Waste Management 29 (2009) 15041513

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

A statistical analysis to assess the maturity and stability of six composts

Dimitrios P. Komilis *, Ioannis S. Tziouvaras
Laboratory of Solid and Hazardous Waste Management, Department of Environmental Engineering, Democritus University of Thrace, Vas. Soas 12, Xanthi 671 00, Greece

a r t i c l e

i n f o

a b s t r a c t
Despite the long-time application of organic waste derived composts to crops, there is still no universally accepted index to assess compost maturity and stability. The research presented in this article investigated the suitability of seven types of seeds for use in germination bioassays to assess the maturity and phytotoxicity of six composts. The composts used in the study were derived from cow manure, sea weeds, olive pulp, poultry manure and municipal solid waste. The seeds used in the germination bioassays were radish, pepper, spinach, tomato, cress, cucumber and lettuce. Data were analyzed with an analysis of variance at two levels and with pair-wise comparisons. The analysis revealed that composts rendered as phytotoxic to one type of seed could enhance the growth of another type of seed. Therefore, germination indices, which ranged from 0% to 262%, were highly dependent on the type of seed used in the germination bioassay. The poultry manure compost was highly phytotoxic to all seeds. At the 99% condence level, the type of seed and the interaction between the seeds and the composts were found to signicantly affect germination. In addition, the stability of composts was assessed by their microbial respiration, which ranged from approximately 4 to 16 g O2/kg organic matter and from 2.6 to approximately 11 g CO2C/kg C, after seven days. Initial average oxygen uptake rates were all less than approximately 0.35 g O2/kg organic matter/h for all six composts. A high statistically signicant correlation coefcient was calculated between the cumulative carbon dioxide production, over a 7-day period, and the radish seed germination index. It appears that a germination bioassay with radish can be a valid test to assess both compost stability and compost phytotoxicity. 2008 Elsevier Ltd. All rights reserved.

Article history: Accepted 17 October 2008 Available online 30 December 2008

1. Introduction Compost is commonly dened as the aerobically stabilized or matured organic matter, although anaerobic processes can also lead to the production of a stabilized (or matured) organic material. A desired but not sole use of compost is its application to crops with the goal to enhance plant growth. A high quality compost should be both mature and stable. A differentiation between the terms maturity and stability has been discussed in the literature (Iannotti et al., 1993; Epstein, 1997; Wu et al., 2000). Maturity is related to the effect of composts to crops and indicates the presence or absence of phytotoxins. Therefore, immature or phytotoxic composts may have a negative impact on plant growth. On the other hand, compost stability is a stage in the decomposition of the organic matter (Epstein, 1997) and is, therefore, a term related to the microbial decomposition or microbial respiration activity of the composted matter. The above differentiation of terms is adopted in this research work as well. A widely used maturity index is the germination index (GI); it is based on relatively simple to perform seed phytotoxicity tests, which are germination bioassays that quantify seed growth upon
* Corresponding author. Tel./fax: +30 25410 79391. E-mail address: dkomilis@env.duth.gr (D.P. Komilis). 0956-053X/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.wasman.2008.10.016

the application of compost liquid extracts to the seeds. The GI was rst introduced by Zucconi et al. (1981), who had used seeds of cress in their experiments; it is calculated by the root length and the percentage of germination of selected seeds compared to a control, which is usually the deionized water. According to Zucconi et al. (1981), GIs allow evaluation of both low levels of toxicity, which affect root growth, as well as of high levels of toxicity, which affect seed germination. Based on that, it would be reasonable to state that GIs lower than 100% indicate a potential phytotoxicity, whilst values greater than 100% indicate a benecial effect on seed growth, and, therefore, indicate a mature compost. However, a widely accepted threshold germination index, above which maturity can be ascertained, does not appear to exist in the literature. In addition to that, several types of seeds have been occasionally used in compost phytotoxicity studies, with cress being the most common (Haug, 1993). The seed phytotoxicity test, which is an indirect test to quantify phytotoxicity, does not reveal the reason of the composts potential phytotoxicity. Phytotoxicity can be usually due to the presence of ammonia, salts, heavy metals and/or organic acids in the compost extract. Several studies have attempted to explain the reason of the phytotoxicity. For example, Tiquia et al. (1996) used six types of seeds to assess phytotoxicity of pig-manure derived compost. The GIs calculated during the composting process varied signicantly among the types of seeds.

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For example, GIs ranged from 30% to 80% for one type of seed (Chinese cabbage) and from 80% to 120% for tomato seeds. A good correlation between the extractable concentration of copper and total ammonium contents with copper of most of the seeds was observed. A range of cress seed GIs from 30% to 120% was reported in a study that evaluated 28 composts in the Greek market (Lasaridi et al., 2006). The above study mentioned that GI values above 80% can indicate maturity. Another study used a cress seed GI of 50% as the limit for compost maturity (Bernal et al., 1998). However, no justication was given for the use of these threshold values by either of the above authors. Other types of seeds that have been used in phytotoxicity tests are lettuce and radish (Mathur et al., 1993a). Mathur et al. (1993b) used cress seeds in their phytotoxicity bioassays. The authors observed that the germination index increased above 100% after day 59, which was an indication of the elimination of phytotoxicity beyond that day. The variable responses of different seeds in germination bioassays can be expected. Some seeds may proliferate and others may have decreased germination with the same compost extract. For example, Marchiol et al. (1999) used 23 types of seeds to assess the impact of municipal solid waste compost and soil leachate onto their germination. The authors found signicant variations among the calculated GIs, as well as among the times of germination. Legume seeds were the most sensitive, followed by seeds from grasses and wild herbs. Germination indices varied from 52.9% to 96.1% for all 11 wild herb seeds, from 47.5% to 92.4% for the six grass seeds and from 62.0% to 96.1% for the six legume seeds. In addition, the aforementioned authors observed that the higher the electrical conductivity of the leachates, the lower the germination indices and the higher the average times of germination for all 23 seeds used throughout the study. Stability indices are usually calculated by quantifying microbial decomposition activity via oxygen consumption, CO2 production or self-heating capacity (Barrena-Gmez et al., 2006; Lasaridi et al., 2006). Therefore, tests such as the dynamic respiration index (DR4), AT4 (static respiration index at four days) or the specic oxygen uptake (SOUR) rate (Lasaridi et al., 2006) are some of the few tests that have been proposed to quantify respiration activity in many countries (Bernal et al., 1998; Barrena-Gmez et al., 2006). In addition to the above, several physical and chemical methods have been often suggested as indirect measurements of biological stability (Riffaldi et al., 1986; Mathur et al., 1993a,b; Baf et al., 2007). As a result, a large heterogeneity of standards for compost quality and application exists in the international legislation (Barrena-Gmez et al., 2006). Haug (1993) mentions, based on his literature review, that an oxygen uptake rate (OUR) less than 0.1 g O2/dry kg/h is acceptable for most compost eld applications, whilst an OUR less than 0.02 g O2/dry kg/h is acceptable for compost application to sensitive plants. According to Barrena-Gmez et al. (2006), oxygen consumption less than 2030 g O2/dry kg or 8 g CCO2/kg C is an indication of a stable compost, whilst static OUR limits can vary from 0.12 to 1.0 g O2/kg LOI/h. The above authors mention various other microbial respiration related parameters that have been adopted as limits in several countries and test methods. Barrena-Gmez et al. (2006) clearly conclude from their literature review that there is not a commonly accepted compost stability index, although O2 uptake, CO2 production and the self-heating tests are the most common. Still, though, different variations of the same test as well as different expressions of the same result may exist (e.g., mass of gas on a dry weight, organic matter or total carbon basis). Mathur et al. (1993b) point out that microbial activity indicators (CO2 production or O2 consumption) should be rather expressed on an organic matter or carbon basis, since the organic matter is responsible for the microbial respiration. This view is also supported by the authors of this manuscript.

Relationships among maturity and stability indices have been occasionally sought by the researchers. For example, Iannotti et al. (1994) observed a relatively high negative correlation between the O2 uptake of MSW compost and a maturity index calculated from ryegrass growth bioassays. On the other hand, the same authors observed that no cress germination occurred in seed phytotoxicity tests with a MSW compost at various maturity levels. Epstein (1997) mentions that poorly stabilized composts correlated adequately with cress germination indices; however, this was not true with highly stable composts. Bernal et al. (1998) found common maturity indices for different composts prepared from various organic wastes. Their results are interesting, since common threshold values were proposed for seven different composts from variable sources. In particular, Bernal et al. (1998) found that the ratio of water extractable carbon to organic N (Cw/Norg) correlated well with most of the other maturity indicators, rendering it the most suitable for evaluating compost maturity. The cress seed GI was among the maturity indices used by Bernal et al. (1998) and, sometimes, correlated well with some of the other compost quality factors. Wu et al. (2000) concluded that compost stability (based on CO2 production) and compost maturity (based on seed germination) are two different parameters of compost quality that should be both measured and reported. These parameters were not well correlated in their study, since the low respiration rate did not necessarily lead to low phytotoxicity for all three biosolids derived composts used. Still, though, the observations by Wu et al. (2000) were based on germination bioassays conducted with tomato seeds only. Cossu and Raga (2008) also observed good correlation among certain stability indices. For example, the oxygen consumption at four days (RI4 expressed in mg O2/g dw) correlated relatively well with the BOD5/COD ratio measured in the leachates and also correlated well with another novel stability index, namely the black index (Cossu and Raga, 2008). Despite the wide use of composted organic matter over the past decades, there is still no widely accepted indicator to differentiate between mature/stable and immature/unstable composts. Therefore, the research presented in this article consists of two parts. The rst part attempts to investigate the germination of several seeds upon application of extracts from composts derived from different source materials. The response of different types of seeds to the same type of compost is evaluated and results are analyzed with the principles of the analysis of variance (ANOVA) to calculate the relative maturity of the six composts used in this study. The analysis, therefore, aims to answer the following questions: (a) can a single type of seed be used to characterize mature composts, and (b) can a consistent threshold GI be used as a maturity index for different composts? The 2nd part of the research studies the potential relation among GIs, the microbial respiration indices and initial compost properties. That is, the 2nd part of the research aims to answer the following questions: (a) is a mature compost stable as well, and (b) does maturity increase as stability increases as well?

2. Materials and methods 2.1. Compost sampling Six types of compost, derived from cow manure, olive pulp, poultry manure, municipal solid waste and sea weeds were used in this study. The cow manure derived compost (CMC) is produced after a composting period of ten months. The manure is aerated in windrows with the addition of bulking agent and geo-worms; thus the process would be classied as vermi-composting. The olive pulp derived compost (OLC) was obtained from a two-phase olive milling facility in Thasos Island (Greece). In the facility, the initial

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mixture to be composted is usually prepared from 1 volume of the olive pulp, which is a solid by-product at a 6570% moisture content, with approximately 1.5 volumes of bulking material, namely olive tree leaves, wood chips and agricultural by-products from rice harvesting. The mixture is placed in windrows and is aerated once weekly with a small compost turner. The active composting period lasts from seven to eight months and then the material is directed to curing in static piles. Two samples were received: one composite sample was obtained from a 5-month old active composting windrow (OLC_A), and the second composite sample was obtained from a 10-month old curing pile (OLC_C) after an 8month active composting period. The poultry manure derived compost (PMC) was obtained from a poultry breeding industry. The industry produces approximately 1520 ton of chicken manure daily. The manure is disposed of in a rotating channel in which aeration is achieved via a mechanical agitator. Retention time in the channel is approximately seven to ten days, followed by a curing time of only eight to ten days. According to technical staff, the composted material, which is packaged in 50-kg bags, is sold at 100 per ton. The composted material obtained from the poultry facility had a strong ammonia odor. The MSW derived compost (MSWC) was obtained from a composting facility in Kalamata (Greece) that accepted commingled MSW, which was manually and mechanically separated within the plant. The separated organic fraction of MSW was mixed with dewatered biosolids from a nearby wastewater treatment plant prior to entering the composting reactors. The compost used in this study was obtained from a 5-year old compost curing pile, since the facility has not been operating for the past few years. The sea weed compost (SWC) is a product of a mixture of 80% sea weeds and 20% cow manure and other agricultural by-products. According to the vendor, the composting process lasts approximately two months, including curing, whilst a thermophilic stage is reached during active composting. All composts were kept at room temperature in sealed black bags after their transport to the university area. Both the CMC and the SWC can be found in the Greek market in bags of 6 L and 5 L, respectively, and at prices of 57 per bag. All composts were stored at room temperature for a period of approximately ten months, until the end of the experimental runs, except for OLC_A, which was stored in a freezer throughout the experiment. Since ve of the products were commercial and mature composts, the storage at room temperature was not thought to affect the properties of the composts. Still, moisture and organic matter measurements were always performed prior to an analysis, during the duration of the experiment, to properly calculate the dry weight and organic matter contents. 2.2. Compost analysis Moisture content, organic matter, total C, total N and pH were measured for all materials. Moisture content was measured through weight difference after drying the material at 75 C until a constant weight was reached; moisture was expressed on a wet weight basis. Dried material was then ground using a commercial grinder (mesh size, 2 mm); measurements of organic matter, total C and total N were then performed. Organic matter was measured in a mufe furnace through the loss on ignition (LOI) at 550 C for 2 h and was expressed on a dry weight (dw) basis. Total C and total N contents were measured using an elemental analyzer (CE Instruments, CHNS-O Model EA-1110). A 7-point calibration was performed during elemental analysis using organic standards (2,5 bis(5-ter-butyl-benzoxazol-2-yl)thiophene or BBOT, cystine, urea) and linear calibration lines were tted through the origin for both C and N. Samples ranged from 2 to 6 mg (dw) during elemental analysis; all measurements were performed in triplicates. The pH of the compost water ltrates that were produced during

the germination bioassay tests described below was measured using a glass electrode and a WTW InoLab pH meter. The electrical conductivity of the ltrates was measured using a portable electrical conductivity analyzer by WTW (Model Cond 197i). 2.3. Types of seeds Seven types of seeds, namely cress, cucumber, lettuce, pepper, radish, spinach, and tomato were used throughout the experiments. All of the above seeds have been often used in germination bioassays to test compost maturity. Seeds were purchased from local agricultural stores and were always pre-wetted for 1214 h prior to the initiation of the experiments to accelerate germination. 2.4. Germination bioassays The germination bioassays used here followed a modied procedure that was based on Zucconi et al. (1981). Approximately 1525 g (ww) of each type of compost was mixed with deionized water in an Erlenmeyer ask at a mixing ratio of 10:1 (water volume, in ml, to dry weight, in g). Mixing was performed for 30 min at room temperature using a magnetic stirrer. The slurry was then ltered under vacuum using a 0.45-lm lter. Approximately 3050 seeds from six of the seven plant species, and ten cucumber seeds, were placed on each 110-mm diameter Petri dish during each run. The above number of seeds resulted in enough space to facilitate seed growth and the measurements that followed. Then, 10 ml of each compost ltrate was added on each Petri dish. Each treatment, i.e. each combination of seed and compost, consisted of triplicate runs. A different compost ltrate was prepared every time during each of the three replicate runs, separately for each type of compost. That is, three ltrations of the same type of compost were performed within each treatment. Controls, run in duplicates for each type of seed, were prepared with deionized (DI) water. All seeds were incubated at 22 C in a WTW incubator model TS 606/2-I. The aforementioned temperature was within the optimum temperature germination range for all seven seeds used throughout the study. Germination percentage and root length were measured after an incubation period of seven days and were expressed as a percentage of the corresponding values of the control (Zucconi et al., 1981). Seeds with root lengths less than 2 mm were not rendered germinated. According to the above description, GI was calculated based on the following formula:

GI

Number of germinated seedssample Total root length of germinated seedssample Total number of seeds in Petri dishsample Number of germinated seedssample Number of germinated seedscontrol Total root length of germinated seedscontrol Total number of seeds in Petri dishcontrol Number of germinated seedscontrol

1
The second fraction in Eq. (1) is usually known as the relative root elongation index, which is not reported separately here. 2.5. Static respiration activity The static respiration activity was measured with manometric respirometers from WTW (equipped with Oxi-Top heads). Respirometers were kept at 35 C over a 7-day period. Oxygen consumption was calculated via the pressure drops recorded and logged separately for each respirometer, using the principles of the ideal gas law. Approximately 50 g (dw) from each compost were placed in each respirometer and water was added to approximately reach 100% of the water holding capacity of each compost. The total CO2 C produced during the 7-day period was calculated after measuring the total and phenolphthalein alkalinities (Komilis and Ham, 2000) of a 1 N 50 ml KOH solution, which was placed in each respirometer to absorb the CO2 produced. Microbial static respiration activity coefcients were expressed in mass of O2 consumed and

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CO2 produced on a dry weight basis, on an organic matter (LOI) weight basis and on a total carbon weight basis. Two replications were performed for the OLC_A, OLC_C, CMC, PMC and MSWC, whilst three replications were performed for the SWC, due to the large variance in the results of that compost. 2.6. Experimental design and statistical analysis The experimental design was based on a 6 7 matrix (six types of composts and seven types of seeds). Statistical analysis was based on a two-factor analysis of variance (ANOVA), with one-factor being the type of compost and the other factor being the type of seed. A one-factor ANOVA was also used to calculate statistical differences among composts based on the GI results of each type of seed separately. All 126 phytotoxicity runs (plus 14 control runs) were performed in random chronological order and lasted approximately 2.5 months. Statistical analysis was performed with MINITAB Release 14. 3. Results and discussion The initial properties of the six composts are included in Table 1. Data in Table 1 are averages, whilst the variance is expressed either as a coefcient of variation or as a standard deviation. As shown, all composts had moisture contents less than 40% (ww). Organic matter (expressed as LOI) had a rather wide range from 15% (dw) for SWC to 79% (dw) for one of the olive pulp derived composts (OLC_C). Sea weed compost had, strangely, rather low organic matter (15% dw) and carbon (10.4% dw) contents, not normally encountered in organic composts. Eyras et al. (2008), who used sea weeds to produce composts, measured an organic matter content of 40% in the original sea weeds, prior to composting, and carbon contents between 2% and 3% (dw) in the sea weed-derived composts, after several months of composting and curing. Therefore, it appears that low organic content values are characteristic of sea weed composts; this could be attributed to the presence of a large fraction of a readily biodegradable organic matter portion in the feed substrate, which is fully mineralized during the composting process, and to the presence of inorganic material, such as sand, in the original feed substrate. All C contents were less than 40% (dw). PMC had the highest N content, namely 2.6% (dw) and SWC had the lowest N content, namely 0.22% (dw). Corresponding C/N values ranged from 11 to 48 for PMC and SWC, respectively. The C/N ratio has been occasionally proposed as a compost quality indicator; for example, a C/N ratio of 10 has been alleged to characterize mature composts according to Mathur et al. (1993a). However, the C/N ratio alone

is not a reliable stability indicator, due to a large variability in values (Epstein, 1997) and, since the C/N ratio of the starting material has to be always accounted for. The pH values ranged from approximately 8.0 to 9.0, all above the upper limit of 8.0 required for MSW derived composts in Greece (Solid Waste Circular, 1997). According to Table 1, OLC_A, OLC_C and SWC were the most alkaline composts, with pH values around 9.0. Alkaline pH values are usually indicators of stable composts, since pH in known to increase in the latter stages of composting. Acids, which are common phytotoxins (Mathur et al., 1993a), are known to be produced in intermediate stages of composting and are, therefore, not expected to be present in mature and stable composts. The OLCs produced ltrates with the lowest electrical conductivities among all compost ltrates, namely from approximately 500 to 800 lS/cm; the PMC produced a ltrate with the highest electrical conductivity value, namely 8520 lS/cm. Electrical conductivity, which is an indirect measure of the total ion concentration, is expected to affect seed growth. 3.1. Germination indices (GIs) The GIs calculated for each of the 126 runs are included in the boxplot of Fig. 1. The lowest recorded GI was 0% and occurred with the PMC for almost all seeds. The highest GI was 262% and occurred with the combination of pepper seed and OLC_C. Table 2 includes the mean germination percentage of all seeds in the control runs and their corresponding variances. The germination percentages in the control are included in Table 2. Pepper and spinach resulted in the lowest germination percentages in the control, 9% and 39%, respectively, whilst all other seeds had germination percentages over 90% in the control. The low germination percentage of pepper might be explained by the fact that, commonly, more than seven days are required for pepper to germinate; all other seeds usually germinate in less than seven days. Table 2 reveals that pepper and spinach were the seeds with the largest variance in their results, as was observed both in the control as well as in the samples. In addition, according to Table 2, it is evident that the variance in the compost samples is greater than the variance in the control for all types of seeds. Due to the low germination of pepper seed in the control, results from the aforementioned seed were not used in the statistical analysis that followed. Therefore, the bioassay tests used here were accepted as long as the minimum germination in the control was approximately 40% and the minimum mean root length in the control was 10 mm/germinated seed. According to Fig. 1, there is a large variance of GIs for the same type of compost. Table 2 also reveals that there is a large variation among the data. Highly variable GIs are calculated for the same compost using the different types of seeds. For example, OLC_C re-

Table 1 Properties of the six composts used throughout the experiment. Compost type CMC+ OLC_A OLC_C PMC+ MSWC SWC+ Moisture content (% ww)a 32 26 15 11 27 27 Organic matter as LOI (dw)b 37 0.51% 59 2.0% 79 1.5% 57 0.38% 33 0.34% 15 0.14% C (dw)c 20.5 2.5% 27.1 5.7% 36.5 9.1% 28.5 3.1% 15.7 2.3% 10.4 10.6% N (dw)d 1.8 1.7% 1.2 23% 1.7 5.3% 2.6 4.0% 1.2 18% 0.22 2.7% C/N 12 22 22 11 13 48 pHe 8.0 0.11 8.9 0.19 9.0 0.12 7.9 0.09 8.1 0.055 8.9 0.10 Electrical conductivity (lS/cm)f 6460 798 494 8520 2910 1220

CMC, cow manure derived compost; OLC_A, two-phase olive mill solid by-product derived compost (sampled from active windrows); OLC_C, two-phase olive mill solid byproduct derived compost (sampled from curing pile); PMC, poultry manure derived compost; MSWC, municipal solid waste derived compost; SWC, sea weeds derived compost. + Commercial product. a n = 2 With coefcients of variation less than 1% (coefcient of variation in % is the standard deviation divided over the mean). b,c,d n = 3; Values are average coefcient of variation. e n = 21; Values are average standard deviation. f The electrical conductivity of the deionized water used during the ltration was 7.1 lS/cm.

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Fig. 1. Boxplot of GIs calculated from seven seeds and six composts (h indicates mean values) (CMC, cow manure derived compost; OLC_A, olive pulp derived compost (sampled from active windrows); OLC_C, olive pulp derived compost (sampled from curing pile); PMC, poultry manure derived compost; MSWC, municipal solid waste derived compost; SWC, sea weeds derived compost).

sulted in a 168% GI when spinach was used, whilst a GI of only 68% was calculated when lettuce seeds were used. OLC_A, on the other hand, resulted in a high GI (242%) when lettuce was used and a relatively low GI (63% when spinach was used. CMC was rendered phytotoxic when cucumber was used, and mature when tomato or lettuce seeds were used. Therefore, sometimes, compost appears to be phytotoxic to a certain seed and, sometimes, the same compost appears to enhance the growth of another seed. Precision is high for the cress, cucumber, radish and tomato, as is concluded from the relatively low standard deviations among replicates of the above four seeds (see Table 2). Spinach had a rather large coefcient of variation (>16%) within a single compost, whilst lettuce showed a large variance when the SWC and OLC_C composts were used. PMC resulted in zero GIs for almost all seeds except cress indicating that it is highly phytotoxic. The pepper seed was the least responsive among all seeds. However, extensive pepper seed germination occurred with the OLCs only and particularly with OLC_C. Apparently, certain constituents of the OLC enhance the growth of pepper or, vice versa, some constituents present in CMC, PMC, MSWC and SWC are potential phytotoxins to pepper.
Table 2 Average germination indices (in %); data are averages coefcients of variation*. Type of seed Compost type CMC** Cress (Lepidium sativum) Cucumber (Cucumis sativus) Lettuce (Lactuca sativa) Pepper (Capsicum spp.) Radish (Raphanus sativus) Spinach (Spinacia oleracea) Tomato (Lycopersicon esculentum)
*

Due to the highly selective germination of the pepper seed and the phytotoxic nature of PMC, they were both excluded from the statistical analysis that follows. 3.2. Two-factor ANOVA Six types of seeds and ve types of compost were nally used and were included in a two-factor analysis of variance (ANOVA); one-factor was the type of seeds and the other was the type of compost. Table 3 is the resulting two-factor ANOVA table. At the 95% condence level (a = 0.05), both main effects and the interaction effect between the composts and seeds were statistically signicant (i.e. all p < 0.05). According to the above, both the seeds as well as the composts signicantly affect the GIs at the 95% condence level (C.L.). This means that different seeds can produce signicantly different GIs, whilst the ve composts are statistically different too. At the 99% C.L., however, the composts appear not to have statistically signicant differences among them (p = 0.027 > 0.01). The most important result, however, is that the interaction effect is highly signicant as well, indicating that each seed will behave differently with different composts. The signi-

OLC_A** 94 9% 88 27% 242 0.4% 54 173% 119 9% 63 16% 50 26%

OLC_C** 62 2% 70 4% 68 77% 198 29% 153 12% 168 25% 125 6%

PMC** 6 0% 0*** 0*** 0*** 0*** 0*** 0***

MSWC** 123 19% 74 9% 165 10% 0** 160 7% 117 73% 63 9%

SWC** 140 28% 87 5% 145 44% 0** 151 4% 105 84% 105 1%

Mean germination percentage in control**** 100 0.0% 90 0.0% 98 2.8% 9 25% 92 0.12% 39 27% 99 1.5%

Mean root length in control (mm/seed)**** 41.7 1.5% 65.6 2.3% 27.1 6.5% 3.4 16% 25.3 3.3% 11.3 61% 32.2 6.6%

86 3% 67 15% 112 8% 0** 101 17% 47 52% 122 6%

Coefcient of variation is the standard deviation divided over the mean (in %). Statistics are based on triplicate samples. *** All samples had zero GIs. **** Statistics for the control are based on duplicate samples.
**

D.P. Komilis, I.S. Tziouvaras / Waste Management 29 (2009) 15041513 Table 3 Two factor ANOVA table to assess effects of seeds and composts on the germination indices. Source Compost type Seed type Compostseed interaction Error Total DF 4 5 20 60 89 SS 11,290 53,861 100,429 57,149 222,729 MS 2822.6 10772.2 5021.4 952.5 F ratio 2.96 11.31 5.27 p value 0.027 0.000 0.000

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using the pair-wise comparisons. As shown by the pooled standard deviations in the last column of Table 4, spinach was the seed that resulted in the largest variance of GIs, whilst tomato was the seed with the lowest variance observed. According to the above, the use of the germination bioassay test is rather unreliable when it is to assess compost maturity. The phytotoxicity test germination indices are highly dependent on the type of seed used. Apparently, composts have variable effects on the different types of seeds. This conclusion was also evident from the two-factor ANOVA test. 3.4. Respiration activity Microbial respiration was quantied via the cumulative O2 consumption, the O2 uptake rate and the cumulative CO2 production during a 7-day period. The O2 consumption and CO2 production were calculated at the 4th day and the 7th day, and are shown in Table 5. In addition, different units of the same property are included in Table 5. Therefore, O2 and CO2 amounts are expressed on a dry weight, organic matter weight and a total carbon weight basis. Fig. 2 includes the cumulative O2 consumption expressed on an organic matter basis. The oxygen uptake rates (OURs) are evident from the slopes of the curves in Fig. 2. According to Table 5, PMC, MSWC and SWC have the lowest 7day O2 consumptions and 7-day CO2 productions, namely less than approximately 2.2 g O2/kg dw and less than approximately 1.0 g CO2C/kg dw, respectively. However, if the values are expressed on a per organic matter (LOI) basis, then SWC becomes one of the least stable composts, with a 7-day O2 consumption and a corresponding CO2 production equal to 15.6 g O2/kg LOI and 5.4 g CO2C/kg LOI, respectively. Based on Table 5, PMC has the lowest respiration activity, which is probably attributed to the potentially toxic environment prevalent in that compost, as was evident from the nearly 0% GIs calculated during the phytotoxicity test; that is, the low respiration of PMC is probably not a result of its stability, but probably due to a highly toxic environment to microorganisms. CMC is the second least stable compost, after SWC, with a microbial respiration activity equal to 12.6 g O2/kg LOI at seven days. The percentage of the oxygen consumed in day-4 in relation to the oxygen consumed in day-7 varies for each compost. It ranges from 55% for PMC, to over 90% for the OLCs and CMC. This is probably a result of the differences in the composition of the organic matter in the six composts. Apparently, OLCs and the CMC appear to have a relatively large readily degradable organic fraction, which resulted in most of the oxygen being consumed during the rst few days. A denition of compost stability is included in a working document by the European Commission (2001), since there is no limit for stable composts set by the EU. According to that denition, stable composts should have, among others, respiration activities less than 10 g O2/kg dry matter after four days (AT4 test). Note that the AT4 test is a static respiration test that is performed at four days and at 35 C incubation temperature, conditions similar to the ones used in this work. Therefore, according to Table 5, all composts have a 4-day respiration activity below the suggested limit mentioned in the EU working document. Barrena-Gmez et al. (2006) also mention that another proposed stability limit is the CO2 production of 8 g CO2C/kg C. Using the 7-day indices included in Table 5, it appears that OLC_C, MSWC and SWC are stable composts, provided that the aforementioned limit is adopted; however, the remaining composts (OLC_A and CMC) can be also rendered nearly stable, since they have CO2 production rates very close to the above suggested limit. The overall-averaged-molar ratio of CO2 to O2 (total mol of CO2 produced over the 7-day period/total mol O2 consumed over the 7day period), a fraction commonly known as the respiratory quotient (RQ), was found to range from 0.86 for PMC, to 1.4 for OLC_C

DF, degrees of freedom; SS, sum of squares of residuals; MS, mean of squares of residuals. p Values are compared to a = 0.05 (at the 95% C.L.) or to a = 0.01 (at the 99% C.L.).

cant interaction effect (p < 0.001) means that the main factor effects cannot be interpreted individually; that is, the main factor effects are not additive. Therefore, according to Table 3, one common threshold GI to indicate maturity cannot be established for all types of composts. 3.3. One-factor ANOVA Since interactions are highly signicant and main factors cannot be interpreted on an additive basis, a one-factor ANOVA was performed for each type of seed, separately. The variable factor was the type of compost. Using the principles of ANOVA, pair-wise comparisons were performed using the Tukey test at the 95% C.L. (Berthouex and Brown, 2004). Table 4 includes all statistically different pairs. Pairs that are statistically similar, at the 95% C.L., are not included in Table 4. According to Table 4, the different types of seeds provide contradictory and inconsistent results on the phytotoxicity of the ve composts. For example, OLC_C is signicantly more phytotoxic to cress than MSWC; however, when tomato is used, the opposite is true. CMC is less phytotoxic to tomato than OLC_A, but when lettuce is used, the opposite is true. OLC_C is less phytotoxic to tomato than OLC_A; on the other hand, OLC_A appears to be less phytotoxic to lettuce than OLC_C. With cucumber and spinach, all composts have statistically equal GIs, which means that they have statistically similar maturities (or phytotoxicities). No common statistically different pair was found for any of the seeds used

Table 4 Pairwise comparisons among composts using the Tukey test at the 95% C.L. Type of seed Cress Cucumber Lettuce Radish Statistically signicant differencesa MSWC > OLC_C SWC > OLC_C No statistically signicant differences among composts OLC_A > CMC OLC_A > OLC_C MSWC > CMC OLC_C > CMC SWC > CMC MSWC > OLC_A No statistically signicant differences among composts CMC > MSWC CMC > OLC_A OLC_C > MSWC SWC > MSWC OLC_C > OLC_A SWC > OLC_A Pooled standard deviation (%) 21 12 38 14

Spinach Tomato

59 8.0

a The term A > B means that compost A has a statistically greater GI than compost B or compost A is statistically less phytotoxic than compost B at the 95% C.L..

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Table 5 Microbial respiration activity indices and respiratory quotients for six compostsa. Compost type Static cumulative O2 consumption g O2/kg dw At four days CMC OLC_A OLC_C PMC MSWC SWC
a b c d

Static cumulative CO2 productionc g O2/kg C At seven days 12.6 2.8% 8.7 7.8% 6.1 0.9% 3.9 8.4% 6.5 4.1% 15.6 19% At four days 20.4 6.3% 18 10% 12.9 2.3% 4.5 40% 8.7 24% 16.5 17% At seven days 22.7 2.8% 19 7.8% 13.1 0.9% 7.9 8.4% 14 4.1% 22.5 19% g CO2C/kg dw At seven days 2.3 4.9% 2.5 1.1% 2.6 5.5% 0.71 18% 1.0 13% 0.72 15% g CO2C/kg LOI At seven days 6.3 4.9% 4.2 1.3% 3.3 5.5% 1.3 14% 3.2 6.4% 5.4 28% g CO2C/kg C At seven days 11.3 4.9% 9.2 1.3% 7.0 5.5% 2.6 14% 6.8 6.4% 7.8 28%

RQd

g O2/kg LOI At seven days 4.6 2.8% 5.1 5.4% 4.8 0.%9% 2.2 12% 2.0 11% 2.1 20% At four days 11.3 6.3% 8.1 10% 6.0 2.3% 2.2 40% 4.1 24% 11.4 17%

4.2 6.3 (90%)b 4.7 7.5 (92%)b 4.7 2.3 (99%)b 1.2 43 (55%)b 1.3 30 (65%)b 1.6 27 (76%)b

1.3 7.7% 1.3 6.5% 1.4 6.4% 0.86 5.4% 1.3 2.3% 0.92 14%

Mean coefcient of variation based on duplicate runs, except for SWC for which triplicate runs were performed. Percentage of respiration attained in day-4 in relation to day-7. Carbon dioxide was measured through titration of the alkaline traps placed in the respirometers at the end of the seventh day. The respiratory quotient (RQ) calculated here is the ratio of the mol of CO2 produced over a 7-day period to the mol of O2 consumed over the same period.

Fig. 2. Cumulative oxygen consumptions from six composts based on static respiration tests.

(see Table 5), with a mean value equal to 1.19 0.25 (mean standard deviation from all six composts). The RQs measured in this study are similar to values reported by other researchers. For example, Klauss and Papadimitriou (2002) used a dynamic aerobic composter to study the change of RQs during the process. They used compost from commercial composting plants in their experiments and dened RQ as the ratio of the CO2 concentration (%v/v) in the exit gas to the difference of the O2 concentration in the exit gas (in %v/v) from the ambient air O2 concentration (in %v/v). Note that the above denition produces the same results as the RQ denition used here, namely the mol of CO2 produced to mol of O2 consumed. According to Klauss and Papadimitriou (2002), RQs ranged from approximately 0.7 to 1.0 and had a slightly diminishing trend during the process. In addition, the above authors observed that the highest degrees of aerobiosis were observed at RQ values less than 0.8. Nakasaki et al. (1985), who used mixtures of raw sludge and compost in their composting experiments, found that RQs slightly varied according to the temperature and ranged from approximately 0.6 to 0.8. The lowest values were recorded at 50 C and the highest values at 70 C, indicating the prevalence of catabolism at higher temperatures. Gea et al. (2004) found that RQs were slightly affected by the type

of the substrate. The authors found that average RQs ranged from 1.00 during composting of dewatered raw sludge, to 1.24 during composting of the organic fraction of municipal solid waste. According to Gea et al. (2004), the higher the RQ, the more readily degradable a substrate is. Interestingly, the same authors observed that variations of RQ during the process were less than 10%, as had been also observed by Klauss and Papadimitriou (2002). Finally, RQs between 0.8 and 0.9 were measured by Schulze (1960), and values close to 1 were measured by Kalamdhad et al. (2008). The variations in the RQs among the different composts are likely due to the differences in the composition of the organic matter and the oxidation degrees of the organic carbon in each compost; this was also commented by Barrena-Gmez et al. (2006). However, the change of RQ vs. time was not studied here, since a single measurement of the cumulative CO2 produced over seven days was performed at the end of the incubation period. 3.4.1. Oxygen uptake rates (OURs) The oxygen uptake rate (OUR) is another commonly used stability index. The OURs calculated here were based on the static cumulative O2 consumptions measured via the specic respirometers

Table 6 Pearson correlation coefcients among various parameters based on n = 5 composts (PMC is not included). Maturity indices GI Crs GI Crs GI Cuc GI Rad GI Spn GI Tom GI Let GI Pep O4d O4L O4C O7d O7L O7C C7d C7L C7C C/N LOI C N EC 1.00 GI Cuc n.s. 1.00 GI Rad n.s. n.s. 1.00 GI Spn n.s. n.s. 0.84* 1.00 GI Tom n.s. n.s. n.s. n.s. 1.00 GI Let n.s. n.s. n.s. n.s. 0.92** 1.00 GI Pep n.s. n.s. n.s. n.s. n.s. n.s. 1.00 Stability indices O4d 0.90** n.s. n.s. n.s. n.s. n.s. n.s. 1.00 O4L n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 1.00 O4C n.s. n.s. 0.87* n.s. n.s. n.s. n.s. n.s. 0.88** 1.00 O7d 0.88** n.s. n.s. n.s. n.s. n.s. n.s. 1.00*** n.s. n.s. 1.00 O7L n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.93*** n.s. n.s. 1.00 O7C n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.96*** 0.86* n.s. 0.93** 1.00 C7d 0.94** n.s. n.s. n.s. n.s. n.s. n.s. 0.98*** n.s. n.s. 0.98*** n.s. n.s. 1.00 C7L n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.95** 0.85* n.s. 0.88** 0.96*** n.s. 1.00 C7C n.s. n.s. 0.98*** 0.86* n.s. n.s. n.s. n.s. n.s. 0.88** n.s. n.s. n.s. n.s. 0.81* 1.00 Initial compost properties C/N n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 1.00 LOI 0.89** n.s. n.s. n.s. n.s. n.s. 0.89** 0.82* n.s. n.s. n.s. n.s. n.s. 0.85* n.s. n.s. n.s. 1.00 C 0.93** n.s. n.s. n.s. n.s. n.s. 0.90** 0.86* n.s. n.s. 0.82* n.s. n.s. 0.88** n.s. n.s. n.s. 0.99*** 1.00 N 0.86* 0.80* n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.81* n.s. n.s. 0.88** n.s. n.s. 1.00 ECa n.s. 0.83** 0.85** 0.81** n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 1.00 D.P. Komilis, I.S. Tziouvaras / Waste Management 29 (2009) 15041513 1511

n.s., not signicant. Crs, cress; Cuc, cucumber; Rad, radish; Spn, spinach; Tom, tomato; Let, lettuce; Pep, pepper; O4d, g O2/dry kg at four days; O4L, g O2/kg LOI at four days; O4C, g O2/kg C at four days; O7d, g O2/dry kg at seven days; O7L, g O2/kg LOI at seven days; O7C, g O2/kg C at seven days; C7d, g CO2C/dry kg at seven days; C7L, g CO2C/kg LOI at seven days; C7C, g CO2C/kg C at days; C/N, total carbon to total nitrogen ratio; C, total carbon content (% dw); N, total nitrogen content (% dw); LOI, loss on ignition (volatile matter) in % dw; EC, electrical conductivity. * Signicant at p < 0.1. ** Signicant at p < 0.05. *** Signicant at p < 0.01. a The correlation coefcients for EC are based on six composts, that is PMC was included.

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used throughout the study; therefore, they are static respiration indices, and not dynamic. As is evident from Fig. 2, there are two dominant OURs for all composts during the 7-day incubation period. An inection is observed at every O2 consumption curve, indicating the shift from a relatively fast to a relatively low or negligible OUR. The shift indicates the change from the degradation of the readily degradable matter to the degradation of refractory organic matter. The inection times ranged from 0.5 days for one of the SWC replicates, to four days for the CMC. Both OLCs had the highest OURs during the rst day, namely approximately 0.20 g O2/kg dw/h (or approximately 0.30 g O2/kg LOI/h). SWC had also one of the highest OURs during the rst days, if this is expressed on a per organic matter basis, i.e. approximately 0.35 g O2/kg LOI/h (approximately 0.05 O2/kg dw/h). All other composts had OURs that did not exceed 0.10 g O2/kg dw/h (approximately 0.160.26 g O2/kg LOI/h) during the initial active microbial degradation phase. All above rates decreased to less than 0.015 g O2/kg dw/h (or less than 0.06 g O2/kg LOI/h) after the 4th day and up to day-7. PMC had the lowest JUR from the initiation of the experiment, which never exceeded 0.05 g O2/kg dw/h (or 0.08 g O2/kg LOI/h). These low OURs for PMC could be attributed to the presence of compounds in the compost, such as salts or other ions, that may inhibit microbial growth; this was indirectly indicated by the relatively high electrical conductivity of the PMC ltrate. It is noted that the relatively high level of N measured in PMC, might be toxic to seeds, but is not necessarily toxic to the microorganisms responsible for biodegradation. The nal OURs for OLC_C and OLC_A were the lowest among all other composts, with the OLC_C being the most stable with an average OUR close to 0.001 g O2/kg LOI/h after day two. It was determined that the nal low microbial activity for almost all composts was not due to material drying. From the above discussion, it becomes evident that it is important to clearly specify the units of microbial respiration (e.g., on a per dry matter or per organic matter basis), since differentiations can exist. Precision of the OUR was relatively good for ve of the composts, but not as good for the SWC. However, the OURs of SWC1 and SWC2 after the 2nd day were relatively close, as is evident from Fig. 2. The two replicates of the OLC_C produced almost identical values during the 7-day period and, therefore, one average curve is shown in Fig. 2. A relatively high precision was achieved for the CMC during the rst four days as well. The nal OUR for the CMC was 0.018 0.0054 g O2/kg LOI/h. Based on the nal OURs, it appears that the SWC was the least stable compost, whilst the OLC_A and OLC_C were the most stable composts. The European Commission (2001) suggests that dynamic OURs less than 1 g O2/kg LOI/h can characterize stable composts. According to the limit, all composts can be, therefore, characterized as stable, since they have O2 consumption rates far lower than 1 g O2/kg LOI/h at both the high and the low rate stages of decomposition. The highest OUR was recorded for the SWCs run, which during the rst 12 h of the experiment maintained an OUR approximately equal to 0.35 g O2/kg LOI/h. However, it is noted that the suggested limit of 1 g O2/kg LOI/h is based on dynamic respirometric tests, whilst the tests performed here were static. According to Adani et al. (2008), a dynamic respirometric index (DRI) of 1 g/kg LOI/h can correspond to a static respirometric index (SRI) equal to approximately 0.4 g/kg LOI/h; based on this, the six composts would still be rendered stable, since they have peak OURs lower than 0.4 g/kg LOI/h. Finally, according to the classication of Table B.1 of ECS (2007), all six composts have a very low potential of microbial self-heating, since they all have respiration activities less than 0.5 g O2/kg dw/h during their initial high microbial degradation phase. However, caution is required, since the above classication is based on dynamic respirometric experiments and not static.

3.4.2. Relation between maturity and stability indices A potential relation among the maturity indices (GIs), the stability indices (microbial respiration coefcients) and the initial properties of composts was sought. The primary goal was to observe whether a low microbial respiration leads to a low phytotoxicity/ high maturity and vice versa. Table 6 includes the Pearson correlation coefcients (r) among 21 parameters, namely seven GIs, nine microbial respiration coefcients and the initial compost properties (C/N ratio, organic matter content (as LOI), C content, N content, pH and EC). Microbial respiration coefcients are expressed in different units, i.e. on a per dry weight, LOI and carbon basis. Note that PMC was excluded from the correlation due to its high phytotoxicity, therefore, correlation coefcients were calculated based on ve composts. Table 6 shows that most correlations among the GIs from the different types of seeds are statistically insignicant. This observation supports the results of the analysis of variance presented earlier. That is, seeds have a variable behavior according to the type of compost used. Some correlation coefcients are negative and some are positive. For example, the 0.92 r value between the GI from the tomato seed and the GI from the lettuce seed, which is statistically signicant at p < 0.05, indicates that these two seeds have completely different responses to the composts; as one compost enhances the germination of one seed, the same compost inhibits the germination of the other. According to Table 6, correlations among GIs and the microbial respiration activity coefcients were not statistically signicant in most of the cases. However, a high negative correlation coefcient was calculated between the cress GIs and the CO2 production expressed on a dry weight basis (0.94) and between the radish GIs and the CO2 production expressed on a carbon basis (0.97). A statistically signicant correlation between the cress seed and the O2 consumption (dry weight basis) at four and seven days was also achieved. The negative signs indicate that as the microbial respiration increases, germination indices decrease, that is phytotoxicity increases. However, this was not observed for all seeds. Cress correlates well only when the respiration indices are expressed on a dry weight basis; on the other hand, radish correlates well when the respiration indices are expressed on a LOI or C basis. Based on the above, a germination bioassay with radish can provide an acceptable indication of compost stability. A linear equation that was developed to describe CO2C production as a function of the radish GIs is

CO2 C 18:3 7:2 GIradish R2 0:96; significant at p < 0:01 2

where CO2C is expressed in g CO2C/kg C over a 7-day period, and GIradish is the germination index from a radish seed bioassay (in %). Statistically signicant correlations existed among some microbial respiration activity indices. Apparently, the 4-day O2 consumption correlated well with the 7-day O2 consumption. Statistically signicant correlations also existed between O2 consumption and CO2 production, when both are expressed on a dry weight or LOI basis. The volatile matter (as LOI in % dw) and the C contents correlated quite well with the carbon dioxide production and the oxygen consumption at four and seven days. This indicates that the higher the organic matter content, the higher the microbial respiration. This linear correlation between organic matter content and CO2 production was also observed by Bach et al. (1984), who performed respirometric experiments with mixtures of biosolids and rice hulls. No signicant correlations were achieved between pH with any of the other parameters and, therefore, pH was not included in Table 6. The initial C/N ratio had an expected negative statistically signicant correlation with the ini-

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tial N content. Statistically signicant correlations were also achieved between the initial N content and the GI from cress and the GI from cucumber, which might be an indication of the sensitivity of those two seeds to composts with high nitrogen. A similar statistically signicant correlation was found between the GI from cress and the organic matter content and the carbon content, which indicates the likely sensitivity of the cress seed to wastes with potentially high biodegradability. EC was not found to have a statistically signicant correlation with any of the other parameters, when PMC was excluded from the analysis. However, when the results from PMC were included in the correlation analysis, then EC was found to have a negative statistically signicant correlation with the germination indices of cucumber, radish and spinach; this is a likely indication of a sensitivity of these seeds to composts with high amounts of salts or other ionic compounds (e.g. metals). Finally, a positive statistically signicant correlation (at p < 0.05 with n = 6) was observed (not shown in Table 6) between the RQ and the CO2 produced over seven days, when expressed as per dry matter basis, indicating that the higher the CO2 production, the higher the RQ of a material.

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4. Conclusions 1. The phytotoxicity test germination indices are highly dependent on the type of seed used. A compost that is phytotoxic to a certain seed can enhance the growth of another seed. Therefore, the use of the germination bioassay test is rather unreliable when it is to assess compost maturity, since a common threshold GI to indicate maturity cannot be established for all types of composts. 2. If the 10 g O2 consumed/kg dw over a 4-day period is used as a stability limit, then all six composts can be rendered stable. Other oxygen consumption values (over a 7-day period) that could be used as stability limits are 5 g O2/dry kg, 16 g O2/kg LOI and 23 g O2/kg C. Carbon dioxide production values (over seven days) that could be used as stability limits are 2.5 g CO2C/dry kg, 6 g CO2C/kg LOI and 11 g CO2C/kg C. The above values, however, indicate stability as long as the compost is not phytotoxic. 3. A stability test, alone, is not able to ensure high compost quality. For example, PMC was highly phytotoxic to most seeds, and, therefore, immature, but resulted in a low respiration activity, which is indicative of stable composts. Therefore, it appears that determining compost quality requires a simultaneous use of maturity and stability tests. 4. A negative statistically signicant correlation was observed between the electrical conductivity of the compost ltrates with the germination of three types of seeds. 5. Static oxygen uptake rates (OURs) ranged from approximately 0.08 to 0.35 g O2/kg LOI/h during the rst one to four days of incubation and later reduced to less than approximately 0.06 g O2/kg LOI/h for all six composts. 6. A good correlation between the radish GI and the carbon dioxide production (in g CO2C/kg C over seven days) was established. Therefore, a radish seed germination bioassay can be a valid test to indirectly assess compost stability and compost phytotoxicity at the same time. 7. The averaged seven day respirometric quotients ranged from 0.86 to 1.4 mol of CO2 produced/mol of O2 consumed.