Water Air Soil PollutDOI 10.1007/s11270-011-0864-z
Characterization of Swine Wastewater by ToxicityIdentification Evaluation Methodology (TIE)
C. Alejandra Villamar & Teresa Cañuta &Marisol Belmonte & Gladys Vidal
Received: 25 January 2011 / Accepted: 13 June 2011
# Springer Science+Business Media B.V. 2011
Abstract Since swine wastewater is used by farmers
(around 100%) and total nitrogen (95.5%). This
for soil fertilization, evaluation of toxic compounds or
finding suggests that part of the toxicity comes from
micro-contaminants of separate streams is required.
anionic compounds, such as chlorine.
This paper uses the toxicity identification evaluation(TIE) procedure for the physicochemical and ecotox-
Keywords Acute toxicity. Daphnia magna .
icological characterization of swine wastewater. To
Swine wastewater. TIE
distinguish the most important toxic compounds, aphysicochemical characterization and phase I-TIEprocedure were performed. The acute toxic effect of
swine wastewater and treated fractions (phase II-TIE)were evaluated using Daphnia magna determining
Intensive swine production, due to its input demand
48-h LC50. Results show a high level of conductivity
and the introduction of its residue (e.g., swine
(23.5 μS cm−1), which is explained as due to the
wastewater) into the soil, has resulted in soils
concentration of ions, such as ammonium (NH +–
saturated with nutrients (e.g., N, P, K, organic matter),
N 1.6 g L−1), sulfate (SO 2−
397.3 mg L−1), and
heavy metals (e.g., Cu and Zn), hormones, antibiotics
chlorine (Cl−1,230.0 mg L−1). The acute toxicity of
(tetracycline, sulfamines and β-lactamase), and salts
the swine wastewater was evaluated on D. magna
(e.g., NaCl), which are mobile and can reach
(48-h LC50=3.4%). Results of the different water
subterranean waters producing environmental impacts
treatments indicate that anionic exchange treatments
(Burton and Turner ; Moral et al.
could reduce 22.5% of swine wastewater's acute
Swine wastewater is a mixture of feces (45%),
toxicity by reducing chlorine (to around 51%) and
urine (55%), and washing water, which are character-
conductivity (8.5%). On the other hand, cationic
ized by a high content of organic matter (14.2–26.3 g
exchange treatment increased acute toxicity on D.
BOD5L−1), nutrients (2.4–6.0 gL−1 of total nitrogen,
magna (% RT= −624.4%), by reducing NH +–
and 0.6–1.4 gL−1 of total phosphorus), ions (2.3–5.1 g Cl− L−1, 1.5–3.8 g NH +–
N L−1 and 0.03–0.07 g
N L−1), and other compounds (Blanes-Vidal et
C. A. Villamar : T. Cañuta : M. Belmonte : G. Vidal (*)
al. ; Moral et al. ; Moral et al. Provolo
Engineering and Biotechnology Environmental Group,
and Martínez-Suller The toxic effect,
Environmental Science Center EULA—Chile, University
expressed as median lethal concentration (LC50), of
swine wastewater on aquatic cladocer organisms, such
P.O. Box 160-C, Concepción, Chilee-mail: [email protected]
as Daphnia magna, demonstrate its high toxicity at
Water Air Soil Pollut
low concentrations of this waste (LC50=1–>10%; De
indicator organism and toxicity identification evalua-
la Torre et al. ). Some earlier studies have related
tion (TIE) as the evaluation tool.
the toxicity of Daphnia spp. with ionic compoundslike those found in swine wastewater, whose toxicitymechanisms on algae and cladocers have not been
2 Materials and Methods
studied (Pretti et al. ). They also speculated thatthe toxicity on D. magna would be related with
2.1 Swine Wastewater
enzymatic inhibition and membrane disruption.
Cationic compounds, such as NH +
Swine wastewater was obtained from a small piggery
um), under certain conditions (pH>8.0, temperature,
farm located in southern Chile. Swine wastewater was
and ammonium concentration) can be transformed
sampled after the primary settling treatment and was
into deionized compound, such as NH3 (or ammonia),
transported in polyethylene containers and immedi-
which is toxic to aquatic organisms (Arauzo and
ately stored at approximately 4°C to avoid degrada-
Valladolid ; Blanes-Vidal et al. ; Leung et al.
tion. When the bioassays could not be executed
A study of the effect of treated swine
within 48 h, samples were filtered through a 0.45-
wastewater (24-h LC
on the reproduction and
m pore size nitrocellulose membrane and adapted to
longevity of Daphnia carinata and Moina austral-
the assay's temperature.
iensis found that D. carinata and M. australiensisadults can tolerate swine wastewater concentrations in
2.2 Toxicity Testing
the order of 2.8 and 8.8 mg L−1 of NH3, respectively,while juveniles can only tolerate concentrations in the
Acute toxicity was determined by exposing D. magna
order of 2.2 and 7.5 mg L−1 of NH3, respectively
juveniles (<24 h) to each sample for 48 h. At the end
(Leung et al. ). However, ammonium presents
of exposure, mortality, defined as lack of organism
acute toxicity on D. magna at values of 48-h LC ≥
mobility when the vessel was shaken, was recorded.
1.5 mg L−1 of NH +–
N (Martins et al. ). In
Organisms were obtained from in-house cultures
species of Ceriodaphnia dubia, the chronic toxicity
maintained according to guidelines given by USEPA
caused by the NH +
depends on the oxidation time of
). Before the experiment, cultures and animal
and NO3 , which affects the reproduction of
exposures were conducted at 20±2°C in a photope-
these crustaceans (Dave and Nilsson ).
riod of 16-h light/8-h dark and fed three times per
Additionally, the toxicity of anionic compounds,
week with a recharge of water each 48 h, permitting
such as Cl− has been studied. In the case of
parthenogenetic conditions. Dilution and control
Daphnia spp., the acute toxicity of this anion has
water was moderately hard, reconstituted water
been studied at concentrations > 0.005 mg L−1
prepared according to USEPA (The solutions
(AQUIRE ; Pretti et al. It has also been
were not renewed and the organisms were not fed
reported that the formation of organochlorides
during the experiments. The 24-h median lethal
(AOX) can produce acute toxicity in organisms
concentrations (LC50) were calculated using the
such as D. magna at concentrations of 24-h EC50 of
Spearman–Karber method (Finney ; ). Esti-
2.2 mg AOX L−1 (Emmanuel et al. ). The
mation of the samples' theoretical toxicity was based
presence of metallic salts, such as CuCl2, CdCl2, and
on the chemical analysis of the individual potential
FeCl2, can present greater acute toxicity for D.
toxicants. Therefore, to express the relative toxicity of
magna (24-h LC50=0.08–172.0 mg L−1) due to the
a certain compound, acute toxic units (TUtheorical) for
synergic effect between chloride and the metals,
this compound were calculated as: nominal com-
contrasting with salts like that present lower toxicity
pound concentration in test solution divided by LC50.
(24-h LC50= 240.0–1020.0 mg L−1) (Martins et al.
Then, assuming that these toxicants were the real
causative agents, they were determined according to
The objective of this study is to provide a
the concentration-addition model of Anderson and
physicochemical and ecotoxicological characteriza-
Weber ). If no interaction between toxicants is
tion of swine wastewater using D. magna as the
expected, then the observed sample toxicity should be
Water Air Soil Pollut
equal to the theoretical toxicity of the toxicant with
(electrical conductivity) were evaluated using electro-
the greatest effect. The sensitivity of the testing is
des. The total alkalinity (TA) was determined by
0.05 mg Hg L−1 (USEPA ).
titration (Vidal and Diez ). In this study,ammonia (NH –
3 N) concentration was calculated for
2.3 Toxicity Identification Evaluation
each test according to Anthonisen et al. (
The baseline test, the pH/adjustment test (pH 3/ad andpH 11/ad), the graduated pH test (pH 6.9/grad and
3 Results and Discussion
pH 8.9/grad), and the pH adjustment/aeration test(pH 3/aer, pH 7.5/aer, and pH 11/aer) were performed
3.1 Physicochemical and Ecotoxicological
as described by Norberg-King et al. (The other
Characterization of the Swine Wastewater
fractionations were based on procedures described byVan Sprang and Janssen (The EDTA (ethyl-
Table shows the results obtained from the physico-
enediaminetetraacetic acid) concentrations (Titriplex
chemical characterization of swine wastewater. High
III, p.a., Merck) in the dilution water and effluent
values were observed for electrical conductivity
sample were based on the 24-h 10% lethal concen-
(13.8–25.6 μS cm−1), alkalinity (TA, 11.4–12.0 g
tration (LC10) on D. magna and were conducted at a
CaCO3L−1), organic matter (COD, 16.2–24.9 g O2
final EDTA of 300 mg L−1. For the pH adjustment/
L−1; BOD5, 4.5–9.8 g O2L−1), nutrients (TN, 1.9–2.5 g
filtration test (pH 3/fil, pH 7.5/fil, pH 11/fil), 2.5 ×
N L−1), and ions (NH +–
N, 0.7–2.0 gL−1; SO4 ,
25-cm glass columns were filled with synthetic
271.9–503.1 mg L−1, and Cl−, 1,210–1,250 mg L−1).
hydrophobic filter wool (Diprolab MR). Additional
These values are characteristic for this type of
column tests consisted in an anion (20 g, amberlite
discharge coming from the fattening phase (Blanes-
IRA-440, Merck), a cation (20 g, amberlite IR-120,
Vidal et al. Moral et al. Moral et al. ;
Merck), and an 8- to 20-mesh activated carbon (15 g,
Provolo and Martínez-Suller ). Moral et al.
Sigma). Baseline toxicity tests were performed in
relates the high electric conductivity in swine waste-
duplicate, whereas TIE manipulations were not. ThepH was adjusted and readjusted by adding dropwise
Table 1 Physicochemical and toxicological characterization
pro-analysis HCl and NaOH of different normalities
(D. magna) of the swine wastewater
(0.1, 1.0, and 2.0 N; p.a., Merck).
For all manipulations, the 24-h LC
verted to toxic units (TUfractionation=100÷24-h LC50)
and were compared with the baseline toxicity test
(TUbaseline). The toxicity reduction percentage (%TR)
due to the applied fractionation was calculated as
%TR ¼ 100 1 TUfractionation
2.4 Analytical Methods
COD (chemical oxygen demand), BOD5 (biological
oxygen demand), ammonium (NH +–
gen (TN), total phosphorus (TP), sulfate (SO 2−
EC electrical conductivity, COD chemical oxygen demand,
chloride (Cl−) were determined according to the
5 biological oxygen demand, TA total alkalinity, TN total
nitrogen, NH + –
N ammonium, NH3-N ammonia TP total
protocols established in Standard Methods (APHA-
phosphorus, SO −2
sulfate, Cl− chlorine, 48 h LC50 median
AWWA-WPCF ). Parameters such as: pH and EC
Water Air Soil Pollut
water to the high salt (e.g., Cl−, p<0.001) and protein
for the cationic exchange fraction, where it increased
content (e.g., NH +
4 , p < 0.001), which is present in the
to 12. This result can be explained by the nature of
diet for animals in the final growth stage, thus
this fraction, which resulted in an increase in the
explaining the results with respect to ionized com-
(non-retained or available) OH- groups during this
pounds (Martínez-Suller et al. Moral et al.
test. A reduction of 51% in Cl− was observed in the
The pH was between the values 7.2 and 7.5. Moral
anionic exchange test without any evidence of
et al. () indicate that a pH> 7.9 favors the
changes in the pH (7.5). In the cationic exchange
3. The NH3 N concentration in this
test, NH4 N was reduced in 100%. It is important to
study fluctuated between 5.5 and 15.9 mg L−1, which
indicate that the cationic exchange fraction had the
is considered to be toxic for D. magna (48-h LC50,
greatest variation in physicochemical characteristics
2.9–6.9 mg L−1) according to Pretti et al. (
of the swine wastewater, presenting an increase in pH
Indeed, the results of the ecotoxicological assays with
and total elimination of NH +–
N. In contrast, SO4
D. magna found acute toxicity of 48-h LC50=3.3–
increased (<55%) in all the treatments except for
3.7%. These values on average are 1.94 times the
anionic exchange, due to the chemical composition of
values reported by De la Torre et al. (for D.
the anionic resin. Reyes et al. ) reported similar
magna (48-h LC50=1.8±0.2%) for pig slurry (e.g.,
results in this test for kraft cellulose effluent.
fattening farm). While the reproductive swine farmsamples have acute toxicity values (48-h LC50)
3.3 Phase II: Reduction of the Toxicity of Swine
between 3% and >10% (De la Torre et al. ).
Wastewater in D. magna
3.2 Phase I: Toxicity Identification Evaluation
Table shows the effect of the different TIE fractions
of Swine Wastewater
for the acute toxicity on D. magna (48 h) of the swinewastewater. For the cationic exchange fraction, an
Table shows the physicochemical characterization of
acute toxicity was observed to increase seven times
the swine wastewater before and after fractioning with
(48-h LC50=0.48%). In contrast, when comparing the
the TIE technique. In all the tests, the pH did not
fractions corresponding to pH 11 (aeration/filtration/
change (7.3–7.4) with respect to the control, except
adaptation) with the control, toxicity increased only
Table 2 Physicochemical characteristics for the different treated fractions from swine wastewater using TIE methodology
EDTA ethylenediaminetetraacetic acid
Water Air Soil Pollut
Table 3 48-h LC50 and TU
Median lethal concentration
on D. magna values below
95% of confidence intervalfor swine wastewater using
Aeration (pH 3.0)
Aeration (pH 7.5)
Aeration (pH 11.0)
Filtration (pH 3.0)
Filtration (pH 7.5)
Filtration (pH 11.0)
Adaptation (pH 3.0)
TU toxic units, TU
standardized toxic units
standardized with respect
1.1–1.5 times. Additionally, toxicity diminished in
organisms such as Daphnia spp. On the other hand,
22.5% for the anionic exchange fraction (48-h LC
species of D. magna exposed to NH4 are observed to
4.45%), 11% in the activated carbon test (48-h LC50=
be less sensitive to Cl− (48-h LC50 = 1.5–
3.83%), and 7% in the EDTA test (48-h LC50=
492.0 mg L−1; Mangas-Ramírez et al. When
3.66%). The results presented in Table demonstrate
comparing the values obtained in the present study
that the reduction in NH +–
N in the cationic exchange
with those reported in the literature, D. magna is
fraction provokes an increase in toxicity (Table
observed to be more sensitive to Cl− than to NH +–
which is reflected in a higher TU value (208.33). In
50: the average value of NH4
contrast, the diminishment in the toxic units in the
4.4 mg L−1 and the average value of Cl− is 43.1±
anionic exchange fraction (TU, 22.47) would be
3.5 mg L−1 which could explain the acute toxicity. It
related with the 51% reduction in Cl−, showing the
is important to note that an increase in pH in the
greater sensitivity of D. magna to anionic compounds.
swine can increase the formation of NH3-N, provok-
In the fractions corresponding to pH 11 (aeration/
ing an increase in toxicity for ammoniac compounds.
filtration/adaptation), a slight increase in toxic units
Figure shows the percentage of TR generated by
(TU, 43.29, 34.72, and 33.78, respectively) was
each fraction of the TIE (Fig. ) and the removal
observed and attributed to the formation of NH
efficiency percentage for NH4 N and Cl− in each test
due to the pH conditions in the swine wastewater.
(Fig. ). It can be observed in Fig. a that toxicity
Table shows the different LC50 values for several
(percentage of TR) was reduced in the following tests:
cladocer organisms that were reported in the literature
anionic exchange (22.5%), activated carbon (9.9%),
to be produced by compounds such as Cl−, NaCl,
and EDTA (5.7%). However, an increase in TR
N, and NH3 N. On the other hand, Cl− is
values, between 4.6% and 624.4%, was observed for
observed to have high acute toxicity for Daphnia spp.
the other fractions and especially in the cationic
between 0.005 and 0.16 mg L−1
exchange fraction. Thus, NH4 N was removed in
(AQUIRE and 48-h LC50 between 0.12 and
the anionic exchange (48.7%), cationic exchange
0.15 mg L−1 (Pretti et al. where the least toxic
(100%), activated carbon (87.2%), and EDTA
is salt (24-h LC50=1,020.0–3,240.0 NaCl mg L−1)
(87.2%) fractions, while Cl− was removed in all the
(Sarma et al. ; Martins et al. ). These results
fractions except for the fraction pH 7.5/aeration where
indicate that chloride is more bio-available for
it increased (−7.1%).
Water Air Soil Pollut
Table 4 Salinity and ammonium effect on aquatics organisms
Sarma et al. Martins et al. ()
Dave and Nilsson
Mangas-Ramírez et al. ()
Mangas-Ramírez et al. ()
Thus, the increase in swine wastewater toxicity is
observed to be related to the reduction of Cl− and thepresence of NH +–
N. First, a diminishment of toxicity
Using the TIE technique, the swine wastewater was
is observed when Cl− and NH +–
N are reduced at the
characterized for each test, finding that the acute
same time. The reduction of Cl− in the anionic
toxicity for D. magna increased seven times in the
exchange fraction would provoke the diminished
cationic exchange fraction. From the estimation of TR
toxicity, and an increase in toxicity would result from
(percent), Cl− (605.0 ±21.2 mg L−1) was found to be
the presence of 78% Cl− in the cationic exchange
the compound provoking this acute toxicity in the
even with a total reduction of NH +–
anionic exchange. The increase in toxicity obtained in
results indicate that NH +–
N in swine wastewater acts
the cationic exchange test (624%) is related to the
as a Cl− immobilizing compound, diminishing toxic-
retention of NH +–
N (0.04±0.01 mg L−1) and a
ity even when the ammonium is more toxic than Cl−
greater bio-availability of Cl− in its ionic form,
when the pH changes.
causing the acute toxicity for D. magna.
-40-600 -610 -620 -630 -640 -650
-140 -120-100 -80 -60 -40 -20
40 60 80 100 120 140
Removal Efficiency (%)
Fig. 1 a Toxicity reduction (TR) percentage for D. magna (48 h) for swine wastewater resulting Phase I TIE fractionation techniques.
b Removal efficiency of NH +–
N (black bar), Cl− (white bar) for each test
Water Air Soil Pollut
This work was supported by CONICYT/
Mangas-Ramírez, E., Sarma, S. S. S., & Nandini, S. (2002).
PBCT (Grant TPI-01) and Innova Bío-Bio (Grant 07-PC S1-
Combined effects of algal (Chlorella vulgaris) density and
198). The authors thank Mr. C. Contreras from Sucesion Yanine
ammonia concentration on the population dynamics of
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