. Antibiotic Resistance Profiles of Isolated Bacteria
2.4. Gram Staining
3. Discussions
3.1. Comparison of Antibiotic Concentrations with Other Studies
3.2. Variability of Antibiotic Removal Efficiencies
3.3. Factors Influencing the Variation of Antibiotic Concentrations
3.3.1. The Influence of Air Temperature and Rainfall on Antibiotic Concentration Variability
3.3.2. Influence of pH and Physicochemical Characteristics on Antibiotic Removal
3.4. Impact of Antibiotic Residues in Effluents of WWTPs on the Aquatic Environment
3.5. Analytical Limitations and Implications
3.6. Seasonal Variations in Estimated Bacterial Loads
3.6.1. SEM Analysis
3.6.2. Analysis of Antibiotic Sensitivity Patterns
4. Materials and Methods
4.1. Location and Collection of Samples
4.2. Description of WWTP and Physicochemical Measurements
4.3. Air Temperature and Rainfall Data
4.4. Chemicals and Reagents
4.5. Analytical Procedures
4.6. Microbiology Assay
- n—Number of colonies in a Petri plate,
- d—The inverse of the dilution of the inoculated sample,
- N—The number of Petri plates considered,
- V—The volume of the sample used, in mL [71].
- Gram staining is a double staining that helps distinguish Gram-positive and Gram-negative bacteria from the samples. This method was used to distinguish coliforms, knowing that most of them are Gram-negative bacteria.
- Antibiogram assay—The diffusimetric method was used, on Mueller–Hinton agar medium [72] with antibiotic disks, which is considered a method with large applicability in practice to test the efficacy of antimicrobial substances. The bacterial strain suspensions were adjusted to 0.5 McFarland turbidity, and their susceptibility for the seven antibiotics (detected through the HPLC method) was tested. Incubation was performed at 37 °C for 18–24 h; thereafter, the diameter of the inhibition zone was measured. The interpretation of the results was carried out in accordance with the EUCAST guidelines [73]. The diameter of the inhibition area is correlated with the sensitivity of the bacterium to the tested antibiotics.
- Scanning electron microscopy (SEM) technique. The microscopic examination was performed to support the observations made by the microbiological procedures. The bacteria were taken from the plates with NA where CFU calculations were made. Then, the samples were fixed with 2.7% glutaraldehyde, washed with phosphate-buffered saline (PBS), dehydrated with 30 to 100% ethanol, and examined using a SEM Hitachi SU8230 (Hitachi, Tokyo, Japan) operated at 30 kV.
4.7. Calculations and Statistical Analyses
4.8. Environmental Risk Assessment (ERA)
Antibiotic | PNEC-ENV (µg/mL) | PNEC-MIC (µg/mL) | Lowest PNEC Value (µg/mL) |
---|---|---|---|
Amoxicillin | N/A | 0.016 | 0.016 |
Piperacillin | N/A | 0.0005 | 0.0005 |
Ciprofloxacin | 0.00045 | 0.00006 | 0.00006 |
Norfloxacin | 0.0012 | 0.0005 | 0.0005 |
Azithromycin | 0.00002 | 0.00025 | 0.00002 |
Clarithromycin | 0.00008 | 0.00025 | 0.00008 |
Doxycycline | N/A | 0.002 | 0.002 |
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Median | Difference (95% CI) | p-Value | ||
---|---|---|---|---|
Influent | Effluent | |||
AMX | 7.11 (<LOQ–16.61) | <LOQ (<LOQ–2.1) | 7.11 (2.84–19.98) | 0.093 |
AZT | 0.56 (<LOQ–1.12) | <LOQ (<LOQ–1.12) | 0.56 (NaN–NaN) | 1 |
CIP | <LOQ (<LOQ–0.26) | 0.41 (<LOQ–1.05) | −0.41 (−1.05–3.55) | 0.854 |
CLT | 1.27 (<LOQ–1.27) | 1.27 (<LOQ–1.27) | <LOQ (−1.27–1.28) | 0.584 |
DOX | 2.18 (<LOQ–2.47) | <LOQ (<LOQ–2.51) | 2.18 (−1.47–2.32) | 0.933 |
NOR | <LOQ (<LOQ–0.79) | 0.89 (0.43–1) | 0.89 (−1–6.02) | 0.824 |
PIP | 0.87 (0.2–1.87) | 0.24 (<LOQ–0.9) | 0.64 (−15.73–1.79) | 0.286 |
Mean | Difference (95% CI) | p-Value | ||
Influent | Effluent | |||
AMX | 9.5 (10.47) | 3.88 (9.12) | 5.62 (−0.28–11.53) | 0.06 |
AZT | 0.56 (0.58) | 0.47 (0.57) | 0.09 (−0.11–0.3) | 0.339 |
CIP | 0.94 (2.64) | 0.51 (0.53) | 0.44 (−1.13–2.01) | 0.553 |
CLT | 0.89 (0.74) | 0.84 (0.69) | 0.05 (−0.31–0.41) | 0.764 |
DOX | 1.5 (1.36) | 1.32 (1.73) | 0.18 (−0.61–0.97) | 0.622 |
NOR | 2.23 (5.09) | 1.05 (1.13) | 1.19 (−2.11–4.48) | 0.445 |
PIP | 1.21 (1.18) | 3.26 (10.02) | −2.05 (−8.53–4.43) | 0.501 |
Median | p-Value | ||||
---|---|---|---|---|---|
Q4 (n = 3) | Q1 (n = 3) | Q2 (n = 3) | Q3 (n = 3) | p {(1, 2)/(1, 3)/(1, 4)/(2, 3)/(2, 4)/(3, 4)} | |
AMX I | n.d. | n.d. | 14.65 (14.43–19.99) | 19.98 (17.74–22.16) | 0.005 {1/<0.001/<0.001/<0.001/<0.001/0.998} |
AMX E | n.d. | n.d. | 10.47 (6.79–20.82) | <LOQ (<LOQ–0.88) | 0.018 {1/<0.001/0.889/<0.001/0.889/<0.001} |
AZT I | 1.12 (1.12–1.12) | n.d. | 1.12 (1.12–1.12) | n.d. | 0.005 {<0.001/1/<0.001/<0.001/1/<0.001} |
AZT E | 1.12 (1.12–1.12) | n.d. | 1.12 (0.56–1.12) | n.d. | 0.044 {<0.001/0.827/<0.001/0.548/1/0.548} |
CIP I | n.d. | n.d. | 1.05 (1.05–5.13) | n.d. | 0.018 {1/<0.001/1/<0.001/1/<0.001} |
CIP E | 1.05 (0.93–1.05) | n.d. | 1.05 (1.05–1.05) | n.d. | 0.007 {<0.001/0.928/<0.001/<0.001/1/<0.001} |
CLT I | 1.27 (1.27–1.27) | 1.27 (0.9–1.54) | 1.27 (0.64–1.63) | n.d. | 0.108 {1/1/<0.001/1/<0.001/0.594} |
CLT E | 1.27 (1.27–1.27) | 0.52 (0.26–0.89) | 1.27 (1.27–1.6) | n.d. | 0.017 {0.629/0.886/<0.001/0.408/0.629/<0.001} |
DOX I | 2.2 (2.18–2.72) | 2.74 (2.56–2.89) | n.d. | <LOQ (<LOQ–1.13) | 0.025 {0.995/<0.001/0.916/<0.001/<0.001/0.895} |
DOX E | 3.9 (3.38–4.17) | 2.23 (1.11–2.31) | n.d. | n.d. | 0.004 {<0.001/<0.001/<0.001/0.629/0.629/1} |
NOR I | n.d. | n.d. | 8.19 (4.1–12.38) | 0.55 (0.28–1.02) | 0.107 {1/0.459/0.459/0.459/0.459/0.878} |
NOR E | 1 (0.89–1) | 1 (0.93–2.48) | 0.92 (0.75–1.7) | n.d. | 0.049 {0.984/1/<0.001/0.988/<0.001/<0.001} |
PIP I | 1.74 (1.29–2.62) | 1.69 (0.85–1.98) | 0.9 (0.58–1.82) | <LOQ (<LOQ–0.28) | 0.228 {0.983/0.983/<0.001/1/0.865/0.638} |
PIP E | 0.9 (0.9–17.98) | n.d. | <LOQ (<LOQ–0.45) | 0.45 (0.24–0.68) | 0.024 {<0.001/0.37/0.37/0.853/<0.001/0.951} |
Location | AMX | PIP | CIP | NOR | AZT | CLT | DOX |
---|---|---|---|---|---|---|---|
WWTP A Q4 | n/a | 74% | n/a | n/a | n/a | n/a | −30% |
WWTP B Q4 | n/a | −315% | n/a | n/a | n/a | n/a | −20% |
WWTP C Q4 | n/a | 48% | n/a | n/a | n/a | n/a | −105% |
WWTP A Q1 | n/a | 100% | n/a | n/a | n/a | 100% | 21% |
WWTP B Q1 | n/a | 100% | n/a | n/a | n/a | 71% | 100% |
WWTP C Q1 | n/a | n/a | n/a | n/a | n/a | n/a | 19% |
WWTP A Q2 | 26% | −237% | n/a | n/a | n/a | 3% | n/a |
WWTP B Q2 | 78% | 100% | 89% | 97% | n/a | n/a | n/a |
WWTP C Q2 | −23% | n/a | n/a | 70% | n/a | n/a | n/a |
WWTP A Q3 | 100% | n/a | n/a | n/a | n/a | n/a | 100% |
WWTP B Q3 | 100% | 96% | n/a | 100% | n/a | n/a | n/a |
WWTP C Q3 | 93% | n/a | n/a | 100% | n/a | n/a | n/a |
Antibiotics | Mean (SD)/Median (IQR) |
---|---|
AMX | 85.74 (39.45–98.19) |
AZT | 0 (0–0) |
CLT | 0 (0–20.34) |
DOX | 18.69 (−25.38–60.72) |
NOR | 98.27 (89.87–100) |
PIP | 95.54 (48.11–100) |
WWTP A | WWTP B | WWTP C | ||||
---|---|---|---|---|---|---|
Influent | Effluent | Influent | Effluent | Influent | Effluent | |
Autumn Q4 | 6.903 | 6.322 | 6.114 | 6.301 | 6.041 | 6.041 |
Winter Q1 | - | 6 | 6.477 | - | 7.491 | - |
Spring Q2 | 8.857 | 6.699 | 9.230 | - | 8.146 | 7.681 |
Summer Q3 | 7.633 | - | 8.447 | - | 6 | - |
WWTP A | WWTP B | WWTP C | ||||
---|---|---|---|---|---|---|
Influent | Effluent | Influent | Effluent | Influent | Effluent | |
Autumn Q4 | - | - | - | - | - | - |
Winter Q1 | - | - | - | - | - | - |
Spring Q2 | G+ | G− | G− | - | G− | G− |
Summer Q3 | - | - | G− | - | - | - |
Variables | B Unadjusted | (95% CI) | p | R2 | B Adjusted | (95% CI) | p |
---|---|---|---|---|---|---|---|
Intercept | 1.66 | (–7.65–10.96) | 0.707 | ||||
Treated | –5.62 | (−13.94–2.69) | 0.175 | 0.08 | −4.5 | (–14.27–5.28) | 0.338 |
Mean monthly temperature (°C) | 0.79 | (0.2–1.37) | 0.012 | 0.37 | 0.79 | (0.19–1.38) | 0.003 |
Variables | B Unadjusted | (95% CI) | p | R2 | B Adjusted | (95% CI) | p |
---|---|---|---|---|---|---|---|
Intercept | 2.11 | −5.07 | 0.574 | ||||
Treated | −5.62 | (−3.48–2.23) | 0.175 | 0.08 | −4.5 | (−4.58–5.59) | 0.398 |
Mean monthly rainfall (L/m2) | 0.15 | (0.06–0.24) | 0.006 | 0.2 | 0.15 | (0.07–0.23) | 0.003 |
Variables | B Unadjusted | (95% CI) | p-Value | R2 | B Adjusted | (95% CI) | p |
---|---|---|---|---|---|---|---|
Intercept | 2.99 | (1.98–4) | <0.001 | ||||
Treated | −0.18 | (−1.5–1.14) | 0.777 | 0.004 | −0.06 | (−1.12–1.01) | 0.907 |
Mean monthly temperature (°C) | −0.14 | (−0.2–−0.08) | <0.001 | 0.62 | −0.14 | (−0.2–−0.07) | <0.001 |
Variables | B Unadjusted | (95% CI) | p | R2 | B Adjusted | (95% CI) | p |
---|---|---|---|---|---|---|---|
Intercept | 3.11 | (1.7–4.51) | <0.001 | ||||
Treated | −0.18 | (−1.5–1.14) | 0.777 | 0.004 | −0.06 | (−1.35–1.24) | 0.924 |
Mean monthly rainfall (L/m2) | −0.03 | (−0.05–−0.01) | 0.006 | 0.43 | −0.03 | (−0.05–−0.01) | 0.008 |
Effluent | AMX | PIP | CIP | NOR | AZT | CLT | DOX | |
---|---|---|---|---|---|---|---|---|
WWTP A | Q1 | 0 | 0 | 0 | 111.1 | 0 | 0 | 16.74 |
Q2 | 9.17 | 0 | 0 | 216.03 | 0 | 337.14 | 0 | |
Q3 | 0 | 12.68 | 0 | 0 | 0 | 0 | 0 | |
Q4 | 0 | 0 | 0 | 0 | 0 | 0 | 20.1 | |
WWTP B | Q1 | 0 | 0 | 0 | 0 | 0 | 91.08 | 0 |
Q2 | 2.73 | 0 | 0 | 16.07 | 0 | 0 | 0 | |
Q3 | 0 | 0.7 | 0 | 0 | 0 | 0 | 0 | |
Q4 | 0 | 98.32 | 0 | 0 | 0 | 0 | 27.35 | |
WWTP C | Q1 | 0 | 0 | 0 | 23.97 | 0 | 0 | 0.02 |
Q2 | 27.33 | 0 | 0 | 69.29 | 0 | 0 | 0 | |
Q3 | 1.54 | 0 | 0 | 0 | 0 | 0 | 0 | |
Q4 | 0 | 0 | 190.2 | 22.13 | 0 | 0 | 27.35 |
Location | AMX | PIP | CIP | NOR | CLT | AZT | DOX |
---|---|---|---|---|---|---|---|
Q4 EWWTP C | IR | S | S | S | S | S | S |
Q4 EWWTP A | S | S | S | S | S | S | S |
Q2 EWWTP A | R | S | S | S | R | R | S |
Q2 IWWTP A | S | S | R | R | S | S | IR |
Q2 IWWTP C | R | R | S | S | S | S | S |
Q2 EWWTP C | R | IR | IR | IR | R | IR | IR |
Q2 IWWTP B | S | S | S | S | R | S | S |
Q3 IWWTP B | R | S | S | S | R | S | S |
WWTP | Mechanical Pre-Treatment | Biological Treatment | Sludge Treatment | Tertiary Treatment |
---|---|---|---|---|
WWTP A | Screening, sand and grease removal, primary sedimentation | Activated sludge process, biological reactors | Anaerobic digesters, sludge thickening, dewatering | Tertiary filtration, disinfection |
WWTP B | Coarse screening, fine screening, grit chamber, primary sedimentation | Aeration tanks | Sludge thickening, dewatering, stabilization | Chemical phosphorus removal, disinfection |
WWTP C | Mechanical bar screen, grit chamber, primary sedimentation | Aeration tanks | Sludge thickening, anaerobic digestion, dewatering | Tertiary filtration, UV disinfection |
Season | WWTP A | WWTP B | WWTP C | |||
---|---|---|---|---|---|---|
Influent | Effluent | Influent | Effluent | Influent | Effluent | |
Q1 | 7.34 | 7.09 | 6.97 | 6.73 | 7.47 | 7.37 |
Q2 | 6.76 | 7.05 | 7.11 | 6.93 | 6.86 | 7.59 |
Q3 | 6.88 | 7.08 | 6.58 | 6.52 | 6.45 | 5.86 |
Q4 | 7.29 | 7.18 | 6.2 | 6.36 | 6.96 | 7.26 |
Season | Recording Month | Rainfall (R24) * | Air Temperature (°C) * | |
---|---|---|---|---|
WWTP A | Q4 | November | 23 | 4.2 |
Q1 | February | 11.4 | 1.8 | |
Q2 | May | 97.4 | 15.4 | |
Q3 | August | 87.8 | 21.3 | |
WWTP C | Q4 | November | 51.4 | 3.6 |
Q1 | February | 13.8 | 0.8 | |
Q2 | May | 52.6 | 15.5 | |
Q3 | August | 77.6 | 21.6 |
Chemical Group | Antibiotic | Use | Chemical Formula | Molecular Weight (g mol−1) |
---|---|---|---|---|
β-lactams | Amoxicillin | Human and Veterinary | C16H19N3O5S | 365.4 |
Piperacillin | Human | C23H27N5O7S | 516.54 | |
Fluoroquinolones | Ciprofloxacin | Human and Veterinary | C₁₇H₁₈FN₃O₃ | 331.34 |
Norfloxacin | Human | C16H18FN3O3 | 319.33 | |
Macrolides | Azithromycin | Human | C38H72N2O12 | 748.99 |
Clarithromycin | Human | C38H69NO13 | 747.95 | |
Tetracyclines | Doxycycline | Human and Veterinary | C22H24N2O8·H2O | 444.4 |
Compound | Linearity (R2) | LOD µg/mL | LOQ µg/mL | Retention Time (min) | Molecular Ion (m/z) |
---|---|---|---|---|---|
Amoxicillin | 0.9989 | 3.139 | 4.240 | 5.5 | 366 [M + H]+ |
Piperacillin | 0.9993 | 0.908 | 1.804 | 13.75 | 518 [M + H]+ |
Ciprofloxacin | 0.999 | 1.060 | 2.104 | 10.8 | 332 [M + H]+ |
Norfloxacin | 0.9991 | 1.008 | 2.001 | 11.3 | 320 [M + H]+ |
Azithromycin | 0.9989 | 1.125 | 2.232 | 12.5 | 749 [M + H]+ 375 [M + 2H]+ |
Clarithromycin | 0.9986 | 1.282 | 2.540 | 15.5 | 748 [M + H]+ |
Doxycycline | 0.9987 | 4.230 | 5.445 | 13 | 445 [M + H]+ |
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Polianciuc, S.I.; Ciorîță, A.; Soran, M.L.; Lung, I.; Kiss, B.; Ștefan, M.G.; Leucuța, D.C.; Gurzău, A.E.; Carpa, R.; Colobațiu, L.M.; et al. Antibiotic Residues and Resistance in Three Wastewater Treatment Plants in Romania. Antibiotics 2024, 13, 780. https://doi.org/10.3390/antibiotics13080780
Polianciuc SI, Ciorîță A, Soran ML, Lung I, Kiss B, Ștefan MG, Leucuța DC, Gurzău AE, Carpa R, Colobațiu LM, et al. Antibiotic Residues and Resistance in Three Wastewater Treatment Plants in Romania. Antibiotics. 2024; 13(8):780. https://doi.org/10.3390/antibiotics13080780
Chicago/Turabian StylePolianciuc, Svetlana Iuliana, Alexandra Ciorîță, Maria Loredana Soran, Ildiko Lung, Béla Kiss, Maria Georgia Ștefan, Daniel Corneliu Leucuța, Anca Elena Gurzău, Rahela Carpa, Liora Mihaela Colobațiu, and et al. 2024. "Antibiotic Residues and Resistance in Three Wastewater Treatment Plants in Romania" Antibiotics 13, no. 8: 780. https://doi.org/10.3390/antibiotics13080780