Long-Term Monitoring of Microplastics in a German Municipal Wastewater Treatment Plant

Citation: Sturm, M.T.; Myers, E.; Korzin, A.; Schober, D.; Schuhen, K. Long-Term Monitoring of Microplastics in a German Municipal Wastewater Treatment Plant. Microplastics 2024,3, 492–502. https://doi.org/10.3390/ microplastics3030031 Academic Editor: Nicolas Kalogerakis Received: 14 June 2024 Revised: 15 July 2024 Accepted: 14 August 2024 Published: 19 August 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Long-Term Monitoring of Microplastics in a German Municipal Wastewater Treatment Plant Michael Toni Sturm, Erika Myers , Anika Korzin, Dennis Schober and Katrin Schuhen * Wasser 3.0 gGmbH, Neufeldstr. 17a-19a, 76187 Karlsruhe, Germany *Correspondence: [email protected]; Tel.: +49-721-1565-9593 Abstract: Wastewater treatment plants (WWTPs) have been identified as important point sources for microplastics (MPs) in the environment; monitoring MP emissions in the WWTP effluent is therefore essential for contamination control. The aim of this study is to acquire a large number of samples (320) over a period of two years and three months to determine the temporal variations in microplastic contamination in the outlet of the municipal WWTP Landau-Mörlheim. The effluent of the third cleaning stage is sampled with a 10 µ m filter cartridge, processed in the laboratory using a hydrogen peroxide treatment, and MPs are then detected by fluorescence staining. The results show high temporal variations in the microplastic concentrations in the effluent of the WWTP. This indicates that high numbers of samples are necessary to obtain a representative assessment of the microplastic emissions; single samples are not representative. The average microplastic concentration in the effluent was 27.8 ± 29.8 MP/L, ranging from 0.6 MP/L to 194.0 MP/L. This leads to a yearly emission of 1.5 × 10 11 MP for the WWTP Landau-Mörlheim, corresponding to an emission of 2.8 ×106MP/inhabitant and year. Statistically significant seasonal variations could not be observed, although there is a trend towards lower MP concentrations in summer. Further, no correlations with other wastewater or weather parameters could be found. Keywords: microplastics; wastewater treatment; WWTP; microplastic detection; microplastic analysis; microplastic analytics; fluorescent dyes; long-term monitoring 1. Introduction Untreated municipal wastewater is a major source of water pollution; it must be treated efficiently to prevent the release of bacteria, viruses, nitrogen, phosphorus, and other pollutants. Therefore, most modern wastewater treatment plants (WWTPs) are equipped with three treatment stages to respond to current and future pollution and adhere to regulations, such as the EU Water Framework Directive (Directive 2000/60/EC) and EU Urban Wastewater Treatment Directive (Council Directive 91/271/EEC). However, the release of MP (MP) has thus far received little attention in policy and regulations for wastewater treatment, although it has been strongly discussed in MP research since 2011 [1,2]. New scientific findings have identified WWTPs as important point sources for microplastics (MP) in the environment [ 3 , 4 ]. Depending on their design and operating conditions, WWTPs can remove between 64% and >99% of MP from wastewater [ 5 , 6 ]. Nevertheless, due to the very high loads of untreated wastewater in combination with very high wastewater volumes, significant amounts of MP are still discharged into the environment. Fourth (advanced) cleaning stages are designed to target the removal of dissolved micropollutants, as in most cases MP removal is insufficient [6–8]. The EU Urban Wastewater Directive (Council Directive 91/271/EEC) is decisive for the monitoring and prevention of pollution caused by wastewaters and thus requires that EU countries have adequate collection and treatment of wastewater. Recently, MP was included as a parameter to be monitored in the revision of the EU Urban Wastewater Microplastics 2024,3, 492–502. https://doi.org/10.3390/microplastics3030031 https://www.mdpi.com/journal/microplastics Microplastics 2024,3493 Directive [ 9 ]. WWTPs with more than 100,000 population equivalents (PE) must therefore take samples to analyze MP levels at least twice a year, with a maximum interval of 6 months between sampling, while plants with more than 10,000 PE must take samples only once every 2 years. WWTPs (>100,000 PE) must also monitor the presence of MP in sewage sludge. A methodology for the associated monitoring and detection must be defined 30 months after the directive comes into force. As soon as this has been adopted by the EU Parliament, it must be transposed into national law. Microplastic monitoring consists of sampling, sample preparation, and the detection method [ 10 , 11 ]. For each of these steps, no standardized methods are available. The used methodology varies among studies, making it difficult to compare results. Further, microplastic detection is often time-consuming and costly due to complex sampling collection processes and sample preparation along with expensive detection methods with long processing times, resulting in low sample throughputs. The fluorescence staining based method applied in this study presents a fast, comparable, and easy to apply alternative requiring only a fluorescent imaging tool [12–14]. Due to the high effort connected to microplastics monitoring, temporal and seasonal variations in microplastics in wastewater treatment plants are rarely investigated and not yet understood [ 15 – 17 ]. Further, studies investigating microplastics on wastewaters often rely on a small number of samples and it is unclear how temporal variations can affect or distort the outcomes [ 18 ]. A deeper understanding of fluctuations is needed to identify appropriate sampling intervals leading to representative results. This study presents the long-term microplastic monitoring at the municipal WWTP Landau-Mörlheim. In total, 320 samples were taken over a period of 2 years and 3 months. The results of this monitoring provide information on the temporal variation in the microplastic contamination and the range in which the contamination is present. Understanding temporal variations in MP concentrations can help to determine which sampling frequencies provide an adequate representation of MP loads in the WWTP effluent. Further, seasonal variations in MP levels and potential correlations with weather data and additional wastewater parameters are investigated. The study also investigates the capability of the applied method based on fluorescence staining for standardized and comparable MP detection. 2. Materials and Methods 2.1. Sample Site—WWTP Landau-Mörlheim The WWTP Landau-Mörlheim, Germany (coordinates: 49.20774, 8.175369) is a municipal WWTP with a capacity of 80,000 population equivalents. It runs three treatment stages: (1) A primary treatment using rakes, a sand trap, and a fat separator, (2) the secondary biological treatment, and (3) the tertiary phosphate elimination. The WWTP receives wastewater from households, industry, and agriculture, mainly viticulture. 2.2. Sampling The sampling is carried out in the WWTP effluent of the 3rd cleaning stage following Sturm et al., 2023 [ 12 ]. The Wasser 3.0 Particle Sampling Unit (PSU) (Figure 1) is used for this purpose. Samples were taken in duplicate at random times and on random days of the week, with the goal to have 1–2 sampling days per week. Limitations were set by the opening hours of the WWTP, which is why samples could only be taken during working days between 7 am and 4 pm. The PSU contains a 10 µ m filter cartridge, which was installed in combination with a pump, hoses, valves, and a water meter to form a compact sampling unit [ 12 ]. The pump takes in water from the WWTP effluent and then pumps it through the filter cartridge under high pressure (max 4 bar). The filter retains particles and MPs larger than 10 µ m, while the water and smaller particles are discharged. The materials used are listed Table 1. Microplastics 2024,3494 Microplastics 2024, 3, FOR PEER REVIEW 3 Figure 1. Wasser 3.0 Particle Sampling Unit (PSU). The PSU contains a 10 µm filter cartridge, which was installed in combination with a pump, hoses, valves, and a water meter to form a compact sampling unit [12]. The pump takes in water from the WWTP effluent and then pumps it through the filter cartridge under high pressure (max 4 bar). The filter retains particles and MPs larger than 10 µm, while the water and smaller particles are discharged. The materials used are listed Table 1. Table 1. Materials used for microplastic sample collection along with the Wasser 3.0 PSU. Part Model No. Supplier Rotary Pump MG80B C-BCMS1B, Grundfos, Erkrath, Germany Filter cartridge (10 µm) 01WTGD Wolftechnik Filtersysteme GmbH & Co., KG, Weil, Germany Water meter Zenner ETKD ZENNER International GmbH & Co. KG, Saarbrücken, Germany Tubing TUFLONPTFE/NW25 Industriebedarf Castan GmbH, Freiberg, Germany The PSU is connected to an inlet hose (black polyvinylchloride) with a suction basket, which is immersed in the middle of the WWTP effluent. The outlet hose is fed into the WWTP effluent behind the inlet hose in the direction of flow. Before each sampling is taken, the PSU is backwashed with tap water. In total, 100 L of treated wastewater are filtered, as 100 L sample volumes can capture the typical ranges of contamination in wastewater treatment plants [19]. After the filtration, the filter cartridge is removed from the housing and the retained solids are rinsed into a 2.5 L glass vessel using a spray bottle, in which the sample is transported to the laboratory for further processing and analysis. Due to its simple assembly and transportability, this method has a high potential as a standard in microplastic detection. 2.3. Sample Processing The sample needs to be processed using a hydrogen peroxide treatment and stained using fluorescent markers specifically designed to bind to and detect MP (Figure 2) [19]. The sample contains all the solids contained in the WWTP effluent, including sewage sludge residues, natural particles such as animal and plant remains or algae, and the MP. Figure 1. Wasser 3.0 Particle Sampling Unit (PSU). Table 1. Materials used for microplastic sample collection along with the Wasser 3.0 PSU. Part Model No. Supplier Rotary Pump MG80B C-B-CMS1B, Grundfos, Erkrath, Germany Filter cartridge (10 µm) 01WTGD Wolftechnik Filtersysteme GmbH & Co. KG, Weil, Germany Water meter Zenner ETKD ZENNER International GmbH & Co. KG, Saarbrücken, Germany Tubing TUFLON-PTFE/NW25 Industriebedarf Castan GmbH, Freiberg, Germany The PSU is connected to an inlet hose (black polyvinylchloride) with a suction basket, which is immersed in the middle of the WWTP effluent. The outlet hose is fed into the WWTP effluent behind the inlet hose in the direction of flow. Before each sampling is taken, the PSU is backwashed with tap water. In total, 100 L of treated wastewater are filtered, as 100 L sample volumes can capture the typical ranges of contamination in wastewater treatment plants [ 19 ]. After the filtration, the filter cartridge is removed from the housing and the retained solids are rinsed into a 2.5 L glass vessel using a spray bottle, in which the sample is transported to the laboratory for further processing and analysis. Due to its simple assembly and transportability, this method has a high potential as a standard in microplastic detection. 2.3. Sample Processing The sample needs to be processed using a hydrogen peroxide treatment and stained using fluorescent markers specifically designed to bind to and detect MP (Figure 2) [ 19 ]. The sample contains all the solids contained in the WWTP effluent, including sewage sludge residues, natural particles such as animal and plant remains or algae, and the MP. During the first step of the sample processing, the number of natural particles is reduced by chemical decomposition. Here, a hydrogen peroxide treatment is chosen as numerous scientific studies have shown that it is effective against natural particles and particularly gentle on MP [20,21]. Microplastics 2024,3495 Microplastics 2024, 3, FOR PEER REVIEW 4 During the first step of the sample processing, the number of natural particles is reduced by chemical decomposition. Here, a hydrogen peroxide treatment is chosen as numerous scientific studies have shown that it is effective against natural particles and particularly gentle on MP [20,21]. Figure 2. Schematic procedure of sample preparation. The sample is filtered through a 10 µm stainless steel sieve and 20 mL hydrogen peroxide and 3–4 grains of iron sulfate are added to the retained solids according to the parameters in Table 2 [12,19]. The processed sample is then filtered again to remove the degraded organic residues. The filtered sample is rinsed from the filter surface with demineralized water into a 100 mL beaker and filled to 100 mL with demineralized water. Table 2. Details for the hydrogen peroxide treatment of the MP samples. Date Procedure Source January 2022– May 2023 • Heat up to boiling temperature for 1 min. • Reduce temperature to 80 °C and stir for 4 h. • Cool down to room temperature (RT) and stir for 20 h. [19] June 2023– March 2024 • Heat to 100 °C for 1 h. • Cool down to room temperature (RT) for 15 min. [12] The used fluorescence marker was specifically designed to stain only MPs and is the result of several years of research. The stained MPs therefore show a strong fluorescence signal under the fluorescence microscope and can be detected (Figure 3) whereas natural particles show no or a very weak fluorescence signal, which means they are not recorded in the measurements [12,19,22]. Figure 2. Schematic procedure of sample preparation. The sample is filtered through a 10 µ m stainless steel sieve and 20 mL hydrogen peroxide and 3–4 grains of iron sulfate are added to the retained solids according to the parameters in Table 2[ 12 , 19 ]. The processed sample is then filtered again to remove the degraded organic residues. The filtered sample is rinsed from the filter surface with demineralized water into a 100 mL beaker and filled to 100 mL with demineralized water. Table 2. Details for the hydrogen peroxide treatment of the MP samples. Date Procedure Source January 2022–May 2023 •Heat up to boiling temperature for 1 min. •Reduce temperature to 80 ◦C and stir for 4 h. • Cool down to room temperature (RT) and stir for 20 h. [19] June 2023–March 2024 •Heat to 100 ◦C for 1 h. •Cool down to room temperature (RT) for 15 min. [12] The used fluorescence marker was specifically designed to stain only MPs and is the result of several years of research. The stained MPs therefore show a strong fluorescence signal under the fluorescence microscope and can be detected (Figure 3) whereas natural particles show no or a very weak fluorescence signal, which means they are not recorded in the measurements [12,19,22]. Microplastics 2024, 3, FOR PEER REVIEW 5 Figure 3. Monthly means of MP contamination measured in the effluent of the WWTP Landau. The staining procedure was optimized throughout the study. The dates of applications and the staining procedures are listed in Table 3. The fluorescent dyes (abcr eco Wasser 3.0 detect mix MP-1, AB930015, and Nile red, AB139346) were purchased from abcr GmbH, Karlsruhe, Germany. After staining for 15 min, the sample was filtered through a black filter membrane and stored in a Petri dish until measured using a fluorescence microscope. Table 3. Details for the fluorescent staining of the microplastic samples. Date Dye Temperature Time Source 01/2022– 12/2022 Nile red 5 °C 24 h [19] 01/2023– 05/2023 abcr eco Wasser 3.0 detect mix MP-1 5 °C 24 h [19] 06/2023– 03/2024 abcr eco Wasser 3.0 detect mix MP-1 80 °C 1 h [8] 2.4. Detection Fluorescent imaging was performed according to Sturm et al., 2023 [19]. A Leica DMS300 (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) was modified for fluorescent imaging. LED flashlights and bandpass filters were used for excitation. Further, a bandpass filter was mounted in front of the lens of the microscope as an emission filter. The images were taken with the following fluorescent parameters (Table 4): Table 4. Details for the fluorescent imaging and particle counting of the MP samples. Date Excitation Emission Counting Source 01/2022– 12/2022 UV light (395 nm) Yellow color foil Manual [19] 01/2023– 03/2024 Blue (430–480 nm) Green (500–570 nm) Automated [12] For image analysis, the particle counting module for the software LAS-X 3.0.1423224 was used. Until 12/2022, particles were counted manually; all samples afterwards were Figure 3. Monthly means of MP contamination measured in the effluent of the WWTP Landau. Microplastics 2024,3496 The staining procedure was optimized throughout the study. The dates of applications and the staining procedures are listed in Table 3. The fluorescent dyes (abcr eco Wasser 3.0 detect mix MP-1, AB930015, and Nile red, AB139346) were purchased from abcr GmbH, Karlsruhe, Germany. After staining for 15 min, the sample was filtered through a black filter membrane and stored in a Petri dish until measured using a fluorescence microscope. Table 3. Details for the fluorescent staining of the microplastic samples. Date Dye Temperature Time Source January 2022–December 2022 Nile red 5 ◦C 24 h [19] January 2023–May 2023 abcr eco Wasser 3.0 detect mix MP-1 5 ◦C 24 h [19] June 2023–March 2024 abcr eco Wasser 3.0 detect mix MP-1 80 ◦C 1 h [8] 2.4. Detection Fluorescent imaging was performed according to Sturm et al., 2023 [ 19 ]. A Leica DMS300 (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) was modified for fluorescent imaging. LED flashlights and bandpass filters were used for excitation. Further, a bandpass filter was mounted in front of the lens of the microscope as an emission filter. The images were taken with the following fluorescent parameters (Table 4): Table 4. Details for the fluorescent imaging and particle counting of the MP samples. Date Excitation Emission Counting Source January 2022–December 2022 UV light (395 nm) Yellow color foil Manual [19] January 2023–March 2024 Blue (430–480 nm) Green (500–570 nm) Automated [12] For image analysis, the particle counting module for the software LAS-X 3.0.1423224 was used. Until 12/2022, particles were counted manually; all samples afterwards were counted automatically. The results are extrapolated to the total surface area of the filter and the sampled amount of wastewater. 2.5. Contamination Control For contamination control, the laboratory was cleaned before each use with a lint-free cleaning rag and lint-free clothing was worn. An air-filter was operated continuously, and glass and plastic laboratory equipment was used whenever possible. Further, the samples were always covered with aluminum foil. To monitor and control any potential sample contamination, regular blanks were measured and subtracted from the measured microplastic concentrations. 2.6. Statistical Analysis For statistical analysis of differences between seasons, a two-way ANOVA followed by a Tukey’s HSD test (p-value 5%) was performed. For a comparison of two groups, a t-test (p-value 5%) was performed. The homogeneity of variance and normality was checked using a residuals versus fits plot and QQ-plot. To evaluate the correlation between weather—and water parameters with MP concentration, a linear regression was performed, and the Pearson correlation coefficient (r) and coefficient of determination (R2) were calculated. 3. Results and Discussion 3.1. Long-Term Data on Microplastic Contamination The monthly means (Figure 3) vary between 107 MP/L in February 2022 and 6.1 ±3.5 MP/L in September 2023. Relatively high standard deviations can be observed, which are caused by fluctuations in the microplastic concentrations. The overall mean of all measurements is 27.8 ±29.8 MP/L. Microplastics 2024,3497 The histogram (Figure 4) shows that 81.7% of the measured MP concentrations are between 0 and 40 MP/L, while 18.3% are above this concentration. Further, the histogram shows that there is a right skewed distribution. The highest microplastic concentration measured in a single sample was 194.0 MP/L on 22 December 2022 and the lowest concentration measured was 0.6 MP/L on 5 September 2023. Samples with high MP concentrations are also visible as outliers in the boxplot. Also, it shows significantly lower MP concentrations in 2023 (19.7 ± 17.9 MP/L) compared to 2022 (33.0 ±33.6 MP/L ) (t-test, t(86) = 2.8, p= 0.006 ). It is notable that the method was adjusted between 2022 and 2023, resulting in a higher sensitivity to MP in 2023 [ 19 ]. Despite the ability to detect more MPs, a lower MP discharge in 2023 was observed and is therefore attributed to lower MP inputs into the sewage treatment plant. Most likely, this is caused increased awareness of the environmental problems associated with MPs, resulting in less MP emissions by industries and private persons, regulations on MPs in products, or a change in the industries present and contributing to the influent of the WWTP. Microplastics 2024, 3, FOR PEER REVIEW 7 Figure 4. Histogram of all MP data and boxplots of all data, data from 2022 and 2023. 3.2. Seasonal Variations To investigate the seasonal variations, the data were grouped by meteorological seasons (Figure 5). Despite the high number of samples, the non-normal distribution of the data and the inhomogeneity variances do not allow the application of statistical comparison by ANOVA, which is why no statistically significant difference could be found. For a statistically reliable conclusion, even higher numbers of samples would have been needed to compensate for the high number of outliers attributed to samples with high microplastic concentrations. Nevertheless, trends can be seen in the boxplots. In all data, the 2022 and 2023 summers show the lowest median concentrations followed by autumn, which shows higher fluctuations in the measured concentrations and the most outliers. Other studies also found lower microplastic pollution in the summer months, due to better microbial-driven coagulation and settling processes during the wastewater treatment. In the winter of 2022, a higher microplastic concentration was observed, which was not reproduced in 2023. In both years, spring had a microplastic contamination similar to winter as well as high fluctuations in the MP contamination levels in spring. Other studies that investigated the seasonal variations in MP in wastewaters came to different results. While some studies could not find any seasonal variations, other studies found higher or lower MP concentrations in summer [15,16,25–27]. This shows that there is not yet a clear understanding of what causes the seasonal variations in MP in wastewaters; more reliable and comparable data are needed. Figure 4. Histogram of all MP data and boxplots of all data, data from 2022 and 2023. The high temporal variations in the monthly means, the high fluctuations in measured concentrations shown in the high standard deviations, and the high abundance of outliers in the form of highly polluted samples highlight the necessity of regular monitoring to achieve representative results. Single samples are not suited to make a representative statement on the MP emissions at WWTPs, as they are unlikely to capture the temporal fluctuations. Therefore, a high number of samples is required. Comparing this to other studies, the concentrations found are within the typical ranges of MPs detected in WWTP effluents, although the varying methodologies make it different to accurately compare results [ 3 , 17 , 23 ]. Other studies investigating MPs in WWTP effluents also capture the high fluctuations and broad range of microplastic concentrations found in the treated wastewater [23,24]. 3.2. Seasonal Variations To investigate the seasonal variations, the data were grouped by meteorological seasons (Figure 5). Despite the high number of samples, the non-normal distribution of the data and the inhomogeneity variances do not allow the application of statistical comparison by ANOVA, which is why no statistically significant difference could be found. For a statistically reliable conclusion, even higher numbers of samples would have been Microplastics 2024,3498 needed to compensate for the high number of outliers attributed to samples with high microplastic concentrations. Microplastics 2024, 3, FOR PEER REVIEW 8 Figure 5. Seasonal variations in the MP contamination in the effluent of the WWTP Landau. 3.3. Correlation Analysis To check for possible correlations with weather conditions and wastewater parameters, a correlation analysis was performed (Figure S1). As wastewater parameters of the effluent water temperature, flow rate, COD, and turbidity were chosen. The weather parameters were air temperature and precipitation. Correlation coefficients and coefficients of determination (Table 5) show that there is no correlation between the microplastic contamination and the listed weather or wastewater parameters. This demonstrates that the microplastic contamination is independent from these parameters and needs to be measured separately. Table 5. Correlation coefficients (r) and coefficient of determination (R2) for MP and weather or WWTP effluent parameters. Parameter r R 2 Water temperature −0.20 0.04 Flow rate WWTP 0.14 0.02 COD effluent 0.08 0.01 Turbidity effluent 0.11 0.01 Precipitation −0.01 0.00 Air temperature −0.21 0.04 Other studies could show correlations between TSS removal and MP and nano-plastics removal, driven by the hetero-aggregation of MP and organic matter [15,28,29]. In addition, it was found that higher water temperatures lead to better flock formation and TSS settling in WWTPs, which also improves hetero-aggregation with MPs and their removal [15]. This correlation could not yet be confirmed. Studies investigating MP in road-runoff during rain events identified it as a source of microplastics into WWTPs, if combined sewer systems for surface-runoff and wastewater are used [30,31]. The MPs mainly originate from tire abrasion and plastic litter [32,33]. The missing correlation with precipitation or flow rate could not confirm higher MP emission caused by road-runoff, which might be due to lower MP concentrations in road-runoff than in the untreated municipal wastewater [34]. One limitation of the fluorescence method is that it cannot detect tire abrasion because of its black color, which is why it is not possible to make a statement about the entry or emission of tire abrasion during rain events [12]. The absence of correlations implies the MP contamination is driven by parameters which were not investigated in this study. Those could for example be industrial activities, Figure 5. Seasonal variations in the MP contamination in the effluent of the WWTP Landau. Nevertheless, trends can be seen in the boxplots. In all data, the 2022 and 2023 summers show the lowest median concentrations followed by autumn, which shows higher fluctuations in the measured concentrations and the most outliers. Other studies also found lower microplastic pollution in the summer months, due to better microbialdriven coagulation and settling processes during the wastewater treatment. In the winter of 2022, a higher microplastic concentration was observed, which was not reproduced in 2023. In both years, spring had a microplastic contamination similar to winter as well as high fluctuations in the MP contamination levels in spring. Other studies that investigated the seasonal variations in MP in wastewaters came to different results. While some studies could not find any seasonal variations, other studies found higher or lower MP concentrations in summer [ 15 , 16 , 25 – 27 ]. This shows that there is not yet a clear understanding of what causes the seasonal variations in MP in wastewaters; more reliable and comparable data are needed. 3.3. Correlation Analysis To check for possible correlations with weather conditions and wastewater parameters, a correlation analysis was performed (Figure S1). As wastewater parameters of the effluent water temperature, flow rate, COD, and turbidity were chosen. The weather parameters were air temperature and precipitation. Correlation coefficients and coefficients of determination (Table 5) show that there is no correlation between the microplastic contamination and the listed weather or wastewater parameters. This demonstrates that the microplastic contamination is independent from these parameters and needs to be measured separately. Table 5. Correlation coefficients (r) and coefficient of determination (R 2 ) for MP and weather or WWTP effluent parameters. Parameter r R2 Water temperature −0.20 0.04 Flow rate WWTP 0.14 0.02 COD effluent 0.08 0.01 Turbidity effluent 0.11 0.01 Precipitation −0.01 0.00 Air temperature −0.21 0.04 Microplastics 2024,3499 Other studies could show correlations between TSS removal and MP and nano-plastics removal, driven by the hetero-aggregation of MP and organic matter [ 15 , 28 , 29 ]. In addition, it was found that higher water temperatures lead to better flock formation and TSS settling in WWTPs, which also improves hetero-aggregation with MPs and their removal [ 15 ]. This correlation could not yet be confirmed. Studies investigating MP in road-runoff during rain events identified it as a source of microplastics into WWTPs, if combined sewer systems for surface-runoff and wastewater are used [ 30 , 31 ]. The MPs mainly originate from tire abrasion and plastic litter [ 32 , 33 ]. The missing correlation with precipitation or flow rate could not confirm higher MP emission caused by road-runoff, which might be due to lower MP concentrations in road-runoff than in the untreated municipal wastewater [ 34 ]. One limitation of the fluorescence method is that it cannot detect tire abrasion because of its black color, which is why it is not possible to make a statement about the entry or emission of tire abrasion during rain events [12]. The absence of correlations implies the MP contamination is driven by parameters which were not investigated in this study. Those could for example be industrial activities, which can lead to high microplastics emissions and can be irregular and not connected to any of the investigated parameters [ 35 ]. To gain a better understanding of MP inputs and being able to apply impact full measures, further investigations of sources and MP inputs into WWTPs are needed. 3.4. Data Extrapolation to Yearly Discharge The yearly discharge is extrapolated by multiplying the average microplastic concentration with the yearly discharge (Table 6). The yearly discharge accounted for 5.8 million m3/year in 2022 and 5.0 million m 3 /year in 2023. As the catchment area has 55,000 inhabitants, this results in an average discharge of 420 million MP/day and 150 billion MP/year into the receiving river. This amounts to 7614 MP/inhabitant per day and 2.8 million MP/inhabitant per year. This interpolation does not consider that the MPs do not only originate from households, but also from industries and surface-runoff (Figure 6). Nevertheless, it makes it clear that measures are necessary to prevent MP pollution [36]. Table 6. Extrapolated microplastic discharge of the WWTP Landau-Mörlheim. Discharged MP [MP] Discharged Water [m3] Per L 27.8 Per day 4.2 ×10814,947 Per year 1.5 ×1011 5.4 ×106 Per inhabitant and day 7614 0.23 Per inhabitant and year 2.8 ×10699.2 Microplastics 2024, 3, FOR PEER REVIEW 9 which can lead to high microplastics emissions and can be irregular and not connected to any of the investigated parameters [35]. To gain a better understanding of MP inputs and being able to apply impact full measures, further investigations of sources and MP inputs into WWTPs are needed. 3.4. Data Extrapolation to Yearly Discharge The yearly discharge is extrapolated by multiplying the average microplastic concentration with the yearly discharge (Table 6). The yearly discharge accounted for 5.8 million m³/year in 2022 and 5.0 million m3/year in 2023. As the catchment area has 55,000 inhabitants, this results in an average discharge of 420 million MP/day and 150 billion MP/year into the receiving river. This amounts to 7614 MP/inhabitant per day and 2.8 million MP/inhabitant per year. This interpolation does not consider that the MPs do not only originate from households, but also from industries and surface-runoff (Figure 6). Nevertheless, it makes it clear that measures are necessary to prevent MP pollution [36]. Figure 6. Sources of MP input into municipal WWTPs [36]. Current scientific studies discuss various methods to remove MPs from WWTP effluents to avoid the discharge into the environment. Also, measures are discussed to stop the MP at its sources before it enters the wastewater streams, such as the treatment of industrial wastewaters, road-runoff, or washing machine effluents [37–39]. Table 6. Extrapolated microplastic discharge of the WWTP Landau-Mörlheim. Discharged MP [MP] Discharged Water [m 3 ] Per L 27.8 Per day 4.2 × 10 8 14,947 Per year 1.5 × 10 11 5.4 × 10 6 Per inhabitant and day 7,614 0.23 Per inhabitant and year 2.8 × 10 6 99.2 4. Conclusions 1. The presented data show that there are high fluctuations in the microplastic concentrations in the effluent of the WWTP. To capture these fluctuations and obtain a representative evaluation of the microplastic contamination, a high number of samples is necessary. Single samples are not representative. The minimum MP sampling interval to capture the yearly emissions is recommended to be between two to four samplings per month. Based on the results, longer sampling intervals do not provide meaningful results; thus the sampling period outlined in the revised EU Urban Wastewater Treatment Directive will not adequately capture the temporal variations in MP contamination levels Figure 6. Sources of MP input into municipal WWTPs [36]. Microplastics 2024,3500 Current scientific studies discuss various methods to remove MPs from WWTP effluents to avoid the discharge into the environment. Also, measures are discussed to stop the MP at its sources before it enters the wastewater streams, such as the treatment of industrial wastewaters, road-runoff, or washing machine effluents [37–39]. 4. Conclusions 1. The presented data show that there are high fluctuations in the microplastic concentrations in the effluent of the WWTP. To capture these fluctuations and obtain a representative evaluation of the microplastic contamination, a high number of samples is necessary. Single samples are not representative. The minimum MP sampling interval to capture the yearly emissions is recommended to be between two to four samplings per month. Based on the results, longer sampling intervals do not provide meaningful results; thus the sampling period outlined in the revised EU Urban Wastewater Treatment Directive will not adequately capture the temporal variations in MP contamination levels and may be misrepresentative of actual pollution loads. To capture both seasonal and monthly variations, higher numbers of samples are needed. 2. The average microplastic concentration was 27.8 ± 29.8 MP/L, ranging from 0.6 MP/L to 194 MP/L. In 2023, a lower MP contamination of 19.7 ± 17.9 MP/L was detected than in 2022 with 33.0 ± 33.6. This may be caused by increased awareness of the problems associated with MP in the environment resulting in reduced emission by industries and households, regulations on MP in products, or a change in the industries present and contributing to the influent of the WWTP. 3. Clear seasonal variations could not be statistically proven, but there is a trend towards lower MP concentrations and lower fluctuations of the concentrations in summer, which was visible in the data. 4. The correlation analysis showed that MPs are not correlated with the investigated wastewater and weather parameters. It should therefore be measured separately as the contamination appears to be driven by other unrelated factors. 5. Further, the data show that WWTPs are clear point sources for MP into the environment and appropriate measures should be taken to prevent this contamination. Advanced treatment stages targeting MP removal at both upstream sources and at WWTPs should be investigated. Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microplastics3030031/s1, Figure S1: Correlation plots for MP concentrations with weather or WWTP effluent parameters. Author Contributions: Conceptualization, M.T.S., E.M., D.S. and K.S.; methodology, M.T.S., D.S. and K.S.; validation, M.T.S. and K.S.; formal analysis, M.T.S., E.M. and K.S.; investigation, M.T.S., A.K. and D.S.; resources, K.S.; data curation, M.T.S. and K.S.; writing—original draft preparation, M.T.S., E.M. and K.S.; writing—review and editing, M.T.S., E.M. and K.S.; visualization, M.T.S.; supervision, K.S.; project administration, K.S.; funding acquisition, K.S. All authors have read and agreed to the published version of the manuscript. Funding: This project has received co-funding from the European Union’s Horizon Europe innovation program under grant agreements 101093964 and 101112877. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use that may be made of the information contained therein. The project has also received part of its funding from the Veolia Foundation (Germany)|Project konti|detect and by Vector Stiftung/Germany (project REC-MP, identification number: P2021-0092). Data Availability Statement: Data are contained within the article and Supplementary Materials. Acknowledgments: The authors thank abcr GmbH, Karlsruhe, Germany, and Entsorgungs und Wirtschaftsbetriebe Landau (EWL, Germany) for the project-related support. The authors thank Oleg Zernikel, Sabrina Polierer, Martin Tashi, Karolin Widera, and Sophia Olbrich for their contribution to the microplastics sampling, sample preparation and detection.