Marine Environmental Research 114 (2016) 1e11

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Marine Environmental Research

journal homepage: www.elsevier.com/locate/marenvrev

Are combined AOPs effective for toxicity reduction in receiving marine

environment? Suitability of battery of bioassays for wastewater
treatment plant (WWTP) efuent as an ecotoxicological assessment
~ o a, *, J.J. Rueda-Ma
B. Daz-Gardun rquez b, M.A. Manzano b, C. Garrido-Pe
rez b,
a
M.L. Martn-Daz
a
Physical Chemical Department, Centro Andaluz de Ciencia y Tecnologa Marina (CACYTMAR), Campus de Excelencia Internacional del Mar (CEIMAR),
diz, Campus Universitario de Puerto Real, 11510, Puerto Real, Ca
Universidad de Ca diz, Spain
b
Environmental Technologies Department, Centro Andaluz de Ciencia y Tecnologa Marina (CACYTMAR), Campus de Excelencia Internacional del Mar
diz. Campus Universitario de Puerto Real, 11510, Puerto Real, Ca
(CEIMAR), Universidad de Ca diz, Spain

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

Article history: Ecotoxicological assessment of three different wastewater treatment plant (WWTP) efuents D1, D2 and
Received 16 September 2015 D3 was performed before and after tertiary treatment using combination of advanced oxidation pro-
Received in revised form cesses (AOPs). A multibarrier treatment (MBT) consisting of microltration (MF), hydrogen peroxide
11 December 2015
photolysis (H2O2/UVC) and catalytic wet peroxide oxidation (CWPO) was applied for all efuents. Sparus
Accepted 12 December 2015
aurata, Paracentrotus lividus, Isochrysis galbana and Vibrio scheri, representing different trophic levels,
Available online 18 December 2015
constituted the battery of bioassays. Different acute toxicity effects were observed in each WWTP ef-
uents tested. The percentage of sea urchin larval development and mortality sh larvae were the most
Keywords:
Acute toxicity
sensitive endpoints. Signicant reduction (p < 0.05) of efuent's toxicity was observed using a classi-
Effects cation pT-method after MBT process. Base on obtained results, tested battery of bioassays in pT-method
Environmental toxicology framework can be recommended for acute toxicity preliminary evaluation of WWTP efuents for the
Fertilization marine environment.
Fish 2015 Elsevier Ltd. All rights reserved.
Larval bioassay
Lethal effects
pT-method
Risk assessment
Waste treatment

1. Introduction WWTP efuent constitutes a special matrix in which it is dif-

In recent years, treatment of wastewater treatment plant
(WWTPs) efuent has been improved in order to avoid the
discharge of hazardous pollutants into receiving ecosystems.
Nevertheless, although they are now less toxic than in the past,
WWTP efuents are still considered a major source of pollution for
marine and freshwater ecosystems. They contain a complex
mixture of chemicals and biological agents, which may harm
receiving aquatic ecosystems and may pose a risk for human health
 et al., 2007; Heberer, 2002; Santos et al.,
(Clara et al., 2004; Gagne
2013; Ternes, 1998).
* Corresponding author. Present address: Centro Andaluz de Ciencia y Tecnologa
Marina (CACYTMAR), Universidad de Ca diz, Campus Universitario de Puerto Real,
diz, Spain.
11510, Puerto Real, Ca
~ o).
E-mail address: beatriz.diaz@uca.es (B. Daz-Gardun
cult to identify the individual behaviour of contaminants released
into receiving ecosystems. Conversely, it is possible to determine
toxic response to the joint action of chemicals in the efuent. Bio-
assays are unsuitable for qualitative or quantitative determination
of contaminants and cannot replace chemical analyses. However,
conventional laboratory bioassays can be very accurate and
reproducible, and it is necessary to include an adequate battery of
these if the aim is to conduct a comprehensive assessment of the
present source of pollution (Krebs, 2005). Suitable bioassay batte-
ries have been described for assessing the toxicity and environ-
mental risk of different sources of contamination, such as dredged
material (Casado-Martnez et al., 2008; Rodrguez-Obeso et al.,
2007), oil spills (Morales-Caselles et al., 2008) and mining activ-
ities (Martn-Daz et al., 2004; Riba et al., 2005). As regards the
legislation concerning WWTP efuent, the Water Framework
Directive (EU WFD) (Directive 2000/60/EC and related acts)

http://dx.doi.org/10.1016/j.marenvres.2015.12.011
0141-1136/ 2015 Elsevier Ltd. All rights reserved.
2 ~o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun
established a list of priority substances. Subsequently, Directive
2008/105/EC laid down environmental quality standards (EQS) for
priority substances (and certain other pollutants), establishing EU
water quality standards for surface water. Besides other specic
measures for better control of WWTP efuent pollution, these
regulatory frameworks require adequate environmental quality
studies based on short-term and long-term exposure of aquatic
species. Notable among these are studies on sh in Switzerland
(Bucher and Hofer, 1993; Burkhardt-Holm et al., 1997), Sweden
(Paxeus, 1996), Germany (Hoeger et al., 2004), Japan (Higashitani
et al., 2003) and the United States (Grizzle et al., 1988), and most
particularly in Canada on bivalve molluscs (Akaishi et al., 2007;
Bouchard et al., 2009; Gagne  et al., 2007, 2011) and sh (Gagne
et al., 2006; Hebert et al., 2008). Nevertheless, few studies have
monitored efuents discharged into marine environments and few
public institutions or environmental agencies have attempted to
standardise tests to analyse the risk to resident organisms posed by
efuent discharges into the marine environment. As indicated in
regulatory frameworks on freshwater quality (Environment
Canada, 2011; USEPA, 2002), such toxicity tests should take
different levels of the trophic chain into account.
Many WWTPs now perform tertiary treatment to further
improve efuent quality and generate reclaimed water in line with
regulations. Tertiary treatment is also frequently used to remove
specic groups of chemicals or pathogens that are not removed by
traditional technologies (e.g. primary settling and secondary bio-
logical treatment). It is well known that advanced oxidation pro-
cesses (AOPs) can remove organic pollutants resistant to
conventional water treatment techniques, through the generation
of highly oxidising species such as hydroxyl radicals.
In UVC/H2O2 systems, hydroxyl radicals are formed when water
containing H2O2 is exposed to UV light in the range of 200e280 nm
(Vilhunen et al., 2010). The photolysis of H2O2 can be described by
the following reaction:
H2O2 UV (l z 200e280 nm) / HO, HO, [1]
One of the main drawbacks of UVC/H2O2 is the presence of re-
sidual H2O2 in the water after treatment, which can be toxic for
some organisms (Dra bkova
 et al., 2007; Heringa et al., 2011). Hence,
granular activated carbon (GAC or AC) can be an alternative to
remove residual H2O2 [2, 3] by means of the catalytic wet peroxide
oxidation process (CWPO) (Rey et al., 2011; Santos et al., 2009):
H2O2 AC / AC 2$OH [2]
AC H2O2 /AC $OOH H [3]
The GAC step of the process is very important not only for
removal of residual H2O2 from the water but also of organic matter
(due to the generation of $OH) (Rueda-Ma rquez et al., 2015b).
Therefore, the combination of these AOPs yields promising results
for organic matter removal and toxicity reduction.
The aim of this research was to establish a suitable battery of
bioassays to assess WWTP efuent toxicity for the receiving marine
ecosystem after conventional treatments. Likewise, a combination
of AOP (H2O2/UVC and CWPO) was applied to evaluate the potential
reduction in efuent toxicity effects on the biota.
2. Materials and methods
2.1. Sampling sites description
Three different WWTPs were tested and named D1, D2 and D3.
The rst efuent tested belongs to D1 (36220 47.9000 N-
6100 54.1800 W). It is located in a residential tourist location and
it receives mainly domestic wastewater. This WWTP shows
important seasonal variations of inuent ow rates
(4000e12,000 m3/d) due the variations of population according the
tourism activity. Regarding D2 (3631029.8900 N-6140 07.9100 W), it
treats 17,000 m3/d of a typical medium-city (urban and industrial
wastewaters) including a medium-size hospital (410 beds). It rep-
resents a signicant source of emergent pharmaceuticals and other
contaminants related to the activity of sanitary facilities. The third
WWTP, D3 (36250 38.2800 N-6090 21.8500 W) efuent, belongs to a
plant which treats the wastewater of a typical medium-city in this
geographical zone. The inuent (7000e9000 m3/d) is composed by
urban and small and medium-sized industrial wastewaters. Sam-
pling sites are shown in Fig. 1.
Technologies of the three WWTPs are similar. First step is a
pretreatment with a screening, desanding and degreasing, followed
by primary settling treatment. Thereafter, the wastewater un-
dergoes biological process (secondary treatment), a conventional
activated sludge (medium daily organic load per aerated sludge
mass) and nally a secondary settling. The WWTP efuent is dis-
charged to coastal waters.
2.2. Sample collection
Samples were collected over 24-h by continuous sampling from
the efuent of each WWTP in winter season of 2014. Equal volume
discrete sample aliquots (10 L) were collected at constant time in-
tervals (1 h) into a refrigerated container with an automatic
sampler. Ambar glass bottles were used to preserve and store
samples. Samples were ltered with glass bre lter (bre glass
lter and pore size 1 mm, Whatman) and stored in a chamber at
4  C in dark conditions to avoid the degradation of the contami-
nants until assays were performed.
2.3. Wastewater characterization
In order to check if the quality of tested efuents is in agreement
with requirements of Directive 91/271/EEC, all collected samples of
wastewater were analysed using physicalchemical methods.
Biological Oxygen Demand (BOD5) and Chemical Oxygen De-
mand (COD) were determined using Standard Method 5220-D and
5210-B respectively (APHA-AWWA-WEF, 1998). The concentration
of H2O2 in water samples was estimated with Merckoquant tests
for hydrogen peroxide concentrations from 0 to 100 mg/L (Colori-
metric test strips methods, 1e100, 0e30, detection limit 1 mg/L).
The total organic carbon concentration (TOC) was measured by
means of total organic carbon analyser Shimadzu TOC e VCPH TOC
in non-purgeable carbon (NPOC) mode. Finally, concentration of
suspended solids was determined gravimetrically (APHA-AWWA-
WEF, 1998).
2.4. Multibarrier treatment (MBT)
The MBT process utilized in this work consisted of following
steps: microltration (bre glass lter and pore size 1 mm, What-
man), followed by photolysis of hydrogen peroxide (H2O2/UVC)
and catalytic wet peroxide oxidation (CWPO) using granular acti-
vated carbon. More details regarding MBT process can be found
elsewhere (Rueda-Ma rquez et al., 2015a, 2015b).
The UVC/H2O2 experiments were conducted in bach operation
mode using a cylindrical photoreactor with working volume of
260 mL, a peristaltic pump (Masterex L-S Digital Pump System,
600 mL min1) and glass mixing tank for wastewater. The low
pressure UVC lamp (Philips TLV-8W, emission peak at 254 nm,
power 8 W, 2 WUVC) was centred along the focal axis of the annular
 ~ o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun 3

diz, and location of the Bay of Ca

Fig. 1. Map of the sampling WWTPs in the Bay of Ca diz in the Atlantic coast of Europe (insert).

reactor and protected by a quartz sleeve. 2.5. Toxicity tests

The H2O2 (30% w/v, Merck, CAS Number 7722-84-1) was spiked
into mixing tank. The concentration of added H2O2 was in accor-
dance with total organic carbon concentration (TOC) of the
wastewater. The H2O2/TOC ratio (R) equal to ve (R5) was chosen as
an optimal for all the experiments based on earlier studies (Rueda-
Marquez et al., 2015a).
After H2O2/UVC step, water was pumped through GAC columns
(CWPO treatment) from coconut shell (Hidrowater, with an
average particle density of 0.47 g cm3, mesh size 4e8 mm and
BET surface area 900e1000 m2 g1) under ambient conditions
(20  C). The MBT process efuent was used for acute toxicity tests.
The total volume of the residual wastewater in all the cases was
7000 ml.
Dilutions with natural ltered sea water from the Laboratory of
Marine Culture at the University of Cadiz (Spain) were used to
performed toxicity tests. Controlled water quality parameters were
temperature (171  C), salinity (38 0.5 g/L), pH (7e7.5) and dis-
solved oxygen (>5 mg/L, 60% sat). For each test, 8 decimal dilutions
from 1:1 (100% efuent, positive control), 1:2, 1:4, 1:8, 1:32, 1:64 to
1:512, and negative control (clean sea water) were used to test the
toxicity effects. All the parameters were controlled at the beginning
and at the end of the experiment to ensure acceptability of tests.
2.5.1. Bioluminescence inhibition
Bioluminescence inhibition of Vibrio scheri test were per-
formed with whole WWTP efuent samples of each WWTP using
4 ~o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun
the standard protocol of Microtox Basic Test (BT) by Azur Envi-
ronmental (Environmental, 1998). The changes in the luminescence
were evaluated after 5, 15 and 30 min. The concentration of a test,
which caused a 50% reduction of bacteria luminescence (EC50) with
95% condence intervals (CIs), was measured to know the response
of the exposure bacteria.
2.5.2. Growth inhibition of microalgae
Growth inhibition test of the microalga Isochrysis galbana
(Phylum Haptophyta) was selected to assay the toxicity of the ef-
uents to primary producers. Inocula of I. galbana were obtained
from the collection of the Laboratory of Marine Culture at the
Marine and Environmental Sciences Faculty of the University of
Cadiz (Spain). The methodology performed was described by
Garrido-Perez et al. (2008). The algal density was determined at 0,
24, 48, and 72 h period in terms of optical density (USEPA, 2002) at
a wavelength of 690 nm using a colorimeter Nannocolor PT-3
MACHEREY-NAGEL. Both stimulation growth (enrichment) and
inhibition growth percentage of the microalgae was obtained from
these data of absorbance. Percentage of response was calculated
through the equation % Enrichment [(TeC)/C] $100; where C was
the control biomass measured as absorbance (690 nm) and T was
the treatment or biomass of each sample measured as absorbance
(690 nm) (Garrido-Perez et al., 2008).
2.5.3. Sea urchin toxicity test
Sea urchin toxicity tests were carried out following protocols
ndez and Beiras (2001); Volpi Ghirardini and
described by Ferna
Arizzi Novelli (2001), USEPA (USEPA, 2002), and Environment
Canada (Environment Canada, 2011). Paracentrotus lividus in-
dividuals were collected from a clean site of the Atlantic coast of
Cadiz (Spain) (3631058.4300 N and 6180 32.9300 W) from a rocky
subtidal environment where sea urchins are found at 1.5e2 m
depth. Individuals were kept in cooler box transportation to the
laboratory. This species was used as bioindicator by previous
studies (Beiras et al., 2012; Carballeira et al., 2012; Casado-Martnez
et al., 2007).
2.5.3.1. Sea urchin fertilization test. Fertilization success was indi-
cated by the presence of a fertilization membrane.
2.5.3.2. Sea urchin embryo-larval development test. The results
expressed as a percentage of normal pluteus stage (dened as those
with four well developed arms and pyramid-shaped) normalised to
the corresponding toxicity control.
2.5.4. Mortality in the sh larvae
Organisms were obtained from the collection of the Laboratory
of Marine Culture at the Marine and Environmental Sciences Fac-
ulty of the University of Cadiz (Spain). Newly hatched seabream
larvae (3 he5 h post-hatching), with the yolk sac larvae were
collected. An amount of 70 newly hatched larvae of Sparus aurata
(3 he5 h post-hatching), with the yolk sac, were taken and intro-
duced in a 1 L glass vessel with 600 mL of dilution volume per
triplicate. After 96 h, the measured endpoint was the survival of the
90% of the larvae comparing with the toxicity control.
2.6. Statistical analysis
Statistical analysis was performed by SPSS/PC statistical
package (15.0). Signicant differences between organisms
exposed to different dilutions of the wastewaters with or without
MBT and the negative and positive control were using a one-way
ANOVA followed by multiple comparisons of Turkey's and Dun-
ett's test. Signicance level was set at 0.05. Correlation analysis to
nd out concentrations-response relationship was performed us-
ing a Spearman rank.
To determinate the toxicity class of the WWTPs analysed, the
pTeMethod [4] was used according to the most sensitive test.
pTeValue was dened with standardised aquatic toxicity tests and
using a geometrical sequence of dilutions factor of two to identi-
fying the rst toxic dilution factor. pTeIndex showed the numerical
classication, regarding the dilution factors, of aquatic toxicity
measured within the most sensitive test of the test battery (Krebs,
2005). The pTeValue indicates the extent to which a sample must
be diluted at a ratio of 1:2 not to produce any toxic effects. Standard
values of the pTeMethod were described by Krebs (Krebs, 2005).
pT log2 dilution factor
dilution factor 2pT antilog2  pT (4)
pT log2 1=x log2 x
3. Results
3.1. MBT process results
The BOD, COD and SS parameters established in European
Directive 91/271 were within acceptable discharge limits for all the
efuents analysed (Table 1). The results of previous research have
demonstrated that application of combined AOPs yields a signi-
cant decrease in efuent organic matter content and reduces
toxicity. Similarly, mechanical biological treatment (MBT) was also
selected for this study, since previous research has shown that it is
an optimal toxicity treatment (Rueda-Ma rquez et al., 2015a, 2015b).
The MBT process was applied in order to decrease WWTP
efuent toxicity. The results of total organic carbon (TOC) reduction
(mineralisation) during MBT are presented in Fig. 2.
The initial TOC in the wastewater (D1, D2 and D3 efuents)
before MBT was around 10 mg/L. Therefore, the initial H2O2 con-
centration was 50 mg/L (R5). After the H2O2/UVC step (the UVC
dose accumulated by water equal to 6 WUVC.s cm2), TOC removal
for D2, D3 and D1 efuent was 30%, 44% and 45.5%, respectively.
The residual concentration of H2O2 in all waters was about 20 mg/L.
The H2O2/UVC treatment efuent was pumped though GAC in
order to remove residual H2O2 and reduce TOC (CWPO). After 5 min
of contact time, the concentration of H2O2 in the efuent was below
the detection limit. At the end of the MBT process, mineralization of
D2, D3 and D1 efuents was 71%, 74.8% and 80.3%, respectively.
3.2. Toxicity results
All the treated efuents tested showed no signicant adverse
effects on the control treatment (clean sea water) (p < 0.05).
However, the positive control (100% efuent) showed signicantly
acute toxicity (p < 0.05) before and after the MBT process. Hormesis
was detected in the 3 efuents before and after the detoxication
treatment using the Microtox Basic Test.
3.2.1. WWTP D1
Results of the I. galbana growth inhibition toxicity test were
expressed as the percentage of stimulation after 72 h of exposure.
I. galbana showed different responses depending on the dilution of
the D1 efuent (Fig. 3). Before MBT, a doseeresponse relationship
was observed, since a decreasing % of enrichment was related to a
decrease in efuent dilution (p < 0.01, r 0.924). The positive
control (100% efuent) and 1:2 dilution presented negative values
for % of enrichment corresponding to microalgae growth inhibition.
After the tertiary treatment, a negative % of enrichment was
 ~ o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun
Table 1
Summarize of the characteristics of parameters in the residual medium on sampling date between January and February (data corresponding of 30 days in the sampling
month).
Total organic carbon (TOC, mg/L) Biologic oxygen demand (BOD) (mg O2/L)
D1 10.0 12 7.96
D2 10.5 19 5.12
D3 13.2 10 5.23
Fig. 2. Mineralization of organic compounds as a function of UVC dose accumulate by
the water (H2O2/UVC) and contact time (CWPO) at conditions: H2O2/UVC (R5, Dose: 6
WUVC.s cm2) and CWPO (5 min of contact time).
observed for all the dilutions (p < 0.01, r 0.794).
Sea urchin fertilisation and larval development test results for
the D1 efuent are shown in Fig. 3. A signicant doseeresponse
relationship was observed for both sea urchin fertilisation and
larval development tests before MBT (p < 0.01, r 0.923; p < 0.01,
r 0.719, respectively) and after MBT (p < 0.01, r 0.874;
p < 0.01, r 0.897, respectively). Less acute toxicity effects of the
efuents were observed after application of the combined process.
No signicant differences were found for the sea urchin fertilisation
test, except for the positive control (100% efuent), or for the larval
development toxicity test (p < 0.05). In accordance with the USEPA
(2002) guidelines for risk assessment, the MBT process reduced the
toxic effects measured using sea urchin tests after exposure to the
WWTP efuent. The toxic effect measured using the larval devel-
opment test was reduced, with toxicity being observed from dilu-
tion 1:32 before MBT and from dilution 1:4 after MBT. The sea
urchin embryogenesis toxicity test proved to be more sensitive
than the fertilisation one.
The results of the S. aurata toxicity test are shown in Fig. 3. A
doseeresponse relationship was observed between D1 efuent
dilution and the % mortality of sh larvae before and after MBT
(p < 0.01, r 0.775; p < 0.01, r 0.924, respectively). Signicant
differences were observed in % mortality of sh larvae before and
after MBT in dilutions higher than 1:4. After the AOP treatment,
higher values were observed for % mortality of sh larvae before the
tertiary treatment. Application of MBT to the D1 efuent for the sh
larvae test was not effective in reducing WWTP efuent toxicity
according to OECD risk guidelines.
3.2.2. WWTP D2
Fig. 3 shows the analytical results of the D2 efuent toxicity test
using I. galbana. A signicant doseeresponse relationship was
5
Chemical oxygen demand (COD) (mg O2/L) Suspended solids (SS) (mg/L) pH
75 9.06 10 6.15 7.42
85 10.21- 20 7.47- 7.32
83 9.32 27 7.23 8.01
observed between a negative % of enrichment (corresponding to a
higher % of growth inhibition), and a higher concentration of
efuent prior to MBT (p < 0.01, r 0.966). However, a signicant
reduction in toxicity was detected after MBT (p < 0.01, r 0.794).
Positive values for % of enrichment were observed after the tertiary
treatment except for the 1:4 and 1:2 dilutions and the positive
control (100% efuent) (p < 0.05).
The acute toxicity measured in sea urchin for D2 efuent
showed a positive doseeresponse relationship between efuent
dilution and toxic responses in fertilisation and larval development
tests before MBT (p < 0.01, r 0.869; p < 0.01, r 0.721,
respectively) and after MBT (p < 0.01, r 0.848; p < 0.01,
r 0.817 respectively). Fertilisation toxicity test results did not
improve after the tertiary treatment. Signicant differences with
the negative control were observed in the positive control (100%
efuent) before and after the detoxication treatment (p < 0.05). In
contrast, according to the USEPA (2002) risk assessment guidelines,
the sea urchin larval development test showed pluteus larvae in
efuent concentrations below 1:4 after MBT; however, before the
tertiary detoxication treatment, pluteus larvae were detected in
dilutions lower than 1:8.
Results of the S. aurata mortality toxicity test showed a signi-
cant doseeresponse relationship between toxic effect and efuent
concentration before and after MBT (p < 0.01, r 0.922, r 0.920,
respectively). In accordance with OECD risk guidelines (2013), no
signicant reduction in toxicity was obtained after applying the
AOP. The results are presented in Fig. 3.
3.2.3. WWTP D3
The results of the I. galbana growth inhibition toxicity test
indicated a signicant difference between the doseeresponse
relationship before and after MBT (p < 0.01, r 0.388, r 0.979,
respectively). Before MBT, signicantly higher values were obtained
for negative % of enrichment (corresponding to % of growth inhi-
bition) (p < 0.05). However, after tertiary treatment, positive values
for % of enrichment were observed except for the 1:2 dilution and
the positive control (100% efuent).
A signicant doseeresponse relationship was observed for sea
urchin fertilisation and larval development tests in D3 efuent
before tertiary detoxication treatment (p < 0.01, r 0.736;
p < 0.01, r 0.910; respectively) and after (p < 0.01, r 0.897;
p < 0.01, r 0.812, respectively). The fertilisation and larval
development toxicity tests did not show reduced toxicity in % of
fertilisation and % of larval development, respectively, after MBT. In
addition, toxicity seemed to be higher after application of the ter-
tiary treatment.
The advanced oxidation process proved effective in the % mor-
tality of S. aurata test for D3 efuent. A signicant doseeresponse
relationship was observed between concentrations and toxic re-
sponses before MBT (p < 0.01, r 0.818) and after MBT (p < 0.01,
r 0.913). In accordance with OECD risk guidelines (2013), the
application of MBT yielded a reduction in toxic effects on sh
larvae. Before application of MBT, the rst toxic dilution was 1:32,
whereas after tertiary treatment, the rst toxic dilution was
reduced to 1:4. The results are presented in Fig. 3.
6 ~o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun

3.3. Toxicity classication and comparison of the sampled efuents
The toxicity tests yielded different results for each WWTP
efuent analysed. Various approaches were assayed in order to
identify the most sensitive toxicity test and the most polluted
WWTP efuent. In line with USEPA and OECD acceptability criteria,
sea urchin larval development was the most sensitive test for D1
and D3 efuents. However, the % of larval mortality was the most
sensitive test for the D2 efuent. The pT-method toxic scale was
used to assess the WWTP efuents, obtaining a pT-value 5
(highly toxic) for the D2 and D3 efuents, which were therefore
the most toxic WWTP efuents tested. After the detoxication
treatment, the pT-value for both WWTP efuents was lower in 3
categories, classifying them as slightly toxic. Meanwhile, the D1
efuent was moderately toxic before MBT (pT-value 3), and this
classication was reduced to very slightly toxic after MBT (pT-
value 1). The pT-method results are shown in Table 2.
4. Discussion
WWTP efuent toxicity/risk assessment depends mainly on the
ecological representativeness of biological species at different tro-
phic levels and the sensitivity of those species to pollutants, in
particular to those affecting the study area. For specic pollutants,

Fig. 3. % Enrichment of I. galbana (negative values corresponding to % growth inhibition), % Fertilization and % Normal pluteus for P. lividus and % Mortality in S. aurata exposed for
different efuent dilutions before (Without treat.) and after MBT process (MBT) in D1, D2 and D3 WWTPs. The a indicates signicant differences (p < 0.01) between the toxicity
control and the dilutions of the efuent; b indicates signicant differences (p < 0.05) in the same dilutions of efuent before and after the detoxication treatment.
 ~ o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun 7

Fig. 3. (continued)

several options have been proposed for different sources of

contamination, such as metals (de Paiva Magalh~ aes et al., 2014),
hydrocarbons (Bellas et al., 2008), pharmaceuticals (Aguirre-
Martnez et al., 2015) and pesticides (Mansour and Gad, 2010).
Nevertheless, most of them focus on freshwater monitoring
(Pandard et al., 2006) or on the effects of single contaminants. Very
few studies have assessed contaminant mixtures (efuent matrix)
discharged into marine environments.
To establish the advantages and the limitations of the chosen
battery of bioassay, those of each acute toxicity test are discussed in
order to determine the marine environmental risk posed by WWTP
efuent to the receiving ecosystem. A tertiary treatment, MBT, was
applied to the discharged efuents to evaluate the capacity of the
process to yield the necessary reductions in acute toxicity of the
wastewater.
The objective of using a battery of bioassays to perform a toxicity
assessment is to determine the presence or absence of a mixture of
noxious compounds in wastewater that is bio-available to biota
resident in the receiving ecosystem. Therefore, a comprehensive
toxicity assessment can only be achieved by using several toxicity
tests within a test battery, and considering not only resident species
also ecologically representative and widespread species in order to
compare studies and results.
The results obtained for the selected battery of bioassays indi-
cated that different acute toxicity effects depended on the WWTP
efuent tested. This nding is in agreement with several studies
showing that the origin of the efuent (industrial, urban, hospital,
8 ~o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun
Table 2
Summarize results of pT-Method for each WWTP.
Water treatment plant Treatment Bioassay
D1 Without treatment Embryo P. lividus
D1 MBT Embryo P. lividus
D2 Without treatment Embryo P. lividus
D2 MBT Embryo P. lividus
D3 Without treatment Larval development S. aurata
D3 MBT Larval development S. aurata
etc.) can induce different toxicity effects in organisms (Arstikaitis
et al., 2014; Escher et al., 2011; Santos et al., 2013).
All samples tested using the Microtox basic test with the bac-
terium V. scheri presented hormesis, which is dened as the
stimulatory benecial effect on organisms of very low concentra-
tions of toxic chemicals (Calabrese, 1999). Normally, Microtox
bacterial bioluminescence is considered an extremely sensitive
endpoint (Park and Choi, 2008). There is some evidence to suggest
that these compounds might be present in the efuent since
Maranho et al. (2014) analysed sediments exposed to the same
residual water treatment plant efuents, and found antibiotic
concentrations. Furthermore, Ferna ndez-Alba et al. (2002), Macken
et al. (2008), Mankiewicz-Boczek et al. (2008) and Schipper et al.
(2010) have already reported the presence of antibiotics in efu-
ents, describing them as common contaminants in a WWTP
efuent matrix.
The microalgae growth inhibition toxicity test showed different
responses (growth inhibitiontoxic response, growth stimulation
trophic response) according to the dilutions and the origin of the
WWTP efuent. The application of AOPs reduced the concentration
of organic matter in the WWTP efuent (Sindelar et al., 2014).
Previous studies (Moreno-Garrido, 2008; Radjenovic et al., 2009)
have suggested that urban and industrial efuents contain large
amounts of organic matter, disinfectants and antibiotics. This
reduction in organic matter in the WWTP efuent matrixes tested
was probably responsible for the positive microalgae % of enrich-
ment results obtained after MBT instead of the negative % of
enrichment (% of growth inhibition) before tertiary treatment.
Conversely, in the case of the D1 efuent, detoxication induced a
negative % of enrichment after tertiary treatment. Nevertheless,
after MBT, the efuents still produced toxic effects at the highest
efuent dilution (1:2) and for the positive control (100% efuent).
Consequently, the results revealed that this selected endpoint for
the microalgae growth inhibition toxicity test showed a reduction
in toxicity. However, this reduction in efuent toxicity was insuf-
cient to be harmless to the organism. Signicant differences be-
tween % of enrichment and toxicity control were observed for
almost all the efuent dilutions, although no signicant tendency
(doseeresponse) or differences between dilutions were observed.
This was due to the initial condition of the bioassay regarding the
concentration of nutrients added to the vials following the standard
protocol (Garrido-Perez et al., 2008). Thus, more research is
required to determine the real behaviour of the microalgae in the
efuent matrix.
In contrast, the results of the sea urchin larval development and
sh larvae mortality tests suggested that these were suitable to
assess the toxicity effects of the WWTP efuent. The results ob-
tained for P. lividus after exposure to the different WWTP efuents
indicated that this was the most sensitive of all the battery tests
selected.
It is widely known that embryonic and larval stages of marine
invertebrates are less tolerant to toxicants than adults (Connor,
1972; His et al., 1997; Marin et al., 1991; Ringwood, 1991), and
pT- Value Toxicity class
pT- Index Designation
3 III Moderately toxic
1 I Very slightly toxic
5 V Highly toxic
2 II Slightly toxic
5 V Highly toxic
2 II Slightly toxic
that embryoelarval tests, in particular with bivalves and sea ur-
chins, have been used for decades as sensitive, simple and reliable
tools for assessing and monitoring marine pollution (His et al.,
1997; Kobayashi, 1981; Woelke, 1972). In addition, newly hatched
larvae constitute a particularly critical and sensitive life stage,
because when they hatch, embryos lose their protective membrane
and are fully exposed to potential toxicants (Beiras et al., 2003).
Similarly, some chemicals such as ammonia or sulphide present
in the majority of WWTP efuents could be the cause of toxic ef-
fects on sea urchin larvae. Previous studies have shown that sea
urchin sperm cells and larval development are not affected by
sulphides (Losso et al., 2007). However, no direct relationship was
observed between toxic effect and ammonia concentration,
although other toxic effects have been observed due to elevated
concentrations of un-ionised ammonia in some cases. In fact, sub-
chronic toxicity tests have indicated a toxic effect caused by
ammonia (Anderson et al., 2001; Losso et al., 2007). In conse-
quence, more specic studies should be performed to determine
the potential toxicity of ammonia in organisms exposed to efuent
discharge in order to avoid false positives for toxic responses.
As regards the organisms and tests selected to perform the
battery of bioassays to assess WWTP efuent, the species were
chosen on the basis of the following aspects. Since a single test is
insufcient to evaluate toxicity (Kokkali and van Delft, 2014), we
considered diversity and variability in sensitivity when selecting
toxicity tests, as recommended by Rizzo (2011). The choice of the
most appropriate tests depends on the needs of the environment
and the possibilities of the study area. Consequently, 4 species were
selected to represent the principal trophic levels (primary pro-
ducers, primary consumer, secondary consumer and decomposers)
for inclusion in a battery of bioassays that would be effective in
southwest Spain but also in other coastal areas worldwide. The rst
species was the decomposer V. scheri, which forms part of a widely
used commercial pack from Azur Environmental (Environmental,
1998) and has been used in numerous studies to test toxicity in
the laboratory in under a few hours (Lopez-Roldan et al., 2012;
Mankiewicz-Boczek et al., 2008). Primary producers were repre-
sented in the battery by the microalgae I. galbana. The distribution
and abundance of this species in marine waters is virtually un-
known, as reports have varied from one author to another
(Marlowe et al., 1990; Parke, 1949), but it is considered a coastal
species with universal distribution in the oceans. Furthermore, this
species has been used in several studies to test the toxicity of
different efuents (Aguirre-Martnez et al., 2015; Fisher et al.,
2014). Two different species were chosen to represent consumers,
P. lividus (sea urchin) and S. aurata (sh). Both are distributed
throughout the Mediterranean Sea and along European Atlantic
coasts (Alarco  n et al., 2004; Boudouresque, 1987; Harmelin et al.,
1981; Kempf, 1962), and have been used in short-term assays to
assess toxicity (Aguirre-Martnez et al., 2015; Arufe et al., 2004;
Dimitriou et al., 2003; Gambardella et al., 2015; Oliva et al.,
2008). In addition, the use of these sea urchin toxicity tests and
these endpoints are among the standard procedures most strongly
 ~ o et al. / Marine Environmental Research 114 (2016) 1e11
B. Daz-Gardun
recommended by environmental agencies (Environment Canada,
2011; USEPA, 2002). Regarding the suitability of the test end-
points, P. lividus fertilisation and larval development have been
studied by several authors (Ferna ndez and Beiras, 2001; Carballeira
et al., 2012; Maranho et al., 2014). Likewise, the sh mortality test
has generally shown good sensitivity in toxicity testing of xenobi-
otics. In this study, S. aurata, a widespread marine species, proved
suitable for assessing WWTP efuents in marine environments.
After a 96 h period of exposure, it showed sufcient sensitivity to
enable classication of the environmental risk of efuents in the
tested samples.
Most of species in the selected battery met all the requirements
to assess the toxicity of the efuents studied. This battery of bio-
assays using different organisms and endpoints is necessary to
obtain a comprehensive toxicity determination of an aqueous
matrix. In addition, organisms selected for a battery should repre-
sent different trophic levels, be widely distributed not only in the
study area but also elsewhere in order to facilitate comparison of
results and also reproducibility, assessing the same problem at
other study areas. The selected species should also have shown
sensitivity to a complementary range of contaminants. In summary,
frequently used, standard bioassays were selected here to assess
their suitability and obtain information about a marine risk
assessment of urban efuents, since existing regulatory frame-
works lack guidance on the battery of toxicity tests suitable in the
case of WWTP efuents discharged into marine environments.
In addition, a tertiary treatment (combination of AOPs) was
applied to assess the toxicity of WWTP efuents. It should be noted
that AOPs are suitable for treating wastewaters with relatively low
COD concentrations (5 g/L) (Andreozzi et al., 1999). In this study, it
was expected that after the MBT process, residual xenobiotics and
other pollutants present in initial WWTP efuent would be
partially degraded and/or converted into nontoxic forms. However,
the reduction in COD after AOP treatments is not always associated
with a reduction in the toxicity of the treated water. By-products
that are more toxic than the initial pollutants can be formed and
this should be taken into account (Heringa et al., 2011; Quero-
Pastor et al., 2014). In the case of the MBT process applied in this
study, the acute toxicity of the WWTP efuent decreased. However,
the water remained slightly toxic after the MBT process. This was
probably due to the presence of trace amounts of H2O2 (below the
detection limit of 1 mg/L), or the possible formation of toxic by-
products (Marrot et al., 1998; Ruggeri et al., 2013; Zorita et al.,
2009).
Once the results of the bioassay battery had been obtained, the
WWTP efuent was classied according to the marine environ-
mental risk it presented using pT-method criteria. The pT-method
has proved a useful tool for assessing environmental risk for ma-
rine ecosystems (Soares and Freitas, 2000). Here, this method was
used to classify WWTP efuent into different toxicity categories
before and after the application of a photobiotreatment. It should
be emphasised that the pT-method was used as a hazard assess-
ment tool for identifying WWTP efuent toxicity and quantifying
the potential risk (Krebs, 2005). The pT-scale could therefore play a
future role as a hazard identication scheme (HIS) pursuant to the
EU WFD. A HIS provides the basis for assessing potential impacts on
an aquatic system. The results of the pT-method could inform de-
cisions regarding the need for improvements to wastewater treat-
ment in marine environments, in this case based on the scientic
criteria described in this study.
The results showed that the highest values measured with the
pT-method corresponded to the D2 and D3 efuents (pT-
value 5, highly toxic). After MBT, a reduction in toxicity was
observed and the pT-value dropped in 3 categories, classifying
them both as slightly toxic. The pT-value for the D1 efuent was 3
9
(moderately toxic) and detoxication processes decreased this
value to 1 (very slightly toxic). This classication corroborates the
results obtained in the different bioassays for each WWTP efuent,
where higher values of % of survival of S. aurata larvae and % of
fertilised eggs and pluteus larvae of P. lividus were observed after
MBT treatment than in the initial toxic categories (moderately
toxic and highly toxic).
To conclude, the selected battery of bioassays proved a suit-
able tool within the pT-method framework to assess WWTP
efuent toxicity in marine ecosystems. Application of the MBT
process reduced acute toxicity and therefore the contamination
risk in receiving marine ecosystems. Nevertheless, it should be
noted that the environmental dilution factor of the WWTP ef-
uents released into the receiving ecosystem was between 1:4 to
1:2 dilutions. Most of the post-MBT application results revealed
signicant toxic effects according to the OECD (2013) and USEPA
(2002) risk guidelines for the organism tested. Thus, although
this tertiary treatment reduces the environmental risk of WWTP
efuent, it does not render it completely harmless. Consequently,
more research is required to identify the groups of chemicals,
natural resources, pathogens or biotoxins that might be poten-
tially resistant to MBT, inducing adverse effects in receiving
ecosystems.
Acknowledgements
The present research work has been supported by the Spanish
Ministry of Economy and Competitiveness (Ref. CTM2012-37591)
and FEDER Funds from European Union. This is the contribution n
116 from the CEIMAR Journal Series. Authors would like to thanks
to Chiclana Natural, Aqualia Gestio n Integral del Agua S.A. and
Depureal 21 S.L. for their collaboration in this research work. This
manuscript has been English grammar edited by Centro Superior de
Lenguas Modernas (CSLM) University of Cadiz.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.marenvres.2015.12.011.
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