Waste Management 54 (2016) 101–109

Contents lists available at ScienceDirect

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

Development of phytotoxicity indexes and their correlation with

ecotoxicological, stability and physicochemical parameters during
passive composting of poultry manure
Brian Jonathan Young a, Pedro Federico Rizzo a, Nicolás Iván Riera a, Virginia Della Torre a,
Valeria Alejandra López a, Cecilia Denisse Molina a, Florencia Estefanía Fernández a, Diana Cristina Crespo a,
Raquel Barrena b, Dimitrios Komilis b,⇑, Antoni Sánchez b
a
Laboratorio de Transformación de Residuos, Instituto de Microbiología y Zoología Agrícola (IMYZA), Instituto Nacional de Tecnología Agropecuaria (INTA),
Las Cabañas y De los Reseros s/n, Hurlingham, Buenos Aires, Argentina
b
Departamento de Ingeniería Química, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain

a r t i c l e i n f o a b s t r a c t

Article history: Both raw and composted poultry manure is applied as soil amendment. The aims of this study were: (1)
Received 2 February 2016 to develop phytotoxicity indexes for organic wastes and composts, and (2) to assess the correlation
Revised 2 May 2016 among phytotoxicity indexes, ecotoxicological endpoints and stability and physicochemical parameters
Accepted 2 May 2016
during passive composting of poultry manure. Six 2-m3 composting piles were constructed and four
Available online 12 May 2016
parameter groups (physicochemical and microbiological parameters, ecotoxicological endpoints, and bio-
logical activity) were determined at four sampling times during 92 days. Extracts were used to carry out
Keywords:
acute toxicity tests on Daphnia magna, Lactuca sativa and Raphanus sativus. Composting decreased aver-
Daphnia magna
Lactuca sativa
age toxicity 22.8% for the 3 species and D. magna was the most sensitive species. The static respiration
Maturity index decreased from 1.12 to 0.46 mg O2 g OM h
1 whilst organic matter reduced by 64.1% at the end
Phytotoxicity of the process. Escherichia coli colonies remained higher than values recommended by international
Raphanus sativus guidelines. The D. magna immobilization test allowed the assessment of possible leachate or run-off tox-
Stability icity. The new phytotoxicity indexes (RGIC0.8 and GIC80%), proposed in this study, as well as salinity,
proved to be good maturity indicators. Hence, these phytotoxicity indexes could be implemented in mon-
itoring strategies as useful ecotoxicological tools. Multivariate analyses demonstrated positive correla-
tions between ecotoxicological endpoints (low toxicity) and biological activity (stability). These two
parameter groups were associated at the final sampling time and showed negative correlations with sev-
eral physicochemical parameters (organic and inorganic contents). The final poultry manure compost
was rendered stable, but immature and, thus, unsuitable for soil amending.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction nutrients (N, P, K), heavy metals (As, Pb, Zn, Ni, Cd, Cu, Mn),
xanthophylls, antibiotics, antiprotozoals, antioxidants, mold inhi-
Poultry production is a quantitatively important agro industry bitors, probiotics, polychlorinated phenols, tetrachlorodibenzo-
worldwide. World production of eggs has increased 24.98% p-dioxin and hormones (Frank et al., 1988; Jackson et al., 2003)
between 2001 and 2011, according to statistics from Food and that may impact negatively the ecosystem through leaching and
Agriculture Organization of the United Nations. During the same runoff. These adverse effects on ecosystem can be circumvented
period, egg production in Argentina soared 101.7% (FAOSTAT, with low-cost composting. Composting minimizes the concentra-
2013). Consequently, a large volume of waste (poultry manure) is tion of phytotoxic substances, controls the spread of pathogens,
an inevitable side effect of this increase. improves storage and handling of waste, and reduces unpleasant
Commonly, raw poultry manure is applied to farmland as odors (Edwards and Daniel, 1992). The quality of the compost
organic amendment (Bolan et al., 2010). This waste contains may have either a positive or negative impact on both soil fertility
and plant health. Nutrients loss depends on several factors such as
aeration, moisture content, temperature and carbon-to-nitrogen
⇑ Corresponding author.
(C:N) ratio (Ogunwande et al., 2008). The initial C:N ratio is the
E-mail address: [email protected] (D. Komilis).

http://dx.doi.org/10.1016/j.wasman.2016.05.001
0956-053X/Ó 2016 Elsevier Ltd. All rights reserved.
102 B.J. Young et al. / Waste Management 54 (2016) 101–109

most widely used parameter for deciding composting methodol- each composting pile and at each sampling time and were kept at
ogy. Poultry manure contains high nitrogen content. Therefore, 4 °C until analysis.
the degradation process may be improved adding carbon-rich
materials (Petric et al., 2009). High initial C:N ratio causes longer 2.1.2. Physicochemical and microbiological characterization
composting time (Tuomela et al., 2000), whereas low initial C:N The following physicochemical parameters were evaluated:
ratio generates higher emission of volatile gases and leachates ambient and pile temperature, moisture content (MC), organic
(Tiquia and Tam, 2000b). Co-composting of poultry manure with matter (OM), total organic carbon (TOC), total Kjeldahl nitrogen
other agricultural wastes improves the physicochemical character- (TN), C:N ratio, soluble organic carbon (SC), total phosphorous
istics and reduces the phytotoxicity (Rizzo et al., 2013). Compost- (TP), soluble phosphorous (SP), major cations, metals, pH, and elec-
ing may be aerated by passive or active systems (Ogunwande, trical conductivity (EC), according to standard methods (USDA and
2011). Passive aeration is an effective inexpensive treatment sys- USCC, 2001). The major cations (Ca, Mg, K, Na) and metals (Zn, Mn,
tem for co-composting poultry manure, poultry litter and sawdust, Cu) were quantified using an atomic absorption spectrophotome-
according to Ogunwande and Osunade (2011), and is more cost- ter (Varian model 220 A). The percentages of OM and TN losses
effective than active aeration systems in terms of initial capital were calculated using Eqs. (1) and (2), according to Paredes et al.
investment, operation, maintenance, and operator training costs (2000).
(Solano et al., 2001).  
The use of toxicity tests on aquatic and terrestrial organisms X1 ð100
X2 Þ
OM loss ð%Þ ¼ 100
100  ð1Þ
allows the integral assessment of the waste before disposal. Sev- X2 ð100
X1 Þ
eral authors have reported acute toxicity on several organisms  
exposed to raw (Gupta and Kelly, 1992; Gupta et al., 1997) and X1 N2
TN loss ð%Þ ¼ 100
100  ð2Þ
composted poultry manure (Komilis and Tziouvaras, 2009). Daph- X2 N1
nia magna has demonstrated a good sensitivity to assess toxicity of
where N1 and N2 are the initial and final TN percentages, and X1 and
landfill leachate (Matejczyk et al., 2011; Pablos et al., 2011), haz-
X2 are the initial and final ash percentages, respectively.
ardous wastes (Pablos et al., 2009) and municipal solid waste lea-
Commercial kits (Rida CountÒ) were used for microbiological
chate (Isidori et al., 2003; Bortolotto et al., 2009). Germination
characterization to determine total coliforms, Escherichia coli and
index (GI) is the phytotoxicity index used commonly to assess tox-
Salmonella spp. (CFU g
1) provided by R-Biopharm AG.
icity from complex solid samples, such as waste or compost. How-
Biological activity was measured using the static respiration
ever, there is a lack of ecotoxicological tools in the literature when
index (SRI) (Iannotti et al., 1993; USDA and USCC, 2001). This tech-
a material demonstrates high toxicity. In addition, integral moni-
nique is a static respiration stability assessment method which is
toring strategies have not previously been used to study a passive
performed in mesophilic temperatures (37 °C) with sealed
aeration composting of poultry manure with low quantities of
500 mL flasks. An electrochemical dissolved O2 electrode is placed
carbon-rich materials. The aims of this study were: (1) to develop
in the headspace of the flask and records the O2 air concentration
phytotoxicity indexes for waste or compost samples, and (2) to
drops within the flask. Oxygen uptake rate (OUR) is finally
assess the correlation between ecotoxicological endpoints with
expressed in mg O2 g
1 OM h
1 and is calculated via the slope of
both stability and physicochemical parameters during passive aer-
the O2 concentration drop. The SRI is the maximum averaged
ation composting of poultry manure. A seed toxicity test was used
OUR calculated during a 24 h period (after the initial lag time).
to assess effects on a terrestrial plant since our objective was to
develop the phytotoxicity indexes. On the other hand, D. magna
test was selected to assess possible leachate or run-off toxicity, 2.2. Toxicity tests
since it is a standard toxicity test widely used in monitoring pro-
grams of different kind of samples. In order to simulate the mixture of water-extractable sub-
stances present in leachate or runoff, aqueous extracts were pre-
pared mixing a dry sample with deionized water (1:10 w/v).
2. Materials and methods These extracts were stirred at room temperature (23 ± 2 °C),
according to a procedure described by Tiquia et al. (1996).
2.1. Composting
2.2.1. Organisms
2.1.1. Experiment Two species of plants and an aquatic crustacean were used as
Poultry manure was collected in an automatized farm of the test organisms. A non-chemically treated seed lot of lettuce (Lac-
ZucamiÒ type, located in Hurlingham, Argentina. Six 2-m3 tuca sativa variety ‘‘Carilauquen INTA”) and radish (Raphanus sati-
(1 m  1 m  1.2 m) composting piles were built mixing poultry vus variety ‘‘Puntas blancas”) were obtained from the
manure with dry grass (7:3 v/v) in an experimental field of the experimental stations of the INTA, located in La Consulta and San
National Institute of Agricultural Technology (INTA), Hurlingham, Juan cities, Argentina, respectively. Seeds were kept in a dry envi-
Argentina. Composting piles had an initial C:N ratio of ronment at 4 °C.
24.6 ± 3.6:1 and moisture content of 70.6 ± 3.2% wb. In addition, The aquatic crustacean D. magna was reared in a laboratory of
wood chips were added as bulking agent. A static pile with a pas- ecotoxicology (IMYZA, INTA). The population of daphnid was fed
sive aeration system and V-shaped pipe configuration was used as 3–4 times per week with a mixture of several species of algae,
composting method, according to Ogunwande (2011). A pipe with under controlled conditions (23 ± 2 °C and 16L:8D). Dechlorinated
35-mm diameter perforations was used, as recommended by and aerated tap water (pH = 8.1 ± 0.3; EC = 642 ± 24 lS cm
1) was
Ogunwande and Osunade (2011). used as culture medium.
Experimental design consisted of a completely randomized sta-
tistical design with 6 composting piles (n = 6) and repeated mea- 2.2.2. Seed germination and root elongation test
surements. Each composting pile was the experimental unit Seed germination and root elongation tests were carried out at
(n = 6 piles). Sampling was done by quartering from each pile at 22 ± 2 °C in darkness for 120-h, according to standardized proto-
days 0, 14, 56 and 92 (n = 24), according to standardized specifica- cols (Sobrero and Ronco, 2004). Experimental design consisted of
tions (USDA and USCC, 2001). Three sub-samples were taken from 10 treatments (i.e. 9 different concentrations of the extracts and
 B.J. Young et al. / Waste Management 54 (2016) 101–109 103

a control group) per composting pile and per sampling time turned bins after a period of 11 weeks. The samples of the
(n = 240) using triplicates. The extract concentrations used in the untreated and treated effluent were collected in the treatment sys-
tests ranged from 0.5 to 100% v/v (0.5, 1; 5, 10, 20, 40, 60, 80, tem from an anaerobic bioreactor, according to Young et al. (2012).
and 100%); deionized water was used as a negative control and The anaerobic bioreactor was loaded daily with 35 kg of cereal
zinc chloride solutions as positive control. A total of 15 tests were residues and 125 L of treated effluents. Untreated and treated efflu-
conducted using lettuce and 18 tests using radish. Fifteen seeds of ents were obtained from the inflow into the first treatment pond
each the species (radish and lettuce) were exposed to 4-mL of each and the recirculated flow to the bioreactor respectively.
of the nine extract concentrations and control water in 90-mm Values of the phytotoxicity indexes (RGIC0.8 and GIC80%) were
diameter Petri dishes lined with filter paper (Munktell AB differentiated into two categories according to the toxicity effects
Box 300, SE-790 20 GRYCKSBO, Sweden). A total of 10,800 seeds observed:
of each species were used in these experiments. The quality con-
trols used were percentage of germination over 90%, coefficient – Inhibitory effects: 6100%.
of variation for root elongation below 30%, in negative controls, – Non-inhibitory effects: >100%.
whilst Zn (zinc chloride) was used as a reference toxic in positive
controls. The zinc chloride concentrations at each positive control 2.2.3. D. magna immobilization test
were: 18.75, 37.5, 75, 150, 300 mg Zn L
1. The Daphnia immobilization test was used to assess the acute
Toxicity endpoints assessed were seed germination and root toxicity from composting extracts (USEPA, 1996). Toxicity tests
elongation (Inhibition concentration 50 [IC50, no-observed-effect were carried out by triplicate. Experimental design consisted in
concentration [NOEC], lowest-observed-effect concentration 10 treatments for each composting pile and sampling time
[LOEC], relative growth index [RGI], and germination index [GI]). (n = 240). Extract concentrations used in the tests ranged from
Alterations in germination and normal development of seedlings 0.1 to 80% v/v (0.1, 1, 4, 8, 15, 25, 40, 60, and 80%), and a negative
were recorded. The root elongation length was used to calculate control. Ten neonates <24-h of hatching were exposed during 48-h
RGI (Eq. (3)) (Alvarenga et al., 2007). RGI values between 0 and in a static system, containing 30-mL of each of nine the extract
0.8 are categorized as inhibition of root elongation (I), values concentration or control water. A total of 7200 daphnids were used
>0.8 and <1.2 as no-significant-effect (NSE), and values >1.2 as in these experiments. Toxicity endpoints assessed were effective
stimulation of root elongation (S) (Young et al., 2012). The number concentration 50 (EC50), NOEC, and LOEC. The quality controls used
of germinated seeds and root elongation length were used to calcu- were immobilization under 10% in negative controls and Cr (potas-
late GI (Eq. (4)) (Zucconi et al., 1981). GI values lower than 80% sium dichromate) as reference toxic in positive controls. The potas-
were considered to indicate inhibition (Tiquia et al., 1996). sium dichromate concentrations at each positive control were: 0.1,
RLS 0.2, 0.3, 0.4 and 0.5 mg Cr L
1.
RGI ¼ ð3Þ
RLC
2.3. Statistical analysis
RLS GSS
GI ð%Þ ¼   100 ð4Þ
RLC GSC The temporal variation of parameters was assessed by one-way
ANOVA. When the F values of the ANOVA were significant
where RLS is the radicle length of the sample, RLC is the radicle
(p < 0.05), means were compared by the Tukey’s pair wise test.
length of the control, GSS is the number of germinated seeds in
The influence of physicochemical parameters on the ecotoxicolog-
the sample and GSC is the number of germinated seeds in the
ical endpoints and biological activity was also assessed by multi-
control.
variate statistical procedures, such as principal component
Two phytotoxicity indexes (RGIC0.8 and GIC80%) are proposed
analysis (PCA) and correlation analysis (Pearson correlation
herein to assess the maturity of composted manure. RGIC0.8 esti-
coefficient).
mates the lowest extract concentration to get an inhibition of root
elongation (RGI = 0.8). GIC80% estimates the lowest extract concen-
tration to get a response of 80% in GI. The validation process of 3. Results and discussion
these new phytotoxicity indexes was conducted using published
and unpublished data of our group from several types of samples. 3.1. Composting
Phytotoxicity indexes were applied to data of four samples of com-
post and two samples of effluents. The poultry manure derived 3.1.1. Physicochemical characterization
compost (PMC) was produced after a period of 12 weeks, according The variation of the ambient and pile temperature profiles
to Rizzo et al. (2013). Poultry manure was mixed with corn bare showed a similar tendency from 40 days (Fig. 1). As was reported
cobs, sawdust and shavings. Composting piles were manually by other authors, two main phases can be seen in the temperature
turned. The poultry litter and horse manure derived compost profile of composting piles. The average maximum temperature of
(PLHMC) was composted in an experimental field of the INTA after the piles ranged between 40 and 46 °C and lasted for five days, as
a period of 16 weeks, according to Riera et al. (2014). Poultry litter shown in Fig. 1 (top). However, some piles reached a maximum of
contained a mixture composed by poultry manure, feathers, spilled 60.5 °C. The maturation phase started from day 40, when the tem-
feed, and bedding material. Active aeration composting was perature of the piles was similar to ambient temperature. Passive
obtained in manually turned bins. The municipal solid waste aeration systems reach lower temperatures than active aeration
derived compost (MSW1) was obtained from a composting facility systems (Barrington et al., 2003). Silva et al. (2009) reported that
in Trenque Lauquen (Argentina). Organic fraction of MSW was sep- co-composting of poultry manure with low quantities of carbon-
arated at home and then composted in the plant for 16 weeks. rich materials (80:20 ratio) reached a maximum pile temperature
Active aeration composting was conducted in manually and lower than 40 °C. However, Ogunwande and Osunade (2011) com-
mechanically turned piles. Other municipal solid waste derived pared three passive aeration composting of poultry manure that
compost (MSW2) was obtained from a composting facility in reached a thermophilic phase above 42 °C that lasted for approxi-
Metropolitan Area of Buenos Aires (Argentina). Organic fraction mately 20 days. However, this longer thermophilic phase could be
of MSW was manually and mechanically separated within the due the initial composition of the composting piles. Ogunwande
plant. Active aeration composting was obtained in manually and Osunade (2011) evaluated composting with sawdust, poultry
104 B.J. Young et al. / Waste Management 54 (2016) 101–109

Fig. 1. (Top): Average temperature of ambient air and of the inside of the composting piles (average based on n = 6) error bars demonstrate standard deviations), (Middle):
Average cumulative losses (±standard error) of the OM and TN (%) and average SRI (mg O2 g
1 OM h
1) during the composting period; (Bottom): Average values (±standard
error) of the phytotoxicity indexes measured during the composting period.

manure and litter. Poultry manure is characterized by a high rela- parameters showed a significant decrease as well, such as MC,
tive density, whereas sawdust and poultry litter are materials with TOC, SC, TN, Ca and Mg (Table 1). The MC was kept above 60% by
low density that could improve the total porosity of the mix. In this manual irrigation. The pH was remained slightly alkaline from
study, the initial composition had a high proportion of poultry day 14, then increased and the final pH was <9. This increase could
manure (70%) which could have affected the porosity and thus be attributable to proteolysis and high microbial activity during
oxygen diffusion. the first days of composting (Bustamante et al., 2008). Authors
Both the organic and inorganic content decreased (OM = 34.8% reported similar pH values using passive and active aeration sys-
and EC = 54.5%; p < 0.05) during the biodegradation period. Other tems (Ogunwande and Osunade, 2011; Rizzo et al., 2013). The high
 B.J. Young et al. / Waste Management 54 (2016) 101–109 105

Table 1
Mean (±SD) physicochemical and microbiological parameters of the six composting piles at each sampling time and limit values of final composts.

Parameter 0-d 14-d 56-d 92-d Target value or range/upper limit Reference
a b b b
pH 6.8 ± 0.3 7.7 ± 0.3 7.6 ± 0.4 8.2 ± 0.4 6–8/9 WRAP (2011)
EC (mS cm
1) 17.6 ± 1.9a 13.3 ± 5.5ab 10.8 ± 2.9b 8.0 ± 1.8b <0.6/1.5 WRAP (2011)
C:N ratio 24.6 ± 3.6a 31.6 ± 3.1a 23.4 ± 3.6a 24.8 ± 3.7a 20:1 SENASA (2011)
OM (%) 70.1 ± 1.3a 62.1 ± 3.0b 51.1 ± 6.8c 45.7 ± 2.7c P15 SENASA (2011)
MC (%) 70.6 ± 3.2a 67.8 ± 3.8a 64.6 ± 4.9ab 60.4 ± 4.2b 35–40/50 WRAP (2011)
TOC (%) 35.0 ± 0.7a 31.0 ± 1.5b 25.5 ± 3.4c 22.9 ± 1.3c – –
TN (%) 1.4 ± 0.2a 1.0 ± 0.1b 1.1 ± 0.2b 0.9 ± 0.1b NPK P 6 SENASA (2011)
TP (mg g
1) 20.3 ± 3.1a n.d. n.d. 24.8 ± 3.0a NPK P 6% SENASA (2011)
SC (%) 2.5 ± 0.3a n.d. n.d. 1.2 ± 0.3b – –
SP (mg g
1) 0.5 ± 0.1a n.d. n.d. 0.5 ± 0.1a NPK P 6% SENASA (2011)
Ca (mg L
1) 79.8 ± 15.4a n.d. n.d. 31.5 ± 40.8b >1% SENASA (2011)
Mg (mg L
1) 125.2 ± 30.3a n.d. n.d. 70.9 ± 47.5b >0.05% SENASA (2011)
K (mg L
1) 1636.6 ± 165.9a n.d. n.d. 1695.3 ± 861.3a NPK P 6% SENASA (2011)
Na (mg L
1) 472.9 ± 39.9a n.d. n.d. 468.9 ± 139.7a <100/150 WRAP (2011)
Zn (mg L
1) 0.9 ± 0.5a n.d. n.d. 0.6 ± 0.6a <150/400 WRAP (2011)
Mn (mg L
1) 2.0 ± 0.5a n.d. n.d. 1.1 ± 0.3b – –
Cu (mg L
1) 1.5 ± 1.3a n.d. n.d. 0.9 ± 0.7a <50/100 WRAP (2011)
Total coliforms (CFU) 8.0  106 ± 1.0  107a n.d. n.d. 7.5  106 ± 9.3  106a – –
E. coli (CFU) 1.9  107 ± 1.1  107a n.d. n.d. 7.5  106 ± 9.1  106a Absent/1000 WRAP (2011)
Salmonella spp. Absent n.d. n.d. Absent Absent/zero WRAP (2011)

Different letters indicate significant differences (p < 0.05) among sampling times.
EC = Electrical conductivity; OM = Organic matter; MC = Moisture content; TOC = Total organic carbon; TN = Total Kjeldahl nitrogen; SC = Soluble carbon; TP = Total phos-
phorous; SP = Soluble phosphorous; n.d. = no data.

initial values of EC could be associated to the poultry diet (Bolan coefficients of variation between the averages of root length in
et al., 2010). Although EC decreased, the compost obtained had the negative controls were 19.6 and 23.3% for lettuce and radish,
restrictions in use due to a high EC final value. Active aeration sys- respectively, lower than that recommended in the test protocols.
tems may reach a higher decrease of EC due to salt loss by higher The IC50 average values of root elongation in the positive controls
leaching (Rizzo et al., 2013). Such high EC values in poultry manure were 55.4 ± 16.9 (n = 15) and 82.6 ± 15.1 (n = 18) mg L
1 of Zn for
compost were found by Komilis and Tziouvaras (2009). lettuce and radish respectively. On the other hand, the average
The highest losses of OM and TN were registered during the first value of immobilized neonates of D. magna in the negative controls
14 days (Fig. 1-middle). The cumulative losses of OM and TN at day was 2.2%, lower than that recommended in the test protocols. The
92 were of 64.1 ± 2.1% and 68.1 ± 10.4% respectively. Also, a posi- EC50 average value in the positive controls was 0.30 ± 0.07 (n = 21)
tive correlation between OM and EC (R2 = 0.77) was found. TN loss mg L
1 of Cr.
was associated with the gradual increase of pH during the first
14 days (52.1%), which could increase the volatilization of N-NH+3. 3.2.2. Exposure to extracts
Ogunwande (2011) compared three passive aeration systems and Toxicity tests carried out on terrestrial plant species (lettuce
reported a TN loss of 38.1% until day 14, lower than those found and radish) allowed determining the quality of the compost as a
in this study. Tiquia and Tam (2002) reported similar losses of soil amendment, whereas on the aquatic organism (daphnid)
TN (58%) using a forced-aeration system for composting of poultry allowed determining the potential toxicity of leachates or runoff.
litter. On the other hand, Parkinson et al. (2004) found a higher TN The three organisms exposed to aqueous extracts showed acute
loss in active aeration system than in passive aeration system. Both toxicity in all samples. Ecotoxicological endpoints of the test
moderate temperatures and presence of microbial groups that organisms at each sampling time can be found in Table 2. The aver-
increase and/or maintain the pool of N, such as N-fixing and nitri- age EC50 or IC50 of the 3 species was 8.29 ± 0.35% (n = 18) in the ini-
fying bacteria (Paredes et al., 1996). It could have caused a decrease tial sampling (day 0) and 31.12 ± 10.99% (n = 18) in the final
in TN loss during the mesophilic phase. sampling (day 92). Composting reduced the average toxicity by
22.8% for the 3 species. The sensitivity of the organisms measured
3.1.2. Stability and microbiological contents in terms of EC50 or IC50 was highest for daphnid, followed by let-
The highest pile temperature and biological activity tuce and then radish. Rizzo et al. (2013) also found lettuce to be
(SRI = 1.12 mg O2 g OM h
1) were recorded during the first 14 days more sensitive to adverse effects than radish. Endpoints of immo-
(Fig. 1). The SRI showed a negative correlation with SC and Mn (R2: bilization (D. magna) and root elongation (L. sativa and R. sativus)
0.92 and 0.76 respectively; Table 3), whereas it showed a slight exhibited toxic response in all samples. Delgado et al. (2013)
positive correlation with TP (R2: 0.65; p < 0.05). Low values of SRI reported high mortality on daphnid exposed to poultry manure
at end of the process (day 92) indicated that biological stability leachates. Root elongation was an endpoint with most sensitivity
(SRI 6 0.5 mg O2 g
1 OM h
1) was reached. Salmonella spp. was than seed germination for the both plant species, as reported by
not detected during composting. However, the high counts of total Fuentes et al. (2004). Seed germination exhibited no toxic response
coliforms and E. coli observed in all piles and at all sampling times in 17 and 33% of the samples for LOEC, NOEC and IC50 respectively
suggest that these pathogens survived the short thermophilic at day 14 and 56 for lettuce and at day 0 for radish. In addition, this
phase of the process, which indicates the low quality of the derived endpoint exhibited no toxic response from day 14 for radish. Sev-
compost. eral authors have reported the genotoxicity of leachate landfill and
compost extracts on terrestrial plants and bacteria (Cabrera et al.,
3.2. Toxicity tests 1999; De Simone et al., 2005; Kwasniewska et al., 2012). Gupta
and Kelly (1992) demonstrated that poultry litter leachate may
3.2.1. Quality controls induce mutagenicity using the Ames test. Further studies could
Results of the toxicity tests were acceptable according to the focus on assessing the capability of composting to reduce the geno-
criteria established by the quality controls. In the seed tests, the toxicity of poultry manure.
106 B.J. Young et al. / Waste Management 54 (2016) 101–109

Table 2
Mean (95% CI) ecotoxicological endpoints of the test organisms at each sampling time.

Endpoint 0-d 14-d 56-d 92-d

Lettuce
Seed germination
IC50 (%) 23.5 [15.6–31.5]a 69.5 [61.5–77.4]**b 60.0 [49.2–70.9]**bc 46.8 [44.4–49.3]c
LOEC (%) 28.3 [17.7–39.0]a 70.0 [54.0–86.0]*b 64.0 [50.6–77.4]*b 50.0 [41.2–58.8]ab
NOEC (%) 14.2 [8.8–19.5]a 50.0 [34.0–66.0]*b 44.0 [30.6–57.4]*b 30.0 [21.2–38.8]ab
Root elongation
IC50 (%) 8.8 [3.6–14.0]a 45.4 [26.5–64.4]b 55.0 [39.8–70.2]b 32.9 [24.7–41.1]ab
LOEC (%) 7.8 [1.9–13.6]a 25.2 [10.7–41.3]a 38.3 [16.6–60.1]a 28.3 [17.7–39.0]a
NOEC (%) 3.6 [0.5–6.7]a 12.3 [3.9–20.7]a 24.2 [6.9–41.4]a 14.2 [8.8–19.5]a
Radish
Seed germination
IC50 (%) 63.3 [47.7–78.8]** n.t. n.t. n.t.
LOEC (%) 88.0 [73.7–100.0]* n.t. n.t. n.t.
NOEC (%) 68.0 [53.7–82.3]* n.t. n.t. n.t.
Root elongation
IC50 (%) 9.7 [4.4–15.0]a 51.8 [34.4–69.2]b 54.1 [39.5–68.7]b 41.6 [20.6–62.7]ab
LOEC (%) 12.0 [0.0–24.3]a 49.0 [21.2–76.8]a 54.2 [33.9–74.5]a 41.0 [11.6–70.4]a
NOEC (%) 5.5 [0.0–11.9]a 32.2 [8.1–56.3]a 36.8 [21.4–52.3]a 26.9 [3.0–50.8]a
Daphnid
EC50 (%) 6.7 [4.3–9.0]a 26.5 [16.6–36.4]ab 29.3 [20.1–38.6]b 28.5 [14.8–42.2]b
LOEC (%) 9.0 [5.0–13.0]a 27.6 [17.0–38.2]a 33.3 [16.4–50.2]a 28.2 [12.4–43.9]a
NOEC (%) 4.3 [1.8–6.8]a 16.8 [9.8–23.8]a 21.0 [9.0–33.0]a 17.3 [6.4–28.3]a

Different letters indicate significant differences (p < 0.05) among sampling times.
n.t.: no toxicity response.
*
A total of 83% of the samples exhibiting a toxic response.
**
A total of 67% of the samples exhibiting a toxic response.

Table 3
Values of the new phytotoxicity indexes on several types of samples to validate the proposed methodology.

Type of sample Waste origin Treatment Seed RGIC0.8 GIC80% Reference

Immature compost (PMC) Poultry manure Active composting Lettuce 19.67 16.11 Rizzo et al. (2013)
Radish 34.40 21.77
Immature compost (PLHMC) Poultry manure Active composting Lettuce 35.99 30.88 Riera et al. (2014)
Radish 24.63 20.32
Immature compost (MSW1) Organic fraction of MSW Active composting Lettuce 40.61 45.56 Unpublished data
Radish 87.88 73.45
Mature compost (MSW2) Organic fraction of MSW Active composting Lettuce 108.76 104.19 Unpublished data
Untreated effluent Cereal residues Anaerobic biodigestion Lettuce 18.02 24.77 Young et al. (2012)
Treated effluent 111.35 121.57

PMC: poultry manure derived compost. PLHMC: poultry litter and horse manure derived compost. MSW1 and MSW2: municipal solid waste derived compost.

Fig. 2. Principal components analysis (PCA) shows the association between physicochemical parameters and ecotoxicological endpoints with respect to the first two
components. Data of IC50 of lettuce and radish are only shown for root elongation. Black dots indicate each composting pile and sampling time.
Table 4
Pearson correlation coefficients among various parameters measured at four sampling times at six composting piles (n = 24).

Physico-chemical parameters Stability D. L. sativa (lettuce) R. sativus (radish)

magna
Ash OM or SC Ca Mg Na Mn Cu SRI NOEC or NOEC or IC50 s.g. NOEC or GRIC0.8 GIC80% NOEC or GRIC0.8 GIC80%
TOC LOEC LOEC r.e. LOEC s.g. LOEC r.e.
Physico-chemical pH n.s. n.s.
0.75* 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.
parameters EC
0.80** 0.77** 0.83** n.s. n.s. n.s. 0.79* n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Ash
0.78** 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.

B.J. Young et al. / Waste Management 54 (2016) 101–109

OM or TOC 0.92** n.s. n.s. n.s. 0.78* n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
TN 0.87** 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.
SC 0.77* n.s. n.s. 0.78* n.s.
0.92** n.s. n.s.
0.86** n.s. n.s. n.s. n.s. n.s. n.s.
Ca 0.94** n.s. 0.75* n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
K 0.97** n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Zn n.s. 0.84** n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Mn n.s.
0.76* n.s. n.s.
0.79*
0.91** n.s. n.s. n.s. n.s. n.s.
Stability SRI n.s. n.s. 0.85** n.s. n.s. n.s. n.s. n.s. n.s.
**
D. magna EC50 0.90 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
L. sativa (lettuce) IC50 r.e. 0.85** n.s. 0.78** 0.90** 0.82** n.s. n.s. n.s.
NOEC or n.s. n.s. 0.84** 0.91** n.s. n.s. n.s.
LOEC r.e.
IC50 s.g. 0.83** n.s. n.s. n.s. n.s. n.s.
GRIC0.8 0.79** n.s. n.s. n.s.
R. sativus (radish) IC50 r.e. 0.89** 0.81** 0.81**
NOEC or 0.87** 0.87**
LOEC r.e.
GRIC0.8 0.98**

n.s.: not significant; r.e.: root elongation; s.g.: seed germination; OM and TOC are shown together because they have the same correlation coefficient values; NOEC and LOEC are shown together because they have the same
correlation coefficient value.
*
p < 0.01 significant parameters are only shown.
**
p < 0.001 significant parameters are only shown.

107
108 B.J. Young et al. / Waste Management 54 (2016) 101–109

3.2.3. Phytotoxicity indexes relationship between electrical conductivity and increasing toxic-
RGI and GI have been used to assess the toxicity of composting ity. Specifically, authors associated conductivity with the inhibition
samples (Tiquia and Tam, 2000a; Tiquia, 2010). The results of root elongation on seeds of lettuce (Young et al., 2012). The IC50
obtained in the present study are similar to those reported by other showed a negative correlation with SC, Mn and TN (R2: 0.86, 0.79,
authors. GI values in the 100% extract concentration were zero in 0.71 respectively), whereas showed a positive correlation with
62.5% of the samples for lettuce (average GI value = 3.88%; both SRI and TP (R2: 0.85 and 0.70 respectively). The lack of strong
n = 24) and in 16.6% of samples for radish (average GI correlation between maturity and stability indexes was also
value = 21.07%; n = 24). Komilis and Tziouvaras (2009), for exam- observed in Oviedo et al. (2015) as well as in Komilis and
ple, found GI values between 0 and 6% in extract concentration Tziouvaras (2009). Komilis and Tziouvaras (2009) reported nega-
of 100% (raw extract) of poultry manure derived compost using tive correlations between GI of cress and both TOC and TN. How-
radish, lettuce, pepper (Capsicum spp.), spinach (Spinacia oleracea), ever, we found a negative correlation between GIC80% of lettuce
tomato (Lycopersicon esculentum), cress (Lepidium sativum) and and Mn (R2: 0.69). Both lettuce (R2: 0.79) and radish (R2: 0.98)
cucumber (Cucumis sativus). If raw extract inhibits germination was obtained a positive correlation between RGIC0.8 and GIC80%.
completely, RGI and GI lose their value as indexes. For this reason, The lower correlation between these phytotoxicity indexes for let-
Komilis and Tziouvaras (2009) had excluded GI data of poultry tuce could be attributable to a higher inhibition of seed
manure derived compost from correlation analysis. An alternative germination.
experimental strategy was proposed by Morel et al. (1985), who
determined GI using three aqueous extract concentrations (10,
20 and 40% w/v) (Silva et al., 2009). However, this methodology 4. Conclusions
cannot be used with any type of sample because the concentra-
tions depend on the toxicity degree. Therefore, we propose to use 1. The proposed monitoring strategy demonstrated the low effec-
RGIC0.8 and GIC80% as cut-off values to indicate the lowest concen- tiveness of passive aeration systems to treat poultry manure
tration that induces inhibitory effects. Also, values lower than or that is present in high percentages in composting piles (>70%).
equal to 100% indicate any toxicity degree from a sample or imma- 2. Although the values of SRI, C:N ratio and OM loss indicated
turity of the compost, whereas values >100% indicate a non- compost stability, E. coli colonies remained higher than the lim-
toxicity degree from a sample or maturity of the compost. These its recommended by international guidelines.
new indexes allow the comparison between samples with different 3. The D. magna endpoints allowed the assessment of possible lea-
toxicity degrees, such as EC50, IC50 or LC50, which are commonly chate or run-off toxicity, which showed positive correlations
used in ecotoxicology. The use of several types of samples allowed with phytotoxicity endpoints.
to analyze the robustness of the indexes during the validation pro- 4. Multivariate analyses demonstrated positive correlations
cess (Table 3). Mature compost (MSW2) and treated effluent between ecotoxicological endpoints (low toxicity) and biologi-
showed non-inhibitory effects, whereas immature compost (PMC, cal activity (stability). A PCA demonstrated that these two
PLHMC and MSW1) and untreated effluent showed inhibitory parameter groups were associated with final sampling time
effects. and showed negative correlations with several other physico-
The RGIC0.8 values showed an increase between the initial and chemical parameters (organic and inorganic contents). The lat-
final sampling time for lettuce from 0.31 to 30.50%, and for radish ter were associated to initial sampling time.
from 0.06 to 52.74% (Fig. 1-bottom). The GIC80% values showed an 5. The RGIC0.8 and GIC80% indexes and salinity indicated that the
increase between the initial and final sampling time for lettuce compost did not reach maturity. As a result, the final compost
from 0.42 to 54.34%, and for radish from 0.06 to 54.76% (Fig. 1- was not considered suitable for use as a soil amendment.
bottom). These values indicate that composting reduced toxicity. 6. The newly proposed phytotoxicity indexes (RGIC0.8 and GIC80%)
However, the maximum values of RGIC0.8 and GIC80% were lower could be used to assess toxicity from complex samples or could
than 100%. Therefore, the composting piles of this study did not be implemented in monitoring strategies as useful ecotoxico-
reach full maturity. Further studies could incorporate these phyto- logical tools.
toxicity indexes to assess several types of samples, such as efflu-
ents, surface water or solid waste extracts.
Acknowledgements
3.2.4. Correlations
Multivariate analysis can suggest a relationship between toxic- The study was funded by the Project PNNAT 1128042 from
ity and the inorganic and organic content. An association between Instituto Nacional de Tecnología Agropecuaria, Argentina. Authors
physicochemical parameters and ecotoxicological endpoints, are grateful to Dr. Carlos Greco for comments on the manuscript.
including the initial and final sampling times (Fig. 2) was detected Dimitrios Komilis thanks Techniospring for the financial support.
after applying Principal Components Analysis (PCA). The results of
this analysis account for 67.6% of the variability of the data matrix.
References
PCA showed two clear groups of parameters associated to each
sampling time. High values of EC, carbon content (TOC and SC), Alvarenga, P., Palma, P., Goncalves, A.P., Fernandes, R.M., Cunha-Queda, A.C., Duarte,
TN, MC, OM and some cations (mainly Mn, Mg and Ca) were asso- E., Vallini, G., 2007. Evaluation of chemical and ecotoxicological characteristics
ciated with the initial time, whereas high values of the ecotoxico- of biodegradable organic residues for application to agricultural land. Environ.
Int. 33, 505–513.
logical endpoints (low toxicity), pH and TP were associated with Barrington, S., Choinière, D., Trigui, M., Knight, W., 2003. Compost convective
the final time. airflow under passive aeration. Bioresour. Technol. 86, 259–266.
A correlation analysis was carried out between physicochemical Bolan, N.S., Szogi, A., Chuasavathi, T., Seshadri, B., Rothrock, M., Panneerselvam, P.,
2010. Uses and management of poultry litter. World Poult. Sci. J. 66, 673–698.
and ecotoxicological parameters (Table 4). The EC50 for daphnid
Bortolotto, T., Bertoldo, J.B., Zanette da Silveira, F., Defaveri, T.M., Silvano, J., Tröger
showed a negative correlation with SC, Mn and Ca (R2: 0.73, 0.72, Pich, C., 2009. Evaluation of the toxic and genotoxic potential of landfill
and 0.69 respectively). Also, correlations were observed between leachates using bioassays. Environ. Toxicol. Pharmacol. 28, 288–293.
phytotoxicity endpoints of lettuce and physicochemical parame- Bustamante, M.A., Paredes, C., Marhuenda-Egea, F.C., Pérez-Espinosa, A., Bernal, M.
P., Moral, R., 2008. Co-composting of distillery wastes with animal manures:
ters. The highest R2 values were obtained for IC50, NOEC and LOEC carbon and nitrogen transformations in the evaluation of compost stability.
of seed germination on lettuce. Pablos et al. (2011) suggested a Chemosphere 72, 551–557.
 B.J. Young et al. / Waste Management 54 (2016) 101–109 109

Cabrera, G.L., Rodriguez, D.M.G., Maruri, A.B., 1999. Genotoxicity of the extracts physicochemical and ecotoxicological approaches to estimate landfill leachates
from the compost of the organic and the total municipal garbage using three toxicity. Waste Manage. 31, 1841–1847.
plant bioassays. Mutat. Res. 426, 201–206. Paredes, C., Bernal, M.P., Cegarra, J., Roig, A., Navarro, A.F., 1996. Nitrogen
De Simone, C., Angelucci, R., Errichetti, M.F., Marconi, S., Rossi, M., Selvi, S., 2005. A transformation during the composting of different organic wastes. In: Van
statistical approach to evaluate compost genotoxicity. Biol. Fert. Soils 41, 9–14. Cleemput, O., Hofman, G., Vermoesen, A. (Eds.), Progress in Nitrogen Cycling
Delgado, M., Miralles de Imperial, R., Alonso, F., Rodríguez, C., Martín, J.V., 2013. Studies. Springer, Netherlands, pp. 121–125.
Ecotoxicity bioassays on leachates from poultry manure. Bull. Environ. Contam. Paredes, C., Roig, A., Bernal, M.P., Sánchez-Monedero, M.A., Cegarra, J., 2000.
Toxicol. 90, 401–404. Evolution of organic matter and nitrogen during co-composting of olive mill
Edwards, D.R., Daniel, T.C., 1992. Environmental impacts of on-farm poultry waste wastewater with solid organic wastes. Biol. Fert. Soils 32, 222–227.
disposal—a review. Bioresour. Technol. 41, 9–33. Parkinson, R., Gibbs, P., Burchett, S., Misselbrook, T., 2004. Effect of turning regime
FAOSTAT, 2013. Statistics of Food and Agricultural Commodities Production. Food and seasonal weather conditions on nitrogen and phosphorus losses during
and Agriculture Organization of the United Nations <http://www.fao.org> aerobic composting of cattle manure. Bioresour. Technol. 91, 171–178.
(verified August 2015). Petric, I., Šestan, A., Šestan, I., 2009. Influence of initial moisture content on the
Frank, R., Stonefield, K.I., Luyken, H., 1988. Monitoring wood shaving litter and composting of poultry manure with wheat straw. Biosyst. Eng. 104, 125–134.
animal products for polychlorophenols residues, Ontario, Canada, 1978–1986. Riera, N., Torre, V.D., Rizzo, P.F., Butti, M., Bressan, F., Zarate, N., Weigandt, C.,
Bull. Environ. Contam. Toxicol. 40, 468–474. Crespo, D., 2014. Composting process evaluation of two mixtures of poultry
Fuentes, A., Lloréns, M., Sáez, J., Aguilar, M.I., Ortuño, J.F., Meseguer, V.F., 2004. manures. Rev. FCA UNCUYO 46, 195–203.
Phytotoxicity and heavy metals speciation of stabilised sewage sludges. J. Rizzo, P., Torre, V., Riera, N., Crespo, D., Barrena, R., Sánchez, A., 2013. Co-
Hazard. Mater. 108, 161–169. composting of poultry manure with other agricultural wastes: process
Gupta, G., Borowiec, J., Okoh, J., 1997. Toxicity identification of poultry litter performance and compost horticultural use. J. Mater. Cycles Waste Manage.,
aqueous leachate. Poult. Sci. 76, 1364–1367. 1–9
Gupta, G., Kelly, P., 1992. Poultry litter toxicity comparison from various bioassays. SENASA, 2011. Manual of Procedures for Registering Products in the National
J. Environ. Sci. Health., Part A 27, 1083–1093. Register of Fertilizers, Dressings, Substrates, Protectors, Conditioners and Raw
Iannotti, D.A., Pang, T., Toth, B.L., Elwell, D.I., Keener, H.M., Hoitink, H.A., 1993. A Materials. Resolution N° 264/11. Argentina. <http://www.senasa.gov.ar/>
quantitative respirometric method for monitoring compost stability. Compost (verified August 2015) (in Spanish).
Sci. Util. 1, 52–65. Silva, M.E., Lemos, L.T., Cunha-Queda, A.C., Nunes, O.C., 2009. Co-composting of
Isidori, M., Lavorgna, M., Nardelli, A., Parrella, A., 2003. Toxicity identification poultry manure with low quantities of carbon-rich materials. Waste Manage.
evaluation of leachates from municipal solid waste landfills: a multispecies Res. 27, 119–128.
approach. Chemosphere 52, 85–94. Sobrero, C., Ronco, A.E., 2004. Ensayo de toxicidad aguda con semillas de L. sativa.
Jackson, B.P., Bertsch, P.M., Cabrera, M.L., Camberato, J.J., Seaman, J.C., Wood, C.W., In: Castillo Morales, G. (Ed.), Ensayos toxicológicos y métodos de evaluación de
2003. Trace element speciation in poultry litter. J. Environ. Qual. 32, 535–540. calidad de aguas: Estandarización, intercalibración, resultados y aplicaciones,
Komilis, D.P., Tziouvaras, I.S., 2009. A statistical analysis to assess the maturity and Mexico, pp. 71–79.
stability of six composts. Waste Manage. (New York, N.Y.) 29, 1504–1513. Solano, M.L., Iriarte, F., Ciria, P., Negro, M.J., 2001. SE—structure and environment:
Kwasniewska, J., Nałe˛cz-Jawecki, G., Skrzypczak, A., Płaza, G.A., Matejczyk, M., 2012. performance characteristics of three aeration systems in the composting of
An assessment of the genotoxic effects of landfill leachates using bacterial and sheep manure and straw. J. Agr. Eng. Res. 79, 317–329.
plant tests. Ecotoxicol. Environ. Saf. 75, 55–62. Tiquia, S.M., 2010. Reduction of compost phytotoxicity during the process of
Matejczyk, M., Płaza, G.A., Nałe˛cz-Jawecki, G., Ulfig, K., Markowska-Szczupak, A., decomposition. Chemosphere 79, 506–512.
2011. Estimation of the environmental risk posed by landfills using chemical, Tiquia, S.M., Tam, N.F.Y., 2000a. Co-composting of spent pig litter and sludge with
microbiological and ecotoxicological testing of leachates. Chemosphere 82, forced-aeration. Bioresour. Technol. 72, 1–7.
1017–1023. Tiquia, S.M., Tam, N.F.Y., 2000b. Fate of nitrogen during composting of chicken litter.
Morel, J., Colin, F., Germon, J., Godin, P., Juste, C., 1985. Methods for the evaluation of Environ. Pollut. (Barking, Essex: 1987) 110, 535–541.
the maturity of municipal refuse compost. In: Gasser, J. (Ed.), Composting of Tiquia, S.M., Tam, N.F.Y., 2002. Characterization and composting of poultry litter in
Agricultural and Other Wastes. Elsevier Applied Science. forced-aeration piles. Process Biochem. 37, 869–880.
Ogunwande, G.A., 2011. Feasibility study of a V-shaped pipe for passive aeration Tiquia, S.M., Tam, N.F.Y., Hodgkiss, I.J., 1996. Effects of composting on phytotoxicity
composting. Waste Manage. Res.: J. Int. Solid Wastes Pub. Cleansing Assoc., of spent pig-manure sawdust litter. Environ. Pollut. (Barking, Essex: 1987) 93,
ISWA 29, 240–248. 249–256.
Ogunwande, G.A., Osunade, J.A., 2011. Passive aeration composting of chicken litter: Tuomela, M., Vikman, M., Hatakka, A., Itävaara, M., 2000. Biodegradation of lignin in
effects of aeration pipe orientation and perforation size on losses of compost a compost environment: a review. Bioresour. Technol. 72, 169–183.
elements. J. Environ. Manage. 92, 85–91. USDA, USCC, 2001. Test Methods for the Examination of Composting and Compost.
Ogunwande, G.A., Osunade, J.A., Adekalu, K.O., Ogunjimi, L.A., 2008. Nitrogen loss in Edaphos International, Department of Agriculture and Composting Council,
chicken litter compost as affected by carbon to nitrogen ratio and turning USA, Houston.
frequency. Bioresour. Technol. 99, 7495–7503. USEPA, 1996. Ecological Effects Test Guidelines OPPTS 850.1010 Aquatic
Oviedo, E., Torres, P., Marmolejo, L., Hoyos, L., Gonzales, S., Barrena, R., Komilis, D., Invertebrate Acute Toxicity Test, Freshwater Daphnids. EPA 712-C-96-114
Sánchez, A., 2015. Stability and maturity of biowaste composts derived by small Environmental Protection Agency, USA.
municipalities: correlation among physical, chemical and biological indices. WRAP, 2011. Guidelines for the Specification of Quality Compost for Use in Growing
Waste Manage. 44, 63–71. Media. Waste & Resources Action Programme, United Kingdom <www.wrap.
Pablos, M.V., Fernández, C., Babín, M.M., Navas, J.M., Carbonell, G., Martini, F., org.uk> (verified August 2015).
García-Hortigüela, P., Tarazona, J.V., 2009. Use of a novel battery of bioassays for Young, B.J., Riera, N.I., Beily, M.E., Bres, P.A., Crespo, D.C., Ronco, A.E., 2012. Toxicity
the biological characterisation of hazardous wastes. Ecotoxicol. Environ. Saf. 72, of the effluent from an anaerobic bioreactor treating cereal residues on Lactuca
1594–1600. sativa. Ecotoxicol. Environ. Saf. 76, 182–186.
Pablos, M.V., Martini, F., Fernandez, C., Babin, M., Herraez, I., Miranda, J., Martinez, J., Zucconi, F., Pera, A., Forte, M., De Bertoldi, M., 1981. Evaluating toxicity of immature
Carbonell, G., San-Segundo, L., García-Hortigüela, P., 2011. Correlation between compost. Biocycle 22, 54–57.