HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 50 pp. 7–10 (2022) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2022-02 A SURVEY OF SARS-COV-2 GENETIC MATERIAL REDUCTION DURING A TRADITIONAL WASTEWATER TREATMENT TECHNOLOGY ORSOLYA ADAMCSIK*1 , RENÁTA GERENCSÉR-BERTA1 , BORBÁLA OLÁHNÉ HORVÁTH1 , NIKOLETTA KOVÁCS1 , VIOLA SOMOGYI2 , ENDRE GÁBOR DOMOKOS2 , GÁBOR KEMENESI3 , ENDRE GÁBOR TÓTH3 , FERENC JAKAB3 , AND ILDIKÓ GALAMBOS1 1Soós Ernő Water Technology Research and Development Center, University of Pannonia, Zrínyi Miklós u. 18, Nagykanizsa, 8800 HUNGARY 2Research Centre for Biochemical, Environmental and Chemical Engineering, University of Pannonia, Egyetem u. 10, Veszprém, 8200 HUNGARY 3National Virology Laboratory, Szentágothai János Research Centre, University of Pécs, Ifjúság u. 20, Pécs, 7624 HUNGARY The transmission of Severe Acute Respiratory Syndrome Coronavirus-2 in a community can be monitored by a wastewater-based epidemiological approach due to fecal shedding. Although sewage surveillance has gained a con- siderable amount of attention over the last 16 months, an indirect issue within the topic is whether traditional wastewater treatment technologies are sufficiently efficient to eliminate the genetic material of SARS-CoV-2. Samples were taken from the Wastewater Treatment Plant in Nagykanizsa before the virus was concentrated, nucleic acid extracted and SARS-CoV-2 detected by RT-qPCR (Quantitative reverse transcription PCR). The influent and primary treated samples tested positive, while after the secondary treatment, all the results were negative. Consequently, the activated sludge process proved to be efficient in terms of the removal of SARS-CoV-2. Keywords: warning system, COVID-19, wastewater-based epidemiology 1. Introduction During the transmission of severe acute respiratory syn- drome coronavirus 2 (SARS-CoV-2) since the start of the COVID-19 pandemic, wastewater-based epidemiol- ogy (WBE) has gained a considerable amount of atten- tion as a surveillance system. Numerous research groups and health authorities are currently engaged in sewage monitoring worldwide. Apart from viruses, various com- pounds can be monitored, e.g. pharmaceuticals or illicit drugs, to indirectly monitor the behavior or habits of a defined population [1]. Furthermore, the approach allows anonymous, area-based monitoring in a community that is cost-, labor- and time-efficient compared to human na- sopharyngeal swab test campaigns [2]. Moreover, fecal shedding can occur earlier than the onset of symptoms [3, 4], should any be expressed. While the ratio and role of asymptomatic and mild-symptomatic cases is debat- able [5, 6], the WBE approach estimates the SARS-CoV- 2 RNA concentration of the total community that is inde- Recieved: 26 August 2021; Revised: 20 September 2021; Accepted: 20 September 2021 *Correspondence: adamcsik.orsolya@sooswrc.hu pendent of patients’ perception of well-being. Although WBE has its own limitations, sewage monitoring and hu- man test campaigns can also support national and mu- nicipal decision-making. Considering the successful de- tection of SARS-CoV-2 from wastewater and during the consecutive steps of wastewater treatment, a considerable amount of aerosol formation has been observed. More- over, since droplets are regarded as the most important transmission route of SARS-CoV-2, the working condi- tions at wastewater treatment plants (WWTP) should be addressed in terms of safety. According to the WHO [7], no special care is required other than the usual safety pro- tocols at such plants since SARS-CoV-2 particles present in untreated and treated wastewater have been proven to be non-infectious. Another side interest within this topic is whether the sewage treatment technology is sufficiently effective to eliminate the SARS-CoV-2 virus particles de- tected in the influent to ensure the WWTP effluents are SARS-CoV-2-free, even if their infectivity has already been ruled out. The aim of this work is to assess the presence and concentration of SARS-CoV-2 RNA in un- treated and treated wastewater samples. https://doi.org/10.33927/hjic-2022-02 mailto:adamcsik.orsolya@sooswrc.hu 8 ADAMCSIK, GERENCSÉR-BERTA, OLÁHNÉ HORVÁTH, ET AL. Table 1: SARS-CoV-2 RNA detection from wastewater in- fluent and effluent samples at Nagykanizsa WWTP. Sample type Date Cq value (E-gene) Influent 30/09/2020 +(35.69) Influent 14/10/2020 +(33.88) Influent 09/11/2020 +(32.39) Influent 10/11/2020 +(33.70) Influent 11/11/2020 +(34.27) Influent 18/11/2020 +(36.67) Influent 25/11/2020 +(37.03±0.11) Effluent 25/11/2020 – Influent 02/12/2020 +(33.70±0.50) Effluent 02/12/2020 – 2. Materials and Methods Sampling was conducted by Délzalai Víz és Csatornamű Zrt. at Nagykanizsa WWTP during the morning peak time. From May until December 2020, samples of influ- ent and occasionally effluent as well were taken at var- ious sampling frequencies. While in December, on three consecutive days, in addition to influent and effluent sam- pling points, primary and secondary treated samples were also checked. The pairs of influent and effluent samples were processed in duplicates, while to assess the efficacy of the WWTP, three repetitions were used over three con- secutive days. 1 L grab samples were taken and placed in sterile bottles, transported to our laboratory at 4 ◦C and immediately processed. The virus was concentrated and nucleic acid extracted according to Meleg et al. [8] with minor modifications. Concentrates were stored and transported at −80 ◦C to the National Virology Labora- tory, University of Pécs for specific SARS-COV-2 detec- tion by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique, targeting the E and RdRp genes. Modular SARS-CoV-2 E-gene and Modu- lar SARS-CoV-2 RdRp-gene detection kits (Roche, Ger- many) on the MyGo Pro real-time PCR instrument were employed. Quantification cycle values (Cq values) were used to compare the SARS-CoV-2 RNA concentration of the above-mentioned samples. 3. Results and Discussion Sewage samples tested negative during the summer months of 2020 (data not shown), which is plausible con- sidering the insignificant number of open cases nation- wide [9] and the temperature of the sewage as one of the most important environmental factors [10]. At the end of September, as the second wave progressed, the concen- tration of SARS-CoV-2 RNA increased above the detec- tion limit of the methods in the influent samples, while all effluent samples tested negative. During the autumn of 2020 - although the first positive result was registered, followed by fluctuating quantification cycles - all the re- sults from influent samples (Table 1) were positive, while Table 2: SARS-CoV-2 RNA concentration at different stages of wastewater treatment at the Nagykanizsa WWTP (due to the rules of PCR amplification, higher quantifica- tion cycle values are linked to lower SARS-CoV-2 RNA concentrations) Sample type Date Cq value (E gene) 08/12/2020 +(28.89) Influent 09/12/2020 +(34.06) 10/12/2020 +(31.04) 08/12/2020 +(40.04) Primary treated 09/12/2020 +(35.43) 10/12/2020 +(37.13) 08/12/2020 – Secondary treated 09/12/2020 – 10/12/2020 – 08/12/2020 – Effluent 09/12/2020 – 10/12/2020 – their effluent sample pairs all tested negative without ex- ception. From this information alone, it can be concluded that the commonly used, complex wastewater treatment technology itself is sufficiently efficient to decrease the SARS-CoV-2 RNA load from wastewater without any ad- ditional steps, e.g. UV light treatment or surplus chemi- cals. Our results concerning the influent-effluent pairs are in good agreement with earlier findings by Haramoto et al. [11] By progressing one step further, samples were tested after primary and secondary treatment at the Nagykanizsa WWTP (Table 2). Over three consecutive days - while the influent samples tested positive, although the primary treated wastewater exhibited a slightly lower concentra- tion of SARS-CoV-2 RNA - the results were still posi- tive (manifesting in higher quantification cycles). From this reduction, it can be seen that the majority of the SARS-CoV-2 particles are not attached to the solid par- ticles sedimented in this step. However, the duration of SARS-CoV-2 particles in wastewater is also an important factor [10] that can be partially responsible for the loss in RNA load compared to the raw influent. After the ac- tivated sludge process, the estimated duration of SARS- CoV-2 particles was longer than 8 hours and all samples tested negative. This loss of signal is reasonable consid- ering the lengthy nature of the secondary treatment, the microbial activity together with the limited resistance of these viral particles to external agents [12] as well as the extreme sensitivity of the RNA. As in the case of the ear- lier tested influent-effluent pairs, all treated effluent sam- ples yielded negative results. Over the last year, numerous other research groups have tested various WWTPs with partially different technological setups [11, 13–16] to that employed at Nagykanizsa WWTP. A general consensus has been reached in all these experiments regarding the negative results of all the effluent material flow (treated Hungarian Journal of Industry and Chemistry A SURVEY OF SARS-COV-2 GENETIC MATERIAL REDUCTION 9 effluent water and sludge line). Even though some studies yielded positive results for some of the secondary treated effluents [11,13] or secondary sludges [16], in these cases all the treated materials exiting the WWTP produced neg- ative results without exception. 4. Conclusion According to our findings, which are in good agreement with other published research on this topic, the com- mon treatment of wastewater is capable of decreasing the SARS-CoV-2 RNA concentration below the complex detection limit of the multi-step measurement process. WWTPs are capable of minimizing the presence of coro- navirus nucleic acid without having to add further steps to the traditional technology. 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