Effects of Organic Chemicals in Sludge Applied to Soil

3. Materials and methods

3.1 Sludge and soil samples
3.2 Biodegradation experiments
3.3 Ecotoxicity experiments
3.4 Chemical analyses

3.1 Sludge and soil samples

Sludge samples
Three types of sludge samples were collected at two Danish WWTPs. One sludge sample contained both activated and anaerobically digested sludge and was obtained from Herning wastewater treatment plant, which is located in mid-Jutland, Denmark. Two other sludge samples were obtained from Lundtofte wastewater treatment plant in the northern part of Zealand, Denmark. One of these samples, which was designated Lundtofte (ds), consisted entirely of anaerobically digested sludge, while the other sample, designated Lundtofte (as), consisted entirely of activated sludge. The sludge samples were designated by the numbers 1 (Herning), 7 (Lundtofte (ds)) and 8 (Lundtofte (as)) in the previous monitoring study (Tørsløv et al. 1997). Selected data for the WWTPs are shown in Table 3.1.

Soil samples
Two agricultural soil samples were used for the experiments addressing the biodegradation and the toxicity of sludge-bound contaminants. A loamy sand (Jyndevad soil) was collected in Jyndevad, Denmark while a sandy loam (Flakkebjerg soil) was collected in Flakkebjerg, Denmark. The soils were sieved (2 mm) and were generally stored in darkness at 4-5° C until use. The Jyndevad soil, which was used for the test with springtails, was defaunated by incubation of the soil at 60° C for 24 hours followed by freezing at -70° C for 48 hours. Flakkebjerg soil samples used for soil nitrification inhibition tests were stored at -20° C.

1) Person equivalent
2) Chemical oxygen demand

3.2 Biodegradation experiments

Biodegradation of sludge-bound contaminants in soil
The biodegradation of selected contaminants in soil-sludge mixtures (soil:sludge ratio, approx. 70:1 w/w) was examined in a laboratory batch experiment. The experimental setup consisted of three replicates for each of the sludge samples from Herning and Lundtofte WWTPs. A quantity of 500 g of Jyndevad soil (dry weight) and 7.5 g sludge (dry weight) from either Herning or Lundtofte (ds) were added to 1-L glass beakers and were thoroughly mixed. Deionized water was added to achieve 40-60% of the soil water-holding-capacity and this moisture level was maintained by five adjustments a week. The depth of the soil layer in the glass beakers was approx. 2.5 cm. The beakers were covered with parafilm and were aerated by a continuous stream of atmospheric air from two glass pipettes. Incubation occurred in the dark at 15° C. Subsamples of 250 g (dry weight) were removed from each of the glass beakers at the start of the experiment (day 0) and after 28 days for extraction and chemical analyses.

Biodegradation of sludge-bound contaminants in sludge slurries
The biodegradation of the sludge contaminants was examined in a test system that permits a higher level of sludge solids and thus a higher concentration of the specific compounds. The experiment was performed with the sludges from Herning and Lundtofte (ds) and included three replicates for each sludge sample. The experimental system consisted of closed 3-L glass beakers, each equipped with a teflon coated stirrer and an inlet for supply of pure oxygen. The sludges were suspended in an extract of the Jyndevad soil which was prepared as previously described (Balkwill and Ghiorse 1985). The suspensions were supplied with mineral salts by addition of 100 ml/L from a 10-fold concentrated mineral medium (OECD 1993). The final volume of suspension in the beakers was 2.5 L, and the concentrations of sludge were 20 g of solids (dry weight) per litre for the Herning sample and 80 g of solids (dry weight) per litre for the sample Lundtofte (ds). The experiment was started by connecting the oxygen supply and setting the stirrers at approx. 150 rpm. The glass beakers were placed in the dark at 15° C. The concentration of dissolved oxygen was measured five times a week by using an oxygen electrode. In order to reduce the costs of analyses, the removal of the organic contaminants in the sludge was determined by chemical analyses of a composite sample of equal aliquots collected from each replicate at day 0 and after 14 days of incubation.

3.3 Ecotoxicity experiments

Two ecotoxicity tests were used for examining the toxicity of sludge samples and of specific contaminants that were added to sludge. A soil nitrification test was used for characterizing the toxicity of fresh samples from Herning and Lundtofte (ds). The nitrification test and a reproduction test with the springtail F. candida were used for determining the effects of aerobic incubation on the toxicity of Lundtofte (ds). For this purpose, equal aliquots were withdrawn from each of the replicate glass beakers in the sludge slurry biodegradation assay at the start of the incubation and after 14 days. The aliquots were mixed and the composite sample was used in the ecotoxicity tests. From the organic chemicals, for which cut-off values have been defined in wastewater sludge (Ministry of Environment and Energy 1996), LAS and 4-nonylphenol (NP) were selected for experiments aiming at determining the adverse effects of specific sludge-bound contaminants on soil nitrification and reproduction of F. candida. Details about the toxicity tests are given below.

Soil nitrification
The effects of the sludges on the nitrification in soil was examined by using the Flakkebjerg soil as the source of nitrifying bacteria. The method used was modified from the Swedish MATS guidelines (Torstensson 1994) and has been described by Winther-Nielsen et al. (1998). The nitrification was determined in a slurry of soil and a medium containing (NH₄)₂SO₄, PO₄^3-, and HCO₃^-. NaClO₃ was added in order to inhibit the oxidation of nitrite to nitrate. The toxicity of fresh sludge samples was examined by the addition of different amounts of an aqueous extract of the sludge (sludge(dry weight):deionized water, 1:10). The effects of specific chemicals were examined by spiking sludge subsamples with different concentrations of either LAS or NP (see preparation of chemical-sludge-complexes below). The subsamples were extracted with deionized water (sludge:water, 1:10 w/v), and a constant amount of sludge extract was applied in the nitrification test. The effects on nitrification were also examined by using the slurries from the biodegradation experiment prior to and after 14 days of aerobic incubation. The testing of pre- and post-degradation sludge slurries was performed with the supernatants after removal of the solids by centrifugation at 2,000 rpm for 5 min. Each concentration of the sludge extracts was tested in at least two replicates. The nitrification was measured as the rate of nitrite formation by oxidation of ammonium over a 6-hour period.

Springtail reproduction bioassay
The toxicity of the sludges to the collembolan F. candida was examined in a 28-day reproduction test (ISO 1996). The sludge slurries were mixed with defaunated Jyndevad soil, which resulted in a series of concentrations corresponding to 8, 12, 16, 20 and 24 g of sludge slurry (dry weight) per kg of soil (dry weight). Juvenile springtails were exposed to the sludge slurries during their development into adults and the initial reproduction period. Ten juvenile F. candida were placed in each of four replicate vials containing 30 g of the sludge mixture (wet weight). Six replicate vials were included as controls receiving only defaunated soil. Toxic effects were determined by enumeration of the number of offspring and survival of adults after 28 days. Freshly spiked sludge samples with different concentrations of either LAS or NP were mixed with soil in order to achieve a sludge concentration of 3 g of sludge (dry weight) per kg of total dry weight.

Preparation of chemical-sludge-complexes
First, the sludge samples used for the ecotoxicity experiments with specific chemicals were pre-incubated in order to reduce the level of organic contaminants. Approx. 30 g of sludge Lundtofte (as) (15% dry weight) was distributed in a layer of 2-3 cm in five trays lined with aluminium foil. The sludge was covered with parafilm and aerated by a continuous stream of atmospheric air. The sludge was left for stabilization at 20° C to allow degradation of organic contaminants. The duration of the stabilization was at least one week. The water content was monitored three times a week and was regulated by addition of deionized water in order to maintain a constant moisture content. Subsamples of the sludge was withdrawn before and after the stabilization for chemical analysis of selected organic contaminants. The stabilized sludge was thoroughly mixed before it received the additions of either LAS or NP.

LAS and NP were added to the stabilized sludge at five concentrations for each compound. Aqueous solutions containing the different concentrations of LAS were added to 2-L glass bottles with 200 g (dry weight) of stabilized Lundtofte (as) sludge. Deionized water was added to a total volume of 2,000 ml. The mixture was tumbled at 10° C for 24 hours. The sorption process was terminated by removal of the liquid phase after centrifugation at 1,270 g. The six 200-g subsamples received 0, 2.5, 4.1, 8.5, 16.7 and 33.3 g of LAS per kg of stabilized sludge (dry weight). Serial dilutions of 4-nonylphenol (NP) were made in acetone. Six 200-g subsamples (dry weight) of stabilized Lundtofte (as) sludge were weighed out. About 50 g (dry weight) of each subsample was removed and allowed to dry for 2-3 days. The dried sludge samples were pulverized and 50 ml of the serial dilutions of NP was added to separate samples. The solvent was allowed to evaporate at room temperature for 1-2 hours. Then the NP containing 50-g subsamples were mixed with the remaining wet subsample (150 g dry weight). From this step, the sorption process was continued by addition of deionized water and tumbling as described for LAS. The six 200-g subsamples received 0, 0.08, 0.8, 2.0, 4.1 and 8.1 g of NP per kg of stabilized sludge (dry weight). The concentrations of LAS and NP in the samples were verified by chemical analyses of one subsample from each of the twelve glass bottles. The sludge samples were frozen until use for the toxicity test. The purpose of the sorption procedure for the addition of LAS and NP to the sludge was to obtain a bioavailability of the chemicals that approached the bioavailability of original sludge contaminants.

Chemicals
The LAS used was Marlon A 390 (90% surfactant), which was purchased from Hüls Chemicals, Germany. The 4-nonylphenol was Pestanal (90% 4-nonylphenol) and was purchased from Riedel de Häen, Germany.

3.4 Chemical analyses

Subsamples for chemical analyses were extracted in dichloromethane, and the extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). Other subsamples were extracted with methanol and analyzed by HPLC for LAS (Tørsløv et al. 1997).