Removal and Effects of Surfactants in Activated Sludge System

DANIEL MITRU, GHEORGHE NECHIFOR, STEFANIA GHEORGHE, ALINA ROXANA BANCIU, LUCIAN IONESCU, CATALINA STOICA, DANIELA LILIANA IONICA, IRINA EUGENIA LUCACIU* “Politehnica” University of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Analytical Chemistry and Environmental Engineering, no. 1-7, Polizu Str., 011061, Bucharest, Roumania National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podu Dambovitei Str., 060652, Bucharest, Romania

During wastewater treatment, especially in the biological process of activated sludge, surfactants are partially aerobically broken down and partially adsorbed on sewage sludge flocs. It has been reported that high concentrations of anionic and nonionic surfactants have a negative influence on sludge flock properties [17,18] and can lead to microorganism's cell degradation [16,17]. The adsorption of surfactants can modify the characteristics of activated sludge flocs morphology and furthermore, surfactants supplied to the sludge interact with carbohydrates and peptides, which may reduce the degradation pattern of these compounds [17,19,20]. Therefore, the fate of surfactants, and particularly their decomposition in activated sludge processes, plays an important role in minimizing environmental impact.
Due to detergents / cleaning products huge consumptions, as well as their complex mixtures of substances and different formulations, the evaluation of surfactants biodegradability and potential toxicity to ecosystems has become a crucial topic in science and policy, in accordance with European Regulations ( [21]. The main goal of the present study was to assess the removal of both anionic and non-ionic surfactants from the activated sludge`s system, taking into account biodegradability and aquatic toxicity. Laboratory experiment carried out according to the Zahn-Wellens method [22]. The biodegradability level (measured as COD %) and the effect on activated sludge biomass and flocs morphology of a mixture containing anionic and nonionic surfactant at increased concentrations were evaluated. Solutions resulting from biodegradation have been tested to estimate the acute toxicity to planktonic crustaceans (Daphnia magna), because these aquatic organisms are indicated to be most sensitive to the effects generated by the anionic and non-ionic surfactants, first after the luminescent bacterium Vibrio fischeri [23].

Experimental part Materials
Activated sludge samples used in biological experiments were taken from a municipal wastewater treatment plant (WWTP). The samples were collected from aeration basin and the biodegradability tests were performed on the same day of sample collection.
Two stock surfactants solutions in distilled water were prepared: anionic surfactant solution-500 mg ̸ L MDBS and 400 mg/L PGN. Experimental solutions (15-25 mg/L anionic surfactant mixed with 10 mg/L nonionic surfactant) were prepared from the stock solution, using synthetic nutrient medium, specific to the biodegradability method applied to assess the removal and the effect of surfactants on activated sludge microorganisms.

Analytical methods
The anionic surfactant was determined according to the methylene blue active substance-MBAS method (standard SR EN 903:2003), while the nonionic surfactant was analyzed by the method of bismuth active substance-BiAS method (standard SR ISO 7875-2:1996).
In MBAS method, methyl dodecylbezene sulfonate in reaction with methylene blue formed ion pairs which are extracted in chloroform and determined spectrophotometrically at 650 nm [24].
The BiAS procedure consists of gas-stripping of non-ionic surfactants from liquid sample to an ethyl acetate layer, evaporation of ethyl acetate, dissolution of the residue in a water/methanol mixture, precipitation of ethoxylates with Dragendorff reagent, filtration and washing of the precipitate with glacial acetic acid, dissolution of the precipitate in sodium tartrate solution and determination of bismuth UV spectrophotometrically at 263.5 nm [25].
Colorimetric analysis of surfactants was performed using the spectrophotometer Specord BU 205 (Analytic Jena, Germany).

Testing methods
To assess the biodegradability of a mixture of two types of surfactants (MDBS mixed with PGN), a standardized method for the evaluation of ultimate aerobic biodegradability -SR EN ISO 9888:2004 (Zahn Wellens test) was applied [22].
The biodegradability experiment was conducted in batch activated sludge system using 3 bubbling stirred reactors : 2 test reactors contained surfactants solutions: A and respectively, solution B and the control reactor containing similar quantities of nutrient medium and active sludge suspension, without addition of any surfactant.
The biodegradation process is monitored by determination of Chemical Oxygen Demand (COD) in filtered samples, taken at regular time intervals. The percentage of ultimate biodegradation was calculated by measuring the decline in COD concentration of the tested mixtures, corrected for the blank, after each time interval (at least 4 occasions in the interval between the 1st and the last two days of the test run). Chemical oxygen demand (COD) was measured by a standard dichromatic method SR ISO 6060:1996 [26] and mixed liquor suspended solids in bioreactor were determined according to standard method SR EN 872:2005 [27].
Additional information on the primary biodegradability and the total elimination of surfactant content by aerobic microorganisms, during the experimental period (21 days), was obtained.
The effect of mixture containing the both anionic and nonionic surfactants on activated sludge flocs and the quality of sludge biocenosis was also assessed using microscopic analysis of activated sludge collected from the 3 test reactors (A, B and control).
Acute static toxicity test on the liquors deriving from biodegradation test was conducted with planktonic crustacean (Daphnia magna), using DaphToxkitF TM bioassay [28] and the method described in Daphnia sp. Acute Immobilization Test (OECD 202 and SR EN ISO 6341:2013) [29,30]. The biological material used was planktonic crustaceans, Daphnia magna species, provided by MicroBioTests Inc. (Belgium) as immobilized preparation -ephippia (strain batch no. DM131218, expired on 31.07.2019).
Prior to start the toxicity test, Daphnia magna ephippia were incubated for three days at 20-22°C and a light intensity of 6000 lux. Daphnia neonates were exposed to a dilution series of both biodegradation surfactants mixtures (A1, B1: 100%, A2, B2: 50%, A3, B3: 25 %) for a period of 48 hours. In parallel with the experimental test solutions, inhibition tests were also carried out for the control sample (nutrient medium + crustaceans, without surfactants). 20 animals, divided into four groups of five animals each, were used at each test concentration and for the control. Bioassay was performed under static conditions, at 20± 1 0 C, with a daily light: dark period of 12:12 hours and there was no feeding and no aeration during the test. The endpoint of the test was immobilization and / or mortality of crustaceans (determined visually after 24 and 48 hours) and the number of immobile specimen, out of 20, was recorded after 48 hours incubation time and compared with control values. Four replicates of each dilution were tested. The results were analyzed in order to calculate the mobility inhibition of 50% of Daphnia population (effective concentration EC50 at 48 h).
The MBT Daphnia Regetox 2.0 software (Microbiotests Inc., Belgium) was used for experimental data interpretation.

Results and discussions Biodegradation test (Zahn-Wellens method)
Current legislation regulates surfactants (anionic and nonionic) concentrations at the entrance to the urban waste water treatment plants (25 mg/l) and maximum concentrations that can be discharged into the aquatic environment (0.5 mg/l).
Taking into account these legislative requirements, two nutrient solutions with increased content of surfactants (25 mg/L and 35 mg L, respectively) were studied to assess the biodegradation and removal of anionic and non-ionic surfactants by aerobic microorganisms from activated sludge.
The results obtained within surfactants and control biodegradation test (Table 1) show that biodegradability is influenced by the initial concentration of surfactants; the degree of biodegradation (expressed as % COD) is higher (above 90%) when the initial total surfactant concentration is 25 mg/L (15 mg/Lanionic + 10 mg/Lnonionic). When the initial total surfactant concentration is 35 mg/L (25 mg/Lanionic + 10 mg/Lnonionic), the degree of biodegradation achieved (% COD) is lower 80%. The percentage COD biodegradation was plotted against time for both solutions A and B, to give the biodegradation curve (Fig. 1). Biodegradation should be considered to have started when it exceeds the 10% level. A high biodegradation level (>80%) was reached after 10-12 days by mixture A (total initial content of anionic and nonionic surfactants: 25 mg/L), while for mixture B (total initial content of anionic and nonionic surfactants: 35 mg/L), the maximum biodegradability level was 77% after 21 days of experiment. Lag period was less than 3 days for mixture A and about 5-6 days for mixture B and time to reach pass level (70% COD removal) was 14 days for mixture A and 16-18 days for mixture B. At the end of experimental period the degree of biodegradation, calculated as COD removal, was higher than 70% for both solutions A and B.
The percentage of COD removal ≥ 70% means that the substance could be 'ultimately' biodegradable, while the percentage < 70% and ≥20%, the substance is 'inherently' biodegradable. Besides of the 70% pass level, also the criteria for removing surfactant content of at least 80% must to be achieved.
Similar with results obtained for COD elimination, the surfactants removal (Table 2) during experimental period (21 days) was higher for mixture A (86% removal of total content of surfactants) comparative with mixture B, where removal was less than 80%, which represents a minimum level of primary biodegradability for surfactants, according to European Regulation No 648/2004 [31]. The values of COD and surfactants removal obtained within Zahn-Wellens test indicated that biodegradation process occurred at concentration of 25 mg/L anionic surfactant, mixed with 10mg/L non-ionic surfactant and this mixture may be considered readily biodegradable in the aquatic environment. Comparing the COD removal during the biodegradability test versus surfactants concentrations from tested mixtures, it can be appreciated that concentration of anionic surfactant greater than 25 mg/L mixed with non-ionic surfactant greater than 10 mg/L might inhibit the substrate uptake and also, affected the process of biodegradation with activated sludge.
At the end of the biodegradability test, analyzes of surfactants concentrations adsorbed on the activated sludge were performed. Total content of surfactants was less than 1 mg/L (0.36 mg/Lanionic surfactant and 0.58nonionic surfactant) for mixture A, while for mixture B were determined concentrations of 1.77 mg/L anionic surfactant and 0.83 mg/L nonionic surfactant.
The effect of surfactants (MDBS and PGN) on the morphology of activated sludge used as inoculum in the biodegradability test was analyzed via microscopic study.
Microscopic examination of activated sludge collected from experimental mixtures allowed the assessment of activated sludge biocenosis. The microorganisms found were mainly Gram-negative organisms (Klebsiella pneumoniae, Raoultella planticola, Pseudomonas, Vorticella), suggesting that they are more tolerant to the surfactant concentrations present in the solutions than the Gram-positive organisms. It has been reported by Higgins and Burns [32] that many Gram-positive bacteria are noticeably affected by surfactant concentrations of 10-20 ppm while several thousand ppm may be without effect on Gram-negative organisms.
Microscopic examination confirmed changes in the morphology and flocs of activated sludge caused by high loads of surfactants in tested mixture (25 mg/L MDBS and 10 mg/L PGN). The vitality and morphology of protozoa highlighted in the blank control was substantial changed by the presence of surfactants and formation of cysts was observed. High concentration of surfactants exerted obvious effect also, to the quality and quantity of filamentous and non-filamentous bacteria observed in sludge from blank control (Fig. 2). Formation of cysts and lysis of cellular walls, as well as total cell damage observed in microscopic images (Fig.2b, c), particularly for solution B, represent type of a defensive response by protozoa to hazardous environmental conditions, in order to survive. Microscopic observation also showed that activated sludge flocs were disintegrated / fragmented, became smaller and some of cells were damaged, in particular anionic surfactant at concentrations of 25 mg/L in mixture with non-ionic surfactant in conc.de 10 mg/L (Fig. 2c). Surfactant solutions obtained at the end of the biodegradability experiment were subjected to an acute planktonic crustacean toxicity test -Daphnia sp. Acute Immobilization Test.
Results on immobilization and / or death of organisms recorded after 24 h and 48 h for each tested solution constituted the basis for calculating the percentage of inhibition effect (no. of dead bodies + no. of immovable organisms) / 20 x 100 =%.
Results are summarized in Table 3, showing for each test solution and control the immobilization /mortality effect after 24 and 48 hours exposure time. The acute toxic concentration value -effective concentration (EC50-48h) was estimated for biodegradability effluent mixture B, based on the linear regression between the mortality / immobilization percentages and the tested concentrations ( fig. 3). The concentration (EC50-48h) with immobility/mortality effect for 50% of the total number of Daphnia magna organisms used in the acute toxicity experiment was estimated using MBT Daphnia Regetox 2.0 software (Microbiotests Inc., Belgium). The concentration EC50-48h about 42.05% dilution of biodegradability effluent (mixture B) represents a total content of anionic and nonionic surfactants of 3.69 mg/L. According to toxicity classification system of effluents discharged into aquatic environment [33,34], the toxicity of surfactants mixture could be correlated with 25 toxicity units, that suppose an acute toxicity for Daphnia magna (Class III). The final aquatic effect could be magnified or reduce depending on WWTP efficiency, water dilution, and also by biotic (aquatic microbial communities) and abiotic factors (physical and chemical structure of aquatic matrices). Also, other aquatic representative species such as algae or fish can highlight different effects leading to a changed toxicity diagnostic [35].
The mean acute lethal concentration was not estimated for mixture A, because the inhibition / mortality percentage was below 50%. Based on the results obtained, it can be appreciated that the mixture A in 50% dilution (total concentration of surfactants less than or equal to 1.5 mg/L; 0.775 mg/L anionic + 0.725 mg/L nonionic) present no acute toxic effects for planktonic crustacean (Daphnia magna).

Conclusions
Commercial anionic and nonionic surfactants (methyl dodecyl benzene sulfonate -MDBS and 4-nonylphenylpolyethylene glycol -PGN), often used as active substances in detergents and cleaning products were examined in this study to assess their biodegradability in activated sludge`s aerobic system and to investigate their effect on activated sludge biomass and flocs morphology. Also, solutions resulting from biodegradation have been tested to estimate the acute toxicity to planktonic crustaceans (Daphnia magna. The following conclusions can be made on the basis of results obtained within Zahn-Wellens test and ecotoxicity test with Daphnia magna: i) Mixture of MDBS surfactant in concentration up to 15 mg/L with PGN surfactant in concentration up to 10 mg/L can be considered readily biodegradable and may not pose a hazard to the aquatic environment. The COD removal and surfactants degradation exceeded the pass level for ready biodegradability, according to testing method, OECD criteria and European Regulation No 648/2004 on Detergents requirements. The sludge microorganisms adapted to the test substrate and a plateau of more than 80% biodegradation was reached within 21 days of batch experiment; ii) Mixture of MDBS surfactant in concentration of 25 mg/L with PGN surfactant in concentration of 10 mg/L reached the pass level for COD removal (≥70%), but the total surfactant decrease was less than 80%. The increased concentrations of surfactants have been affected biocenosis and active sludge morphology, causing fragmentation of flocs and lysis of protozoa cells; iii) Acute toxicity value estimated to the aquatic organism tested (planktonic crustacean -Daphnia magna) was 3.69 mg/Ltotal content of surfactants (2.25 mg/Lanionic surfactant and 1.44 mg/Lnonionic surfactant. The mixture of anionic and non-ionic surfactants in concentration ≤ 1.5 mg/L (0.775 mg/L anionic + 0.725 mg/L nonionic) will not pose a hazard to the aquatic organisms.
We consider that a more accurate evaluation of the potential impact of anionic and nonionic surfactants mixture on aquatic environment requires a battery of tests to assess total degradation (mineralization), as well as ecotoxicological effects on different aquatic organisms. Our study will continue with new experiments to establish the effect of anionic and nonionic surfactants on activated sludge oxygen uptake rate and their environmental compatibility on the basis of ecological data.