December 13, 2006

Phytotoxicity of Biosolids Compost at Different Degrees of Maturity Compared to Biosolids and Animal Manures

By Zubillaga, Marta Susana; Lavado, Ral Silvio

Biosolids compost is a good organic amendment but immature compost can exhibit phytotoxic behavior which can be attributed to different toxic substances. Our objective was to determine the phytotoxicity of: i) Biosolids; ii) Mix of biosolids and wood sawdust sampled a day after composting started; iii) The same material sampled at the end of the thermophilic stage; iv) cured compost; v) cow manure and vi) horse manure. A germination bioassay was carried out using Lolium perenne (ryegrass) seeds: germination and root growth percentage were determined as well as electrical conductivity, pH, phenol content and volatile organic acids. In three treatments, Ni, Pb, Zn, Cu and Cd were also determined. Ammonia volatilization was determined during biosolids composting. The germination percentage varied from 67% to 95% but the inhibition of root growth appears to be a more sensitive phytotoxicity indicator (18% to 74%. Phytotoxic effects on germinating ryegrass were mainly related to extract pH and electrical conductivity. Potentially toxic elements, volatile organic acids, phenolic compounds and ammonia were not related to germination.


Organic residues are a valuable source of organic matter, nitrogen, phosphorus and other nutrients. Therefore, their usage in agriculture is an efficient way for them to be utilized. Biosolids (anaerobically digested sewage sludge) are organic residues with some limitations that include: potentially toxic elements and organic chemical contents, pathogenic organism occurrence and so on (Smith 1996). Composting them does not change the potentially toxic elements composition substantially but it reduces pathogenic organism populations. Thus, a product free from sanitary restrictions is obtained (Sweeten 1998). During the compost process, several transformation and degradation reactions occur in organic compounds, including contaminants. Such reactions change contaminants to less toxic substances (Hartlieb et al. 2003).

However, immature or nonstabilized compost can show phytotoxic behaviour and, therefore affect crops. This is due to the occurrence of toxic substances because of an insufficient biodegradation of organic compounds (Brodie et al. 1994, He et al. 1995, Keeling et al. 1994). Several authors have mentioned unknown substances but others have attributed phytotoxicity to short chain fatty acids (Helfrich et al. 1998). Phenolic compounds which are used in different industries are found in biosolids, and they are also toxic and recalcitrat (Polymenakou and Stephanou 2005). The occurrence of such substances has led to several investigations to establish parameters with the aim of evaluating stability and maturity of compost before its use (Wu and Ma 2001). Thus, compost maturity, to detect potential harmful effects on plants is currently determined by different seed or plant tests (Zucconi et al. 1981, Iannotti et al. 1994, Warman 1999).

Biosolids are also rich in proteins and nitrogen compounds of low molecular weight, such as urea and uric acid. That is why large quantities of ammonia are released during composting which can bring about ammonia volatilization losses whose phytotoxic effects are widely known (Britto and Kronzucker 2002). Our objective was to determine the phytotoxicity of biosolids compost at different moments of the composting process and its causes. A comparison with other organic amendments has been included.

Materials and Methods


There were six organic materials investigated in this study: i) Biosolids, provided by the city water operator (Aguas Argentinas S:A.); ii) Composting biosolids, sampled a day after composting started (Compost I); iii) The same material sampled at the end of the thermophilic stage, 10 days after the process started (Compost II); iv) compost sampled 90 days after composting started (Compost III) v) Air dried cow manure, (Cow) and vi) air dried horse manure (Horse). Both manures were taken from pasture fed livestock.

The compost production was performed in a pile. Biosolids were air-dried and thoroughly mixed with pine sawdust in a 1:1 volume proportions The pile was removed fornightly and periodically watered. Temperature and water content were recorded weekly.

Germination Test

A germination bioassay based on Zucconi et al., (1981) was carried out. Water extracts of the six above mentioned materials were prepared by adding 5 g dried sieved material to 50 ml distilled water. The extract was shaken and remained at 60C for 60 hours and then it was filtered. Forty Lolium perenne (ryegrass) seeds (Iannotti et al. 1994) were placed in 8 cm diameter petri dishes lined with filter paper containing 6 ml of the six obtained water extracts, or 6 ml of deionized water used as a control. The Petri dishes were incubated in the dark at 26C. At 72 hours all the treatments germinated. The exception were the seeds in biosolids and horse treatments which geminated between 72 and 120 hours. The fact that there was no additional germination in the first four treatments, allowed all data to be evaluated statistically.

Seeds were considered to be germinated when the primary root reached 5 mm long (USEPA 1982) and its length was measured. Germination (G%= G% in each extract/G% in control x 100) and root growth percentage (RG%= mean root length in each extract/mean root length in control x 100) was determined. The germination index (GI) was calculated by multiplying G% and RG% (Zucconi et al. 1981). Each treatment was replicated four times.

Chemical Determinations

pH and electrical conductivity, and phenol content were determined by means of the Prussian blue technique (Das 1994) in 1:10 water extracts. Volatile organic acids (VOA) were determined in 1:50 water extracts by titration with NaOH 0.1N. In the Biosolids, Compost III and Horse treatments Ni, Pb, Zn, Cu and Cd were extracted by acids (McGrath and Cunliffe 1985) and determined using inductively coupled argon plasma emission spectrometry (ICPES).

Ammonia volatilization was determined throughout 70 days, during biosolids composting. An adaptation of the Nommik method was used (Zubillaga et al. 2005) by using cylindrical chambers (0.15 m diameter and 0.20 m height) located on the compost and biosolids surface. Evolved ammonia was trapped in polyurethane discs soaked in 1M sulfuric acid-glycerol solution 2:1 (v/v). Discs and the inner surface of the chambers were rinsed with 2 M KCl, and the extract was removed. Ammonia concentration was determined by using airstream distillation (Page et al. 1982).

Statistical Determinations

Analysis of variance and the least significant differences (LSD) at p

Results and Discussions

Seed Germination and Root Growth

A germination delay was observed in the treatments with biosoilds and horse manure. This can be attributed to the high salt content in both of them (Table 1), and the widely known effect of salinity on germination (Myers and Couper 1989). Apart from such effect, the germination trial showed significant differences among treatments (Figure 1). The germination percentage varied in a range from 67% to 95%. According to Zucconi et al. (1981) when the germination percentage is higher than 80-85%, phytotoxicity will disappear from compost. Only the treatments which surpassed the thermophilic stage (Compost II and Compost III) exceeded this limit. However, the germination percentage of the three compost treatments (Compost I, II and III) ranging from 78% to 95% did not differ significantly among them (p


Characteristics of the treatment extracts, compared with distilled water

FIGURE 1. Ryegrass germination percentage (G%), root growth percentage (RG%) and germination index (GI) as compared with germination on distilled water. Different letters indicate significant differences between treatments (p

The germination percentage of the Biosolids treatment was low (67%) but it did not differ considerably from the Compost I treatment (p

The inhibition of root growth appears to be a more sensitive phytotoxicity indicator. In our case, the tendency found in germination percentage was maintained but root growth percentage showed more noticeable differences, varying between 18 to 74%. Again, treatments which passed compost thermophilic phase (Compost II and III) showed a significantly longer root length (p

Wu et al. (2000) attributed heavy metals, pH and EC to the causes of phytotoxicity decreases as compost maturations proceed. In our case, potentially toxic element concentrations in Biosolids and Compost III were similar (Table 2) and lower than limits of most common regulations (European Union 1986, USEPA 1993). Additionally, in a previous study with similar biosolids compost (Zubillaga and Lavado 2002), it was observed that potentially toxic element concentration on lettuce leaves was low. The potentially toxic element concentration in Horse treatment was very low as compared with the other two treatments. This behavior precludes potentially toxic element as responsible for the result obtained here. Conversely, there were significant linear regressions among pH and EC of extracts and the three germination indexes studied (Table 3).


Heavy metal content in biosolids, Compost III and horse manure (mg kg MS^sup -1^)


Analysis of regression of germination assay and chemical characteristic of the extract studied

Ammonia Volatilization During Biosolids Composting

Ammonia volatilization daily losses varied from 0.0035 g N-NH m^sup -2^ from the biosolids before composting to 0.0158 g N-NH^sub 4^ m^sup -2^ on Compost at stage III (Figure 2). The phy to toxic effect of ammonia on germinating seeds is known (Britto and Kronzucker 2002) and during compost production ammonia could represent a real danger to seed germination. However, there were not significant relations (p

FIGURE 2. Evolution ammonia released during biosolids compost process. Vertical lines show the standard error.


Phytotoxic effects on germinating ryegrass from compost of different maturity degree were observed. Phytotoxicity was related mainly to extract pH and electrical conductivity. Potentially toxic elements, volatile organic acids, phenolic compounds and ammonia were not related to the germination test.


Britto, D.T. and HJ. Kronzucker. 2002. NH^sub 4^^sup +^ toxicity in higher plants: a critical review. J. Plant Physiol., 159:567- 584.

Brodie, H.L., R.G. Francis and B.C. Lewis. 1994. What makes good compost? BioCycle, 35:66-68.

Das, D.K. 1994. Naturally occurring Flavonoids: structure, chemistry and high-performance liquid chromatography methods for separation and characterization. In: Methods in Enzymology, vol. 234. Academy Press Inc. pp:432-433.

European Union. 1986. Council directive of the European Communities. On the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture. N^sup o^ 86/ 278/EEC.

Hartlieb, N., T. Ertunc, A. Schaeffer and W. Klein. 2003. Mineralization, metabolism and formation of nonextractable residues of ^sup 14^C-labelled organic contaminants during pilot-scale composting of municipal biowaste. Environmental Pollution, 126:83- 91.

He X.T., T.J. Logan and S.J. Traina. 1995. Physical and chemical characteristics of selected U.S. municipal solidwaste composts. J. Environ. Qual., 24:543-552.

Helfrich P., B. Chefetz, Y. Hadar, Y.Chen and H. Schnabl. 1998. A novel method for determining phytotoxicity in composts. Compost Sci. & Util., 3:6-13

Iannotti, D.A., M.E. Grebus, B.L. Toth, L.V.Madden and H.A.J. Hoitink. 1994. Oxygen respirometry to assess stability and maturity of composted municipal solid waste. J. Environ. Qual., 23:1177- 1183.

Keeling, A.A., LK. Paton and J.A.J. Mullett. 1994. Germination and growth of plants in media containing unstable refuse-derived compost. Soil Biol. Biochem., 26:767-772.

McGrath, S.P. and C.H.Cunliffe. 1985. A simplified method for the extraction of the metals Fe, Zn, Cu, Ni, Cd, Pb, Cr, Co and Mn from soils and sewage sludges. J. Sci. Food Agric., 36:794-798.

Myers, B.A. and D.I. Couper. 1989. Effects of temperature and salinity on the germination of Puccinellia ciliata (Bor) cv. Menemen. Australian J. of Agri. Res., 40(3): 561-571.

Page, A.L., R.H. Miller and D.R. Keeney (Ed.). 1982. Methods of Soil Analysis, Part 2, 2nd Ed., Madison, Wiscosin.

Polymenakou, P.N. and E.G. Stephanou. 2005. Effect of temperature and additional carbon sources on phenol degradation by an indigenous soil Pseudomonad. Biodegradation, 16:403-413.

Smith, S.R. 1996. Agricultural recycling of Sewage Sludge and the Environment, CAB International, Wallingford, U.K., p.382.

Sweeten, J.M. 1998. Composting manure sludge. In Nacional Poultry Waste Management Symp., Columbus, OH. Dep. Of Poultry Sci., Ohio State Univ., Columbus, p. 38-44.

USEPA. 1982. Seed germination/root elongation toxicity test. EG- 12, Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, DC.

USEPA. 1993. Standards for the use or disposal of sewage sludge. Fed Regist. 58:9248-9415.

Warman, P.R. 1999. Evaluation of seed germination and growth tests for assessing compost maturity. Compost Sci. & Util., 3:33- 37.

Wu, L., L.Q. Ma and G.A. Martinez. 2000. Comparison of methods for evaluating stability and maturity of biosolids compost. J. Environ. Qual., 29:424-429.

Wu, L. and L.Q. Ma. 2001. Effects of sample storage on biosolids compost stability and maturity evaluation. J. Environ. Qual., 30:222- 228.

Zubillaga, M.S. and R.S. Lavado. 2002. Heavy metal content in lettuce plants grown in compost of biosolids. Compost Sci. & Util., 10:363-367.

Zubillaga, M.S., H. Rimski-Korsakov, G. Traveria and R.S. Lavado. 2005. Ammonia volatilization from different organic amendments during storage and after soil application. Agrochimica, 49:169-174.

Zucconi F, A. Pera, M.E. Forte and M. de Bertoldi. 1981. Evaluating toxicity of immature compost. BioCycle, 22:54-57.

Marta Susana Zubillaga and Ral Silvio Lavado

Ctedra de Fertilidad y Fertilizantes, Facultad de Agronoma, Unwersidad de Buenos Aires, Buenos Aires, Argentina

Copyright J.G. Press Inc. Autumn 2006

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