Effects of Organic Chemicals in Sludge Applied to Soil

4. Results and discussion

4.1 Characterization of sludge samples
4.2 Biodegradation of sludge-bound contaminants in soil
4.3 Biodegradation of sludge-bound contaminants in sludge slurries
4.4 Effects of biodegradation on toxicity of sludge samples
4.5 Effects of sludge-bound LAS and NP
4.6 Assessment of the effects of selected sludge contaminants in soil

4.1 Characterization of sludge samples

The chemical analyses of the fresh sludge samples from Herning and Lundtofte (ds) showed that LAS, DEHP and nonylphenolic compounds (NP + NPEs) were present at high concentrations. The levels of these contaminants generally exceeded the cut-off values defined by the Ministry of Environment and Energy (1996). The two sludge samples differed in their contents of contaminants as the Herning sludge contained the highest level of nonylphenolic compounds while the Lundtofte (ds) sludge had the highest concentrations of LAS and PAHs (Table 4.1).

Table 4.1
Occurrence of selected organic contaminants in fresh sludge samples
Forekomst af udvalgte, organiske, miljøfarlige stoffer i friske slamprøver

1) Nonylphenols and nonylphenol ethoxylates with 1 and 2 ethoxylate units
2) Above the prescribed cut-off value in the year 2000 (i.e. 50 mg/kg)
3) Above the current cut-off value (i.e. 50 mg/kg)
4) Sum of PAH above the current cut-off value (i.e. 6 mg/kg)
5) Above the prescribed cut-off value in the year 2000 (i.e. 1,300 mg/kg)
6) Above the current cut-off value (i.e. 2,600 mg/kg)
7) BDL, below detection limit

4.2 Biodegradation of sludge-bound contaminants in soil

The main part of the selected contaminants were generally slowly degraded at 15° C in soil. The results in Table 4.2 show that DEHP decreased to about 80% of its initial concentration when the sludge sample from Herning was used while a reduction to 91% was seen for the Lundtofte (ds) sludge although the reduction was insignificant. The concentration of NP + NPEs was constant during the experimental period of 28 days. However, the analyses of nonylphenolic compounds did not include NPEs with more than two ethoxylate groups or nonylphenoxy carboxylates that are typical products from aerobic biodegradation of NPEs. It is therefore possible that some mineralization of NP and NPEs (with 1-2 ethoxylate units) is concealed by the transformation of long-chained to short-chained NPEs. The concentrations of LAS were reduced to 47% and 48% of the initial values during 28 days. A few other contaminants, e.g. di-n-octylphthalate and fluorene, were apparently also degraded but the initial concentrations of these compounds were low and close to the analytical detection limits.

Table 4.2
Degradation of sludge-bound contaminants in Jyndevad soil. Concentrations are expressed in µg/kg soil (dry weight)
Nedbrydning af slambundne, miljøfarlige stoffer i Jyndevadjord. Koncentrationerne er opgivet i µg/kg jord (tørvægt)

1) Standard deviation of three replicates
2) Nonylphenols and nonylphenol ethoxylates with 1 and 2 ethoxylate units

The biodegradation of LAS observed in this study is in agreement with the results of previous studies of the fate of LAS in sludge amended soil (Table 4.3). An estimation of the mean half-life (T_½) for degradation of LAS in the soil system assuming first order kinetics gives a T_1/2 value of 26 days which is similar to the T_1/2 obtained in a study of Berna et al. (1989). The few available biodegradation data for DEHP in soil indicate that the compound may be degraded in sludge amended soil although the level of degradation differs between individual experiments. The extensive mineralization of DEHP observed by Fairbanks (1985) contrasts with the poor removal of the phthalate in the present study (Table 4.3). In a recent study, ¹⁴C-labelled pyrene was spiked to sludge, and the mineralization was followed in mixtures of sludge and soil (soil:sludge ratio, approx. 50:1). Compared to the degradation rates that may be seen in PAH contaminated soil (Madsen and Kristensen 1997), the mineralization of pyrene in the sludge-soil mixtures was relatively slow although up to 30% of the compound was mineralized during approx. 110 days at 15°C [C. Klinge and B. Gejlsbjerg, unpublished results].

Table 4.3
Degradation of LAS, DEHP and pyrene in sludge-amended soil
Nedbrydning af LAS, DEHP og pyren i jord tilført slam

4.3 Biodegradation of sludge-bound contaminants in sludge slurries

Partial biodegradation was also seen for the contaminants in the sludge slurries. In this experiment, no significant loss was observed for DEHP while the concentration of NP + NPEs decreased to 70-80% of the initial level. There was a remarkable degradation of LAS with 85-97% reduction of the initial level during the 14-day experiment (Table 4.4). The higher concentrations of contaminants in the slurries permit the identification of a number of other chemicals which were at least partially degraded in one or both of the sludge slurries: Diethylphthalate (only Herning sludge); butylbenzylphthalate and di-n-octylphthalate (only Lundtofte (ds) sludge); di-n-butylphthalate (Herning and Lundtofte (ds) sludges); naphthalene and phenanthrene (Herning and Lundtofte (ds) sludges); and fluorene, anthracene, and pyrene (only Lundtofte (ds) sludge). The concentrations of dissolved oxygen in the test vessels were measured five times a week and were adjusted to a level of approx. 20-30 mg O₂/l. The conditions for biodegradation in the sludge slurries may have been more favourable compared with the conditions in the soil. First of all, the main biomass in the slurry consisted of sludge bacteria that had previously been exposed to the contaminants. Secondly, the slurries were stirred during the incubation, which implies that the desorption of the sludge-bound chemicals was more efficient than that under normal conditions in soil.

Table 4.4a
Degradation of sludge-bound contaminants in slurries prepared of Herning sludge (20 g sludge dry weight per litre)
Nedbrydning af slambundne, miljøfarlige stoffer i suspensioner fremstillet af Herning slam (20 g slam tørvægt pr. liter)

1) Nonylphenols and nonylphenol ethoxylates with 1 and 2 ethoxylate units

Table 4.4b
Degradation of sludge-bound contaminants in slurries prepared of Lundtofte (ds) sludge (80 g sludge dry weight per litre)
Nedbrydning af slambundne, miljøfarlige stoffer i suspensioner fremstillet af Lundtofte (ds) slam (80 g slam tørvægt pr. liter)

1) Nonylphenols and nonylphenol ethoxylates with 1 and 2 ethoxylate units

4.4 Effects of biodegradation on toxicity of sludge samples

The toxicity tests with fresh sludge samples indicated that the sludge sample from Herning was more inhibitory to nitrification in soil than was sludge from Lundtofte (ds) (Table 4.5). However, the sludge from Lundtofte (ds) was selected for post-degradation toxicity test because of the higher concentration of sludge dry weight, which could be used in the sludge slurries (Tables 4.4a and 4.4b). Aerobic incubation of slurries of the sludges for 14 days and, hence, biodegradation of some of the organic contaminants reduced the toxicity of the sample from Lundtofte (ds) to soil nitrifying bacteria. This sample was, however, still toxic to the reproduction of F. candida after 14 days of aerobic incubation (Table 4.6). The initial toxicity of the sludge slurry to nitrification was higher than the toxicity of the fresh sludge sample (Tables 4.5 and 4.6). This may be explained by additional effects of the mineral salts that were added to the slurry.

Table 4.5
Toxicity of contaminated sludge samples
Forurenede slamprøvers toksicitet

1) The nitrification tests were performed with aqueous extracts (sludge:water, 1:10, w/v) of the sludge samples. Effect concentrations are expressed as ml extract per kg dry weight.

Table 4.6
Effects of biodegradation on toxicity of the sludge sample from Lundtofte (ds). The sludge sample was incubated in slurries that were stabilized under aerobic conditions for 14 days.
Effekten af bionedbrydning på toksiciteten af slamprøven fra Lundtofte (ds). Slamprøven blev inkuberet i suspensioner, som blev stabiliseret under aerobe forhold i 14 dage.

1) The nitrification tests were performed with the aqueous phase of the slurry (80 g dry weight per litre). The solids were removed by centrifugation at 2,000 rpm for 5 min. Effect concentrations are expressed as ml extract per kg dry weight.
2) Effect concentrations are expressed as g dry weight per kg dry weight

4.5 Effects of sludge-bound LAS and NP

The stabilization procedure, which was performed prior to the addition of LAS or NP to the sludge, resulted in an efficient removal of organic contaminants. Table 4.7 shows that a significant decrease of PAHs, NP + NPEs, DEHP and LAS was achieved during aerobic stabilization of a sample from Lundtofte (as) for 14 days. The reduction of the concentrations of especially NP + NPEs and DEHP was extensive compared to the results that were obtained in the experiments with the sludge slurries (Table 4.4). This may be explained by the fact that the sample from Lundtofte (as) consisted entirely of activated sludge and, therefore, contained a microbial community, which may have been well adapted to the aerobic degradation of the specific contaminants.

Table 4.7
Degradation of sludge-bound contaminants during 14 days of aerobic stabilization of sludge sample from Lundtofte (as)
Nedbrydning af slambundne, miljøfarlige stoffer ved 14 dages aerob stabilisering af slamprøve fra Lundtofte (as)

The concentrations of a number of contaminants decreased during stabilization of the sludge samples that were used for examining the toxicity of spiked LAS or NP (Table 4.7). The chemical analyses performed in order to verify the concentrations of added LAS and NP showed that the freshly spiked sludge samples contained: 0, 1.5, 2.3, 4.3, 9.2 and 19 g per kg sludge (dry weight) for LAS and 0, 0.11, 1.0, 2.1, 5.3 and 10 g per kg sludge (dry weight) for NP. The soil nitrification tests with sludge samples that were freshly spiked with LAS or NP showed no additional effects of these chemicals compared to the controls (Figures 4.1 and 4.2). This was also the case for the toxicity of the spiked sludge samples to the survival of adult F. candida. There were no significant effects on the survival of adult F. candida in the examined concentration range, which implies LC50 values >60 mg/kg (dry weight) for LAS and >30 mg/kg (dry weight) for NP. However, the reproduction of F. candida was severely affected (Table 4.8). The results with NP are in agreement with LC50/EC50 values from other tests with either F. candida or Folsomia fimetaria. However, the LC50/EC50 values obtained with LAS in the present study are much lower than the effect concentrations that were previously reported for collembolans (Table 4.8). These differences may be due to a different sensitivity of F. candida and F. fimetaria, to the methods used for the addition of LAS or to the characteristics of the soils.

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Figure 4.1
Inhibition of nitrification in soil after exposure to aqueous extracts of sludge samples spiked with LAS
Nitrifikationshæmning i jord efter eksponering med vandige ekstrakter af slamprøver, som forinden var tilsat LAS

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Figure 4.2
Inhibition of nitrification in soil after exposure to aqueous extracts of sludge samples spiked with nonylphenol
Niktrifkationshæming i jord efter eksponering med vandige ekstrakter af slamprøver, som forinden var tilsat nonylphenol

Table 4.8
Effects of LAS and nonylphenol to the collembolans F. candida and F. fimetaria. The chemicals were added to either sludge or soil.
Effekter af LAS og nonylphenol på springhalerne F. candida og F. fimetaria. Kemikalierne var blevet tilsat enten til slam eller jord.

4.6 Assessment of the effects of selected sludge contaminants in soil

The biodegradation experiment, which was performed with sludge-amended Jyndevad soil, showed that DEHP and PAHs of medium to high molecular weight, i.e. phenanthrene, pyrene, and benzo(b,j,k)fluoranthene, were poorly degraded or recalcitrant during aerobic incubation for 28 days (Table 4.2). In the experiments with sludge slurries a more extensive degradation was seen for some of the phthalates and PAHs as e.g. the concentration of phenanthrene decreased from 3.1 to 0.2 mg/kg during 14 days. No significant removal of DEHP was observed in the experiments with sludge slurries (Table 4.4b). On the basis of the results obtained in the present study, it is reasonable to anticipate that some phthalates and PAHs with more than two fused aromatic rings may persist for long periods of time in sludge-treated soils. The possible adverse effects of these contaminants to soil-living organisms and the culturing of edible plants were not examined but these issues have been given priority in the Danish Environmental Research Programme (Centre for Sustainable Land Use and Management of Contaminants, Carbon and Nitrogen).

The nonylphenolic compounds, i.e. NP and NPEs with 1-2 ethoxylate units, were also recalcitrant in the sludge-amended Jyndevad soil (Table 4.2) although it is possible that removal of these compounds was balanced by the transformation of long-chained NPEs to short-chained NPEs and NP. The concentrations of NP + NPEs decreased to 70-80% of the initial levels during the 14-day experiment with the sludge slurries (Tables 4.4a and 4.4b). However, this decrease may be due to the formation of degradation products (e.g. nonylphenoxy carboxylates) and only to a minor extent a result of mineralization. The toxicity tests with spiked sludge showed that the lowest EC50 for NP was 16 mg/kg dry weight (Table 4.8). This value may be used for estimating a Predicted No Effect Concentration (PNEC) for NP in soil The PNEC is usually related to the estimated exposure concentration, the Predicted Environmental Concentration (PEC). If the PEC is higher than the PNEC (PEC/PNEC>1) after the application of the sludge on soil, there may be a risk of negative effects on soil-living organisms. For NP + NPEs in soil, a PNEC may be estimated by applying an assessment factor of 40 in relation to the lowest EC50 value for NP (16 mg/kg), which was obtained for the reproduction of F. candida (Table 4.8). Although NPEs are normally less toxic than NP, it is considered to be acceptable to use the EC50 for NP because NPEs are potential precursors for the formation of NP in soil. The assessment factor of 40 is applied here in order to establish PNECs that are comparable to those that were previously estimated for wastewater sludge (Tørsløv et al. 1997). The assessment factor depends on the amount and quality of the available data (Pedersen et al. 1994). Because of the two endpoints that were included in the present study (soil nitrification and reproduction of F. candida), it seems reasonable to apply an assessment factor of 40 for the estimation of PNEC. PECs for NP in soil were estimated by assuming an application of either Herning or Lundtofte (ds) sludge with the levels of contaminants indicated in Table 4.1. It was assumed that NP + NPEs were brought into the soil by a single addition of 6 tons of sludge (dry weight) per hectare. This corresponds to a concentration in the upper 10 cm of the soil of 4 g sludge (dry weight) per kg soil (dry weight) (Tørsløv et. al. 1997). The Danish regulation of the use of sludge on agricultural soil (Ministry of Environment and Energy 1996) prescribes that 6 tons of sludge per hectare is the maximum amount which can be used over a period of three years.

On the basis of the above assumptions, a PNEC for NP + NPEs may be estimated to 0.53 mg/kg (dry weight) while the PECs for the application of Herning and Lundtofte (ds) sludges may be estimated to 1.5 and 0.6 mg NP + NPEs per kg soil (dry weight), respectively. In both cases, the risk quotient PEC/PNEC >1 indicates that negative effects on the soil-living organisms may follow the application of sludge containing high levels of NP + NPEs. The biodegradation experiments indicate that NP + NPEs are slowly degraded under normal conditions in soil.

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Figure 4.3
The development in PEC/PNEC ratio for LAS after application of Herning sludge or Lundtofte (ds) sludge on soil
Udvikling i forholdet mellem PEC og PNEC for LAS efter tilsætning af Herning slam eller Lundtofte (ds) slam til jorden

LAS were rapidly degraded in the experiment with sludge-amended Jyndevad soil (Table 4.2), and an even more efficient degradation of LAS was observed in the sludge slurries (Table 4.4a and 4.4b). These results were expected as LAS are readily biodegradable in OECD tests. A PNEC for sludge-bound LAS and PECs related to the application of Herning and Lundtofte (ds) sludges were estimated as described for NP + NPEs. The PNEC for LAS in sludge-treated soil was estimated to 0.27 mg of LAS per kg soil (dry weight) by using the EC50 (8 mg/kg) obtained for the reproduction of F. candida (Table 4.8). The PECs were estimated to 9.2 mg LAS per kg soil (dry weight) for the Herning sludge and 22 mg LAS per kg soil (dry weight) for the Lundtofte (ds) sludge. The PEC for LAS is expected to decrease because of the rapid degradation of LAS in soil. As the half-life for LAS may be estimated to 26 days from the biodegradation experiment (Tables 4.2 and 4.3), the PECs for LAS may be expressed as a function of time. The PECs were estimated from the time of application (t=0) and until a year after the sludge application (t=365 d) by using a half-life (T_1/2) of 26 d, which corresponds to a first order-rate constant k_bio of 0.027 d^-1. The ratios of PEC/PNEC were calculated from the first day of sludge application until day 365. The development of PEC/PNEC over time (Figure 4.3) indicates that sludge-bound LAS at the levels identified in Table 4.1 may lead to negative effects on soil-living organisms immediately after the application of sludge, where PEC/PNEC>>1. However, the PEC/PNEC ratios are expected to decrease to a level below 1 after 141 days (Herning sludge) and 174 days (Lundtofte (ds) sludge). The calculations that are illustrated in Figure 4.3 indicate that the toxic effects of LAS will not persist in sludge-amended soil, and that such effects will probably be reduced to non-detectable levels before the soil receives the next application of sludge.