Disinfection of Hospital Wastewater

Disinfection of hospital wastewater

Prepared for Aarhus University Hospital

Prepared by Danish Technological Institute Kongsvang Allé 29 DK 8000 Aarhus C Life Science Caroline Kragelund Rickers, Cand Scient.PhD Sabine Lindholst, Dipl.-Ing.

May 2019

Page 2 DANISH TECHNOLOGICAL INSTITUTE

1. Table of contents

2. Summary and conclusion ...... 4 3. Aim ...... 4 4. Laboratory analyses and results...... 4 4.1. Residual PAA concentration ...... 5 4.2. Antibiotic model organisms for untreated wastewater from AUH hospital ...... 6 4.3. Required reduction of ciprofloxacin-resistant bacteria ...... 7 4.4. Required PAA doses for reduction of ciprofloxacin-resistant bacteria ...... 7 5. Pilot-scale plant and results...... 11 5.1. Description of pilot plant ...... 11 5.2. Results obtained using the pilot plant ...... 13 6. Literature references ...... 13 7. Appendices ...... 15 7.1. Appendix A: Brief description of the applied Compact Dry method ...... 15 7.2. Appendix B: Detailed results of PAA concentrations and reaction times ...... 16

2. Summary and conclusion

The aim of this project was to identify the required concentration of peracetic acid (PAA) to reduce the ciprofloxacin-resistant bacteria present in wastewater from the AUH hospital, to the levels observed in municipal wastewater. Different lab scale experiments were conducted to evaluate reaction time of PAA and the corresponding concentration. Both long reaction times (more than 10 min.) and short reaction times (between 2-5 min.) have been investigated and the corresponding PAA concentrations have been determined. The required concentration of PAA for the long reaction times was determined between 30 mg/l – 50 mg/l. For shorter reaction times (5 min) more than 70 mg/l PAA was required and for the very short times (2 min) more than 90 mg/l PAA was needed. In addition, pilot scale experiments were conducted at the container plant situated at the Aarhus Uni- versity Hospital, where the laboratory results for the short reaction times and corresponding high PAA concentrations were confirmed. Concentrations between 83-115 mg/l PAA reduced the number of ciprofloxacin-resistant bacteria to level at Viby WWTP.

3. Aim

The aim of this disinfection project was to clarify and document whether it is possible to reduce the number of antibiotic resistant bacteria present in untreated wastewater from Aarhus University Hos- pital (AUH) by using peracetic acid (PAA).

Since there are no requirements for controlling the presence of antibiotic resistant bacteria in the wastewater, the consortium has been in dialogue with both the waterworks (Aarhus Vand) as well as Aarhus municipality (Aarhus Kommune) to determine the required reduction of antibiotic bacteria. It was decided to use ciprofloxacin-resistant bacteria as model organisms, because ciprofloxacin is a commonly used broad spectrum antibiotic and therefore is expected to result in high numbers of ciprofloxacin-resistant bacteria. Ciprofloxacin is consumed both at hospitalized patients but also in private homes. Here, it was decided that the content of ciprofloxacin-resistant bacteria present in the untreated wastewater from AUH, should be reduced to the level of ciprofloxacin-resistant bacteria in normal household wastewater. Here, the average level of 6 samples of ciprofloxacin-resistant bacteria obtained from the municipal wastewater treatment plant Viby in Aarhus, was identified as the desired reduction level.

Experimental work has been carried out in the laboratory to determine the required concentration of PAA for the reduction of the ciprofloxacin-resistant bacteria as well as the total number of bacteria present in the untreated wastewater. Therefore, different concentrations of PAA and corresponding reaction times have been tested to identify the optimal concentration and time. Two pilot-scale experiments have been conducted using the equipment from Norlex, and here PAA concentration and reaction time identified from lab-scale experiments have been applied.

4. Laboratory analyses and results

In the lab-scale experiments, different PAA doses and corresponding reaction times have been applied to untreated wastewater from AUH. In Table 1, an overview of different doses and reaction times of PAA are illustrated.

Table 1. Overview of PAA doses and reaction time

Peracetic acid (PAA) doses [mg/l] Reaction times [min] 30 10, 30 50 10, 30 60 30, 60 70 30, 60 80 30, 60 100 10, 30, 60

The effect of PAA is affected by sodium-thiosulphate and catalase, and any residual PAA concentration is removed when sodium-thiosulphate is added. This means that it is possible to determine different reaction times of PAA and to control exact reaction times.

The different concentrations of PAA and the corresponding reaction times were initially decided based on the data obtained from Chhetri et al., 2014. However, this study investigated sewer overflow which was simulated in the laboratory by diluting untreated wastewater with fresh water before PAA treatment. In our case, untreated undiluted wastewater was to be treated using PAA, and therefore higher PAA doses were investigated. Different reaction times corresponded to the flow and different sampling positions at the pilot scale equipment from Norlex. It was anticipated that reaction times of 30 min. and 60 min. would be required to obtain the desired reduction degrees of the ciprofloxacin-resistant bacteria.

4.1. Residual PAA concentration

Two methods were applied to determine the residual concentration of PAA. The first one was a method described by Chhetri et al., 2014.

As shown in Figure 1, the residual PAA concentration is plotted as a function of the absorbance (405 nm) measured on different time points. The applied PAA concentration is 100 mg/l, and as observed the absorbance is significantly reduced already after 10 min., suggesting that any residual concentration of PAA is reduced fast when applied to untreated wastewater from AUH.

Figure 1. Overview of residual concentration of PAA at different time points measured with the Chhetri method.

The second method, a test strip method (Quantofix, Macherey & Nagel), was used to further investigate how fast the residual PAA concentration was reduced over time, an experiment was initiated using 30 mg/l PAA added to untreated wastewater from AUH. As evident in Figure 2, the residual PAA concentration is below the detection limit of 5 mg/l after 165 sec., e.g. less than 3 min. after addition. 30 mg/l PAA added to untreated wastewater 30

Quantifix PA 50 - 15

PAA conc [mg/l] conc PAA [mg/l] 0 0 20 40 60 80 100 120 140 160 180 sek

Figure 2. Residual PAA concentration measured at different time points using Quantofix PA 50 test strips.

4.2. Antibiotic model organisms for untreated wastewater from AUH hospital

There is no public information available on which antibiotic microorganisms should be measured in untreated wastewater from AUH, and it was thus proposed to use ciprofloxacin-resistant bacteria. The reason for this is directly related to the consumption of antibiotics at the AUH, as published in an environmental survey of medicine consumption at AUH (Møller et al., 2014). The consumption of ciprofloxacin, also commonly used antibiotics in the private sector, at AUH has a significant environmental impact when discharged with wastewater. Therefore, it was expected that there would be a high concentration of ciprofloxacin-resistant bacteria in the untreated wastewater from AUH, and the concentrations of PAA to reduce these bacteria would be sufficient to reduce other antibiotic bacteria present in lower concentrations. To determine the presence of all bacteria and ciprofloxacin-resistant bacteria, compact dry methods were applied. Samples were investigated with or without addition of ciprofloxacin, and the compact dry plates were incubated at 22 °C for 24 hours prior to bacteria counts. For further details, please see Appendix A.

Other problematic pathogenic antibiotic bacteria as MRSA (methicillin-resistant Staphylococcus aureus), VRE (vancomycin-resistant Enterococcus), ESBL (extended spectrum beta-lactamase E. coli) and CPO (carbapenemase-producing organisms, E. coli and other gram-negative bacteria), which are monitored and reported each year in DANMAP reports (DANMAP 2017). However, these pathogens are isolated from both humans and animals and have a potentially larger impact on spreading antibiotic resistance to the environment. Only limited data exist on the presence of these pathogenic bacteria in wastewater, but reports from e.g. DANMAP surveys (2013, 2017) indicate that they are detected in small quantities at the hospitals. Several studies have shown that some of the above-mentioned pathogens are present in much lower quantities compared to ciprofloxacin-resistant bacteria in wastewater (Danish Nature Agency 2011, Kragelund et al., 2018, Jørgensen et al., 2017, Boopathy et al., 2017).

4.3. Required reduction of ciprofloxacin-resistant bacteria

As a result of the dialogue between Aarhus municipality and the waterworks, Aarhus Vand, the required reduction degree of ciprofloxacin-resistant bacteria present in the untreated wastewater from the AUH has been decided. Since no controlling limits have been proposed within this area, it was decided to aim for a reduction degree of ciprofloxacin-resistant bacteria corresponding to their presence in untreated wastewater from a water treatment plant receiving only household wastewater, such as the Viby wastewater treatment plant (Viby WWTP) previously investigated for the presence of ciprofloxacin- resistant bacteria. It was thus decided to use the obtained results from untreated inlet wastewater, see Table 2.

Table 2. Overview of bacteria counts in untreated wastewater from Viby WWTP (total count and ciprofloxacin- resistant bacteria). Date of investigation Inlet total bacteria count Inlet ciprofloxacin-resistant (cfu/ml) bacteria (cfu/ml)

16-11-2016 12200 1383 18-11-2016 38433 2603 23-11-2016 34400 1553 26-11-2016 20367 1273 30-11-2016 36533 2410 02-12- 2016 39667 7768

Average 30267 2832

4.4. Required PAA doses for reduction of ciprofloxacin-resistant bacteria

Several laboratory experiments were carried out to determine the required PAA dose and corresponding reaction time.

The number of total bacteria counts and ciprofloxacin-resistant bacteria were determined in different samples of the untreated wastewater from AUH, see Table 3. As evident, the number of both total bacteria as well as the ciprofloxacin-resistant bacteria vary slightly between the different sampling dates.

Table 3. Overview of bacteria counts present in untreated wastewater from AUH investigated at different time points. Sampling date Ciprofloxacin-resistant Total counts Percentage of cipro- bacteria (CFU/ml) (CFU/ml) floxacin-resistant bacteria (%) 02-10-2018 1.26E+05 5.15E+05 24 10-10-2018 7.35E+05 3.40E+06 22 15-10-2018 1.28E+05 8.20E+05 16 25-10-2018 1.40E+05 1.01E+06 14

Reduction of ciproflox. res. bacteria [%]

100,00

99,99 99,99

99,98

99,98 99,98 99,98

99,97 99,97

99,96 99,96

99,96

99,95

99,95 99,95 99,95

99,93

99,91

99,89

99,88

99,61 99,61

99,57

99,41

99,39 99,39

98,97

98,89

98,83 98,73

100 98,40

98 97,14 96 94 92 90 88 86 84 82

K60-30min K70-30min K90-20min K70-20min K80-20min K60-20min K70-10min K50-20min K90-10min K30-20min K60-10min K30-10min K50-10min K80-60min K50-30min K30-10min K50-10min K30-30min

K80_10min

K60-60 min K60-60 K80-30 min K80-30 min K70-60

K100-30min K100-10min K120-60min K100-60min K100-10min K120-30min K120-10min K100-30min K100-60min K100-30min

Figure 3. Overview of PAA doses (K90 = 90 mg/l) and reaction time experiments. The different colour bars repre- sent different sampling days.

As shown in Figure 3, different PAA concentrations and reaction times have been tested using laboratory analyses. A large reduction of the ciprofloxacin-resistant bacteria can be observed using all tested PAA concentrations. The effect of PAA and reaction times for each concentration can be seen in Ap- pendix B.

Reduction [%] of ciprofl. res. bacteria after 10 min reaction time with PAA

99,97

99,95 99,95

100,00 98,73 99,00 98,40 98,00 97,00 96,00 95,00 94,00 93,00 92,00 91,00 90,00 K100-10min K100-10min K120-10min K30-10min K50-10min

Figure 4. Overview of reduction of ciprofloxacin-resistant bacteria in percentage at different PAA doses after 10 min. reaction time. Different colours reflect different sampling days.

Reduction degrees between 98 and 99.9 % of ciprofloxacin-resistant bacteria were obtained at different PAA concentrations after a reaction time of 10 min. (Figure 4). However, these results must be compared with the acceptable level of ciprofloxacin-resistant bacteria present in household wastewater (Viby WWTP). When reduction levels and reaction times are compared with the proposed reduction level in Viby (average 2832 CFU/ml), it is evident that lower doses of PAA meet this controlling requirement listed above, see Figure 5.

Figure 5. Overview of ciprofloxacin-resistant bacteria viable after PAA treatment. Blue line represents the level of ciprofloxacin-bacteria present in Viby WWTP.

However, after the discussions with Aarhus municipality, there is a desire to reduce the number of ciprofloxacin-resistant bacteria present in the raw wastewater at the premises of AUH before the wastewater is mixed with wastewater of a different origin. Therefore, additional experiments have been planned with rather low reaction times, for example, 2, 3 and 5 min. The first results with 5 min. reaction time are depicted in Figure 6. As expected, a larger dose of PAA is required to reduce the number of ciprofloxacin-resistant bacteria to the acceptable level from Viby WWTP. More experiments are required to verify the correct dose of PAA. Additionally, shorter incubation times will be tested during pilot scale experiments.

Shorter reaction times (2, 3 and 5 min.) were also investigated in laboratory studies, see Figure 7.

Ciprofloxacin-resistant bacteria after 5 min. of PAA treatment 1,0E+06

1,0E+05

1,0E+04

1,0E+03

1,0E+02

1,0E+01

1,0E+00 Raw wastewater+ Cipro PE K30-5min + E K50-5min + Ciprofloxacin PE K70-5min + Ciprofloxacin Ciprofloxacin

Ciproflox. Resistant bacteria Guiding limit from Viby 2832

Figure 6. Overview of PAA treatment on ciprofloxacin-resistant bacteria after 5 min. of treatment (logarithmic scale) including guiding limit from Viby (2832 CFU/ml)

As evident below in Figure 7, a correlation between required PAA dosage and reaction time exists. Therefore, significantly higher PAA doses are required to reduce the ciprofloxacin-resistant bacteria below the proposed threshold. As demonstrated, the required dosage for the 2-min. treatment is approx.110 mg/l , but more data is necessary to validate the exact concentration.

48,3 % 60.000

50.000

40.000

30.000

20.000 85,4 % 91,3 % 10.000

96,9 % 98,3 % 99,0 % 98,9 % 98,8 % ciprofloxacin resistant bacteria [CFU/ml] 0

Figure 7. Overview of PAA treatments on ciprofloxacin-resistant bacteria after 2, 3 and 5 min.

5. Pilot-scale plant and results

The pilot plant was installed at the AUH premises, see Figure 8. Electricity was obtained from a gener- ator, and the pilot plant was not operated continuously. Instead, experiments were carried out at different periods of time, so both the dose of PAA and reaction times could be changed and compared.

Figure 8. Overview of the pilot plant container at the AUH premises.

5.1. Description of pilot plant

The pilot plant has been designed by Norlex Systems, which applies advanced technology for the re- moval of unwanted substances in wastewater (e.g. micropollutants and antibiotic resistant bacteria). In this project, the pilot plant has been designed to disinfection untreated hospital wastewater. The untreated wastewater is pumped directly from the sewer and is led through a closed-circuit where the treatment with peracetic acid is carried out, see Figure 9. In the plant, it is possible to obtain samples at different places to be able to evaluate the current treatment. This is possible as several sampling sites are included in the closed-circuit system, and therefore different reaction times can be obtained depending on flow of wastewater. After treatment, the wastewater is discharged directly to the wastewater sewer.

Figure 9. Schematic drawing of pilot plant with pumps and sampling points.

Figure 10. The interior of the pilot test plant

The present setup of the pilot plant requires manual operation, but a fully automatically operation can be achieved with only a few adjustments. Then, all operational parameters could be monitored online. The final version of the disinfection plant will have a similar function, which will ensure a continuous operation of the disinfection with a high degree of safety.

5.2. Results obtained using the pilot plant

Pilot-scale experiments were carried out with short reaction times of PAA disinfection, relevant for AUH. The following Figure 11 shows the results from the pilot scale test.

Figure 11. Reduction of ciprofloxacin-resistant bacteria with PE treatment at different concentrations and approximately 2 min reaction time.

The test was conducted with a short reaction time of approximately 2 minutes. The results confirm the results shown in the laboratory tests, where quite high concentrations of PAA were necessary to reduce the ciprofloxacin-resistant bacteria to a level comparing to the Viby level without hospital wastewater contribution. A PAA concentration of 83 mg/l was enough in the pilot scale test to obtain satisfactory disinfection in the pilot scale test, which was quite similar in the laboratory test. Slight dif- ferences can be caused by different total bacteria concentrations in the raw wastewater and inaccura- cies in the manual flow operation of the test setup. In a readily designed automated disinfection setup a safety margin for applied PAA concentration will be implemented.

6. Literature references

Chhetri RK, Thornberg D, Berner J, Gramstad R, Öjstedt U, Sharma AK, Andersen HR. Chemical disinfection of combined sewer overflow waters using performic acid or peracetic acids. Sci Total Envi- ron. 2014 Aug 15;490:1065-72. doi:10.1016/j.scitotenv.2014.05.079. Epub 2014 Jun 9. PubMed PMID: 24918873. Møller, T (2014). Environmental report from Aarhus University Hospital, technical department, Region Midt. (in Danish)

Kragelund et al., 2018. Environmentally friendly treatment of highly potent pharmaceuticals in hospital wastewater – Mermiss. Environmental project No. 1988. https://www2.mst.dk/Udgiv/publica- tions/2018/03/978-87-93614-81-9.pdf Danish Nature Agency: Hospital Wastewater – BAT and development of treatment technologies. June 2011. Report prepared by DHI (in Danish) DANMAP 2017. - Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. ISSN 1600-2032. Grundfos Biobooster, 2016. Full scale advanced wastewater treatment at Herlev Hospital. Report prepared by DHI. https://www.dhigroup.com/-/media/shared%20content/global/news/2016/08/evalu- ation%20report.pdf Jørgensen SB, Søraas AV, Arnesen LS, Leegaard TM, Sundsfjord A, Jenum PA. A comparison of extended spectrum β-lactamase producing Escherichia coli from clinical, recreational water and wastewater samples associated in time and location. PLoS One. 2017 Oct 17;12(10):e0186576. doi: 10.1371/journal.pone.0186576. eCollection 2017. PubMed PMID: 29040337; PubMed Central PMCID: PMC5645111. Boopathy R. Presence of Methicillin Resistant Staphylococcus aureus (MRSA) in sewage treatment plant. Bioresour Technol. 2017 Sep;240:144-148. doi: 10.1016/j.biortech.2017.02.093. Epub 2017 Feb 23. PubMed PMID: 28262305.

7. Appendices

7.1. Appendix A: Brief description of the applied Compact Dry method

Protocol for Compact Dry • Both antibiotic resistant’s strains as well as control strains with no antibiotic resistance are diluted 10-fold in SPO. Also, a dilution series containing the antibiotics ciprofloxacin is made • 1 ml of each dilution is added to Compact dry plates • All plates are incubated at 37 degrees overnight • The number of colonies are counted and the initial concentration of bacteria are calculated-

Concentration of antibiotics Initial literature screening revealed appropriate concentrations of ciprofloxacin to be added. However, initial screening using strains isolated from patients containing ciprofloxacin-resistance, resulted in in- creased the ciprofloxacin concentration.

Table 1. Overview of literature concentration of ciprofloxacin and adjusted applied concentration Antibiotics Concentration from BAT report1 Modified concentration after lab (mg/l) experiments (mg/l) Ciprofloxacin 3 4 The Compact Dry plates were counted and multiplied with the dilution factor to determine the quantity of viable bacteria present in the sample with and without ciprofloxacin.

1 Nielsen et al. 2011. Hospitalsspildevand – BAT og udvikling af renseteknologier

7.2. Appendix B: Detailed results of PAA concentrations and reaction times

First laboratory tests with long retention times (corresponding to Figure 5):

Date PAA conc. Remaining ciprofl. resistant Reduction of (K90 = 90 mg/l) bacteria after PAA treatment ciprofl. and reaction [CFU/ml] resistant bacteria time [%] 25-10-2018 K90-20min 0 100.00 25-10-2018 K70-20min 10 99.99 25-10-2018 K80-20min 10 99.99 02-10-2018 K100-30min 20 99.98 25-10-2018 K60-20min 30 99.98 25-10-2018 K70-10min 50 99.96 25-10-2018 K50-20min 50 99.96 25-10-2018 K80_10min 60 99.96 02-10-2018 K100-10min 60 99.95 25-10-2018 K90-10min 70 99.95 25-10-2018 K30-20min 100 99.93 25-10-2018 K60-10min 120 99.91 10-10-2018 K120-60min 150 99.98 10-10-2018 K100-60min 160 99.98 25-10-2018 K30-10min 160 99.89 25-10-2018 K50-10min 170 99.88 10-10-2018 K100-10min 220 99.97 10-10-2018 K120-30min 230 99.97 10-10-2018 K120-10min 360 99.95 10-10-2018 K100-30min 370 99.95 15-10-2018 K80-60min 500 99.61 15-10-2018 K100-60min 500 99.61 15-10-2018 K100-30min 550 99.57 15-10-2018 K80-30 min 760 99.41 02-10-2018 K50-30min 770 99.39 15-10-2018 K70-60 min 780 99.39 15-10-2018 K60-30min 1320 98.97 15-10-2018 K70-30min 1420 98.89 15-10-2018 K60-60 min 1500 98.83 02-10-2018 K30-10min 1600 98.73 02-10-2018 K50-10min 2020 98.40 02-10-2018 K30-30min 3600 97.14

Laboratory test 27/11-2019 with 5 min. reaction time.

PAA konc. Remaining ciprofl. Resistant Reduction of (K30 = 30 mg/l) bacteria after PAA treatment ciprofl. and reaction [CFU/ml] resistant bacteria time [%] 27-11-2018 K30-5min 25200 96.71 27-11-2018 K50-5min 23000 96.99 27-11-2018 K70-5min 1130 99.85 27/11-2019: Ciprofloxacin resistant bacteria in raw wastewater: 7.7E5 CFU/ml.

Laboratory test 8/1-2019 – short reaction times (2, 3 and 5 minutes) Date PAA konc. Remaining ciprofl. Resistant Reduction of (K70 = 70 mg/l) bacteria after PAA treatment ciprofl. and reaction time [CFU/ml] resistant bacteria [%] 08-01-2019 K70-2min 61000 48.31 08-01-2019 K70-3min 10300 91.27 08-01-2019 K70-5min 3700 96.86 08-01-2019 K90-2min 17200 85.42 08-01-2019 K90-5min 1950 98.35 08-01-2019 K110-2min 1190 98.99 08-01-2019 K110-3min 1250 98.94 08-01-2019 K110-5min 1360 98.85 Ciprofloxacin resistant bacteria in raw wastewater: 1.2E5 CFU/ml

Date PAA konc. Remaining ciprofl. Resistant Reduction of (K30 = 30 mg/l) bacteria after PAA treatment ciprofl. and reaction [CFU/ml] resistant bacteria time [%] 27-11-2018 K30-5min 25200 96.71 27-11-2018 K50-5min 23000 96.99 27-11-2018 K70-5min 1130 99.85 27/11-2019: Ciprofloxacin resistant bacteria in raw wastewater: 765.000 CFU/ml.