Accepted Manuscript

Algae toxicological assessment and valorization of energetic-laden wastewater

streams using Scenedesmus obliquus

Juliana Abraham, Yanxia Lin, Abhishek RoyChowdhury, Christos Christodoulatos,

Matthew Conway, Benjamin Smolinski, Washington Braida

PII: S0959-6526(18)32486-7
DOI: 10.1016/j.jclepro.2018.08.148
Reference: JCLP 13932

To appear in: Journal of Cleaner Production

Received Date: 22 March 2018

Revised Date: 20 July 2018
Accepted Date: 14 August 2018

Please cite this article as: Abraham J, Lin Y, RoyChowdhury A, Christodoulatos C, Conway M, Smolinski
B, Braida W, Algae toxicological assessment and valorization of energetic-laden wastewater streams
using Scenedesmus obliquus, Journal of Cleaner Production (2018), doi: 10.1016/j.jclepro.2018.08.148.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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Algae Toxicological Assessment and Valorization of Energetic-laden Wastewater

Streams Using Scenedesmus obliquus.
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Juliana Abrahama*, Yanxia Lina, Abhishek RoyChowdhury , Christos Christodoulatosa,
Matthew Conwayb, Benjamin Smolinskib, and Washington Braidaa

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a
Center for Environmental Systems, Stevens Institute of Technology, Hoboken, NJ 07030, USA
b
RDECOM-ARDEC, Picatinny Arsenal, NJ 07806, USA

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*
Corresponding author: jabraham@stevens.edu

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Abstract

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The manufacturing of energetic compounds (explosives), such as 1,3,5-trinitroperhydro-1,3,5-
triazine (RDX) and 3-nitro-1,2,4-triasol-5-one (NTO), generates large volumes of wastewater
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containing organic pollutants, which are also rich in nutrients, mainly nitrogen. These
wastewaters are subject to regulatory compliance and require treatment prior to their release into
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local water bodies. An approach is proposed to recover nutrients and water from wastewaters
prior to treatment by growing microalgae with the aim to reduce the energy footprint of the
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munitions production facility by producing algae-based biofuels. In order to assess the

feasibility of implementation of the proposed approach, ten different fully characterized
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untreated wastewaters with various levels of organic and inorganic carbon and nitrogen content
were assessed for microalgae toxicity using Scenedesmus obliquus ATCC® 11477 as target
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microorganism. Toxicity experiments were performed in microplates for six days at 25 ºC, 120
rpm orbital mixing speed, under a 14:10 hours light: dark photoperiod, and 68 µmol photons/m2s
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of light intensity. The results indicate that the majority of the wastewater streams assessed could
be used as nutrient media with no pretreatment while only three of the streams required chemical
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treatment due to their high growth inhibition characteristics. From the toxicological results
obtained, a site specific mixture prepared by blending of the different untreated waste streams
was tested for Scenedesmus obliquus growth. It was observed that this microalga can reach 5x107
cells/mL over six days of incubation in the wastewater mixture containing up to 24 mg/L RDX
and 28 mg/L NTO. This strain exhibited similar growth patterns in the wastewater mixture as
compared to the control sample, suggesting the potential feasibility of using untreated, energetic-

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laden industrial wastewater along with carbon dioxide [CO2 (atmospheric or flue gases)] for
microalgae biomass production.

Keywords: energetic-laden wastewater, microalgae, algae toxicity, valorization, sustainability,

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bioenergy.

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1. Introduction
Energetic compounds (ECs) are a group of organic nitro group-containing compounds, which

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include, among others substances, 1,3,5-trinitroperhydro-1,3,5-triazine (Research Department
Explosive, RDX), 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX), 1-methoxy-2,4-dinitrobenzene

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(DNAN), 3-nitro-1,2,4-triasol-5-one (NTO) and 1-nitroguanidine (NQ). The production of ECs
in munition production facilities generate wastewater, often containing the aforementioned ECs
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and other nitrogen species, which require physical-chemical and/or biological treatment prior to
their discharge. The concentrations of the ECs in the wastewater streams prior to treatment will
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vary depending on their water solubility, presence of organic solvents and the manufacturing
processes (Koutsospyros et al. 2012). Discharge limits are generally facility specific and are
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regulated by the facility’s NPDES (National Pollutant Discharge System) permits.

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The biochemical, physiological and toxic effects of the ECs on algae growth are not well-
understood, and only limited information is available in the current literature (Reddy et al. 2011;
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Dodard et al. 2013). In addition, it is widely documented that the sensitivity of microalgae to a
particular compound depends on the algal species being exposed, and to date no data describing
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the aquatic toxicity of the EC investigated in this research, on the freshwater microalgae
Scenedesmus obliquus (S. obliquus) have been found in the literature.
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Marine and freshwater microalgae are unicellular, autotrophic organisms that can fix CO2 from
the atmosphere to generate both organic compounds and oxygen via photosynthesis. Thus, they
are the main primary producers in most aquatic habitats, providing both carbon compounds and
oxygen to higher trophic levels organisms (heterotrophic organisms).

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The U.S Environmental Protection Agency (U.S EPA) Standard Methods, the International
Organization for Standardization (ISO) and the Organization for Economic Co-operation and
Development (OECD) have developed testing protocols to evaluate potential toxicity on algal
growth. Several microalgae species have been widely used for various applications including
human and animal nutrition, cosmetics, pharmaceuticals, CO2 capture, bioenergy production

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(such as biodiesel by transesterification, biogas by anaerobic digestion, direct burning after
drying and compressed into pellets), and nutrient removal from wastewater. Related to the latter,

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several studies have demonstrated favorable results for biomass growth and lipid production in
primary and secondary treated municipal wastewater, activated sludge and dairy wastewaters

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(Quiroz Arita et al. 2015, Reyimu and Özçimen 2017). In particular, studies with the freshwater
microalgae S. obliquus have demonstrated its ability to grow in different types of media ranging

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from natural habitats to industrial wastewater, its high lipid content along with its capability of
growing at elevated concentration of CO2 (Ho et al. 2010, Xin et al. 2010, Tang et al., 2011); for
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these reasons, this algae specie was selected for further testing.
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In this study, an approach is proposed to use a biological method to recover nutrients from
untreated energetic-laden industrial wastewater streams via algal biomass growth. The proposed
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approach will, in turn, reduce the wastewater’s nutrient concentration and, at the same time,
enable the production of renewable energy such as biodiesel or biogas. It should be noted that the
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wastewater streams utilized for the production of algal biomass may still contain EC
concentrations exceeding regulatory levels and, therefore, they may require further
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physicochemical or biological treatment prior to their discharge.

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The objectives of the work presented herein were: 1) to evaluate and characterize the
composition - in terms of carbon, nitrogen, metals and EC - of ten untreated energetic-laden
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wastewater streams generated at a munitions facility, 2) to carry out algae toxicity assessments of
these untreated wastewater streams using the microalgae S. obliquus as biomarker organism, 3)
to establish the links between the composition of the samples and the observed algae toxicity,
and 4) to assess the feasibility of developing a mixture of available wastewater streams which
can support, without or with minimum treatment, the growth of freshwater strain, S. obliquus to
produce suitable amounts of biomass that could support the production of bioenergy.

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2. Materials and Methods

2.1.Unicellular Algae Strain, Medium and Wastewater Streams

The freshwater microalgae Scenedesmus obliquus ATCC® 11477 was used as target

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microorganism for all algae toxicity tests. Experiments were carried out using inoculant from
seven day old (exponentially growing) cultures in sterilized BG-11 medium with the following
per liter composition: 1,500 mg NaNO3, 75 mg MgSO4, 40 mg K2HPO4, 36 mg CaCl2.2H2O, 20

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mg Na2CO3, 6 mg citric acid (vitamin C), 6 mg ferric ammonium citrate, 2.86 mg H3BO3, 1.81
mg MnCl2.4H2O, 1 mg EDTA disodium magnesium, 0.39 mg Na2MoO4.2H2O, 0.079 mg

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CuSO4.5H2O, 0.22 mg ZnSO4.7H2O, 0.05 mg Co(NO3)2.6H2O.

From the five species cultured at the laboratory (2 freshwater and 3 marine water), S. obliquus

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was selected due to its fast growth capacity (specific growth rate, µ = 0.6 day-1). Untreated
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wastewater streams from ten different collection points within a munitions production facility
were used for this study. The collected effluent samples were labeled with numbers from 1 to 10.
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2.2.Culture Conditions and Algae Toxicity Assessment

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Algae toxicity tests were performed as described in the OECD Guidelines (2011). The
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procedure was adapted to 24 well-microplates using the BG-11 medium. The samples were
incubated at 25°C in a Caron® growth chamber under continuous shaking at 120 rpm under a
fluorescent light intensity of 68 µmol photons/m2s with a 14:10 hours light:dark photoperiod.
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Light intensity was measured with a Fisher Scientific® illumination-meter. A 5% CO2

atmosphere was also provided during the lighting period. The duration of the test was six days.
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The pH of the samples was adjusted to 7.0 prior to the test assessment (with NaOH or HCl, as
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needed). Fluorescence at 685 nm was measured on a microplate reader (Cytation 3, Biotek®) as a

surrogate for the estimation of the biomass at days 0, 1, 2, 3 and 6. Moreover, the development
of green color due to algae growth was pictorially documented. Microscopy analysis was
performed, using an imaging reader (Cytation 3, Biotek®), to assess the differences in
morphology of the cells due to sample’s toxicity. Tests were executed in quadruplicates.
Biomass results are expressed as a mean of cell density (cells/mL) ± SD. Initially, growth media
amended with 10, 20, 40 and 60% (v/v) of untreated wastewater were tested. At the same time, a

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0% sample, containing only medium and microalgae, was used as control. Table 1 shows the
protocols used for algae toxicity tests. A 10x medium is also added to the system to guarantee
that results are due to toxicity of the wastewater and not due to the lack of nutrients for growth.
A blank with just medium was added to the protocol to check for any background fluorescence.
Subsequently, if results were satisfactory with these amendments levels, higher amendment

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levels (80%) were tested. The system’s response is the reduction of growth (or inhibition) in the
sample cultures due to a chemical substance compared to unexposed control cultures. The

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growth was evaluated using the biomass obtained (as cell density) and by calculating the average
specific growth rate (µ) using the exponential growth equation for each control and treatments as

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follows:

Ct = ‫ܥ‬଴ exp(ஜ௧ )

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Where µ is the average specific growth rate, a constant related to the growth curve of the
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microorganism, expressed in units of inverse time (i.e. per day), t is time, C0 is the algae biomass
at time 0, and Ct is the biomass at time t in number of algae cells per mL. The specific growth
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rates were estimated by non-linear regression.

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2.3.Analytical Methodology

Total organic carbon (TOC) was measured with a UV-Persulfate TOC Analyzer Phoenix 8000
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instrument from Teledyne Tekmar (Mason, Ohio). Nitrate (NO3-) and nitrite (NO2-) were
measured using ion chromatography (IC) with IonPac ®AS16 (4 mm×250 mm, DIONEX),
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equipped with a guard column IonPac®AG16 (4 mm×50 mm, DIONEX). Test kits (HACH®)
were used to measure total nitrogen (TN), ammonia (NH3) and total phosphorus (TP). The pH
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was measured by an Oakton® PC 700 pH meter. Identifications of different chemical moieties of

compounds present in the samples, which were not necessarily quantified, were performed by
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HePI-MS as described by Yang et al. (2012). EC including RDX, HMX, DNAN, NTO and NQ
were quantified by HPLC and HPLC-MS based on the U.S EPA Standard Method 8330.
Acetone concentration was measured by GC-FID. Metals were analyzed by inductively coupled
plasma optical emission spectroscopy (ICP-OES) following U.S EPA 200.7 Method and atomic
absorption spectroscopy (AAS) for samples previously acid digested (U.S EPA 3051A) and
filtered without digestion (soluble metals).

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2.4.Statistics
Toxicity tests were carried out in quadruplicates. Data of cell density at the end of the test (6d)
were analyzed by one-way analysis of variance (ANOVA) at a significance level of 0.05 using
Sigmaplot® Software Version 13.0 to assess the effect of the industrial wastewater as well as the

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blend formulated from selected wastewater streams on microalgae growth. When significant
differences were detected, the Holm-Sidak post-hoc test was applied for comparison of

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individual amendments.

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3. Results and Discussion
3.1.Characterization and Algae Toxicity Assessment of Energetic-Laden Wastewater

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Streams
A full characterization was performed for all samples in order to assess their composition, the
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levels of nutrients that potentially could be recovered, as well as the presence of toxic
compounds. The detailed characterization results are presented in Tables 2, 1S and 2S. All
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wastewater streams tested had different chemical compositions depending on the industrial
processes from which they originated within the munitions production facility. The majority of
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the samples were found to contain nutrients such as carbon, nitrogen and small amounts of
phosphorus, which are relevant macronutrients for microalgae growth and, consequently, can be
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reclaimed from the wastewater for biomass production (Table 2). Some samples had high levels
of TN (total nitrogen) due, in part, to the high quantity of NO3- or NH4+; moreover, some of them
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contain substantial amounts of organic nitrogen. The two primary nitrogen species utilized by
microalgae are NH4+ and NO3-. NH4+ is the preferred form for microalgae growth, as the
incorporation of NO3- requires additional metabolic energy and enzymatic activity (Perez-Garcia
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et al. 2011). Conversely, both species have been reported toxic to microalgae growth above
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certain concentration threshold (Chen et al. 2009).

With regards to other important macronutrients for microalgae growth, the amount of
phosphorous (expressed as total phosphorous, TP) found in all untreated wastewater samples is
low compared with other nutrients (Table 2). However, the phosphorous requirements for algae
growth are not as high as compared to nitrogen. For instance, the nitrogen: phosphorous ratio for
normal algae growth can fluctuate from 6:1 to 16:1 (Geider and La Roche, 2002). Therefore, if

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any of these wastewaters streams are reclaimed for algal growth a source of phosphorous, albeit
small, should be added.
Tables 1S and 2S present the concentration of total metals and soluble metals respectively found
in all ten samples. As evident from the data, the levels of total metals in the untreated wastewater
samples are higher than those of soluble metals. This indicates that the majority of metals in the

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samples are present in insoluble form, which reduces their potential bioavailability.
Some trace metals such as iron, zinc, magnesium and copper are necessary for microalgae

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growth, as described above in section 2.1, since they are essential micronutrients or cofactors for
enzymatic activity. On the other hand, these metals can be toxic above certain concentrations.

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The effective concentration of a substance that inhibits 50% of the microalga population after a
specified exposure time is called EC50 and it is commonly used as a measure of a substance

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potency. For instance, studies such as Monteiro et al. (2011) reported that the zinc 96h EC50 for
S. obliquus is 16.99 mg/L and Yan & Pan (2002) showed that the copper 96h EC50 for S.
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obliquus is 0.05 mg/L. Consequently, none of these low concentrations of soluble metal appears
to be toxic to S. obliquus.
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3.1.1 Samples with high concentration of nitrogen species

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Samples 2 and 3 contain high amounts of nitrogen in the form of either NO2-, NO3-, and/or NH4+
(Table 2); thus, to check for potential nutrient toxicity, the samples were diluted several fold, and
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the complementary medium used in the test protocol did not contain any additional source of
NO3-. Figure 1a shows Sample 2 toxicity results. In general in these tests, the control follows the
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growth trend of any microorganism in normal batch conditions: there is a small lag phase, which
corresponds to the time necessary to start replicating followed by an exponential phase
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characterized by its fast growth due to the availability and abundance of nutrients. Depending on
the composition of the sample tested and its toxicity, the different amendment levels will show
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different patterns of growth, meaning a reduction in the cell density measured. It is clearly
observed that Sample 2 is highly toxic to microalgae growth at the 40% and 60% amendment
levels due to the reduction of cell density. The observed algae toxicity is likely due to high
content of NO3- (11,480 and 17,220 ppm, for 40% and 60% amendments respectively, Table 3S).
Sample 2 was then diluted 2-times and further dilutions were prepared from this half-strength
sample (Table 3S). Algae toxicity test results of the diluted Sample 2 are presented in Figure 1b,

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which demonstrates that algae growth increased for 40% and 60% amendments and that the toxic
effect observed was related to high NO3- concentration in this sample. The results confirmed that
although NO3- is an essential macronutrient for microalgae, it could also be potentially toxic for
algal growth over a certain concentration range. These results (showed in Figure 1a and 1b and
Table 3) are in accordance with Taziki et al. (2015) who demonstrated that 3000 mg/L of nitrate

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are toxic for microalgae growth. The results suggested that up to its 10% amendment level (v/v)
wastewater Sample 2 could be considered as a potential source of NO3- for algae growth without

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presenting inhibition in the growth of S. obliquus.

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Based on the toxicity results obtained with Sample 2, untreated wastewater Sample 3 was
initially diluted 500 and 1,000 times before performing toxicity test, as it contains an even higher
concentration of NO3- and NH4+ (Table 2). Figure 1c shows the growth curves of Sample 3 when

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tested with different amendments of the sample previously diluted 500 times, while Figure 1d
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shows the growth curves of different concentrations of wastewater Sample 3 initially diluted
1,000 times. As expected, 1,000X dilution exhibited lower inhibition than the 500X dilution;
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nevertheless, there was still some algae toxicity/cell growth reduction observed, likely due to
other compounds found in this sample such as acetone and dimethylformamide. Both compounds
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were detected in HePI-MS scans of this sample (Table 2) and are well known to be toxic to
microalgae (Cho et al. 2009). Wastewater Sample 3 also contains a significant amount of metals
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such as chromium, copper, iron and nickel (Table 1S and 2S). However, none of them should be
toxic at the concentration levels found in the samples at such dilutions (500X and 1,000X).
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Hence, a 5% content of Sample 3 diluted 1,000 times could be utilized as a source of nitrogen for
biomass production without imparting any toxicity to the mixture.
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3.1.2 Samples with high amount of TOC and TN

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Other samples had high amounts of TOC and TN owing to the presence of EC. In fact, three of
the samples showed high toxicity to microalgae, there is no cell growth measured compared to
the control, due to high concentration and the type of EC they contained. This is the case for
Samples 1, 6 and 7 (Table 2). Sample 1 exhibited a similar toxic behavior as Sample 7, showing
high toxicity to microalgae at all the percentages tested (Figure 2a and 2c) while Sample 6 is
toxic at concentration levels exceeding 20%. Conversely, the 10% amendment showed low

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toxicity to S. obliquus (Figure 2b). These results indicate that these three samples should be
treated before they can be utilized as growth media for the production of algal biomass or
previous to their discharge. Prior to its use in energetic formulations, DNAN was used as an
insecticide (Xing et al. 2012). 2,4-dinitrophenol (DNP) was also used as an antiseptic and
pesticide. Dodard et al. (2013) studied the eco-toxicological effect of DNAN in aqueous and

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terrestrial samples for bacteria, green algae, earthworms and plants, and reported that the EC 50
for Pseudokirchneriella subcapitata (green algae) was 4 mg/L, while Hayley et al. (2009)

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reported an EC50 of 3,465 mg/L for the same strain tested in NTO solutions after pH
adjustments. Potential treatments for these wastewater streams include Mg-based bimetal

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reduction as described by Hadnagy et al. (2018) and Fe-based bimetals reduction as described by
Koutsospyros et al. (2012) and Kitcher et al. (2017).

3.1.3 Samples with low or no toxicity

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The remaining samples exhibited a wide range of results, but no high levels of toxicity to S.
obliquus were observed. For example, Samples 4 and 9 showed no algae toxicity at the 10%,
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20% and 40% v/v amendment levels. However, they induced a significant growth inhibition at
the 60% v/v amendment level. Figure 3a shows the results obtained for sample 4. Sample 9
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(figure not shown) exhibited an identical (similar) trend. In other words, the cell density achieved
in the control sample compared with the cell density obtained with the 10%, 20% and 40 %
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amendments showed no differences whereas the 60% amendment did exhibit differences in algae
growth (Sample 4: 1-way ANOVA, d.f = 4, F = 16.191, p = 0.001, with Holm-Sidak’s post-hoc,
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p = 0.001; Figure 3a and Sample 9: 1-way ANOVA, d.f. = 4, F = 42.343, p = 0.001, with Holm-
Sidak’s post-hoc, p = 0.001). Both samples contain measureable amounts of TN and TOC due to
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the presence of several EC and solvents. One of the EC present, RDX, has a water solubility of
about 42 mg/L and higher solubility in organic solvents. For instance, acetone concentrations
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were 1,961 and 1,148 mg/L for Samples 4 and 9, respectively, which might increase RDX
solubility. Information on the toxicity of RDX to microalgae is limited (Mukhi et al. 2005,
Stanley et al. 2015). Moreover, 42 mg/L of RDX showed no toxicity for S. obliquus (data not
shown). Burton et al. (1994) stated that due to relatively low solubility of RDX in water, no acute
toxicity was observed to green microalgae S. capricornutum and EC50 could not be determined.
Hence, the observed algae toxicity could be due to the presence of RDX in higher concentrations

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exceeding the 40 mg/L aqueous solubility (cosolvency effects, colloidal micro particles), the
acetone concentration present, or potential synergistic interactions amongst the existing EC in
the sample. As a result of all these observations, it was concluded that a maximum amendment
level of 40% of both samples (Samples 4 and 9) could be used as media for microalgae growth
without observable inhibitory effects.

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Sample 8 showed no differences in the cell density obtained for all the amendments tested (1-

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way ANOVA, d.f. = 4, F = 2.149, p = 0.125; Figure 3c) while sample 10 showed very low
inhibition of growth up to the 60% (v/v) amendment level (1-way ANOVA, d.f. = 4, F = 11.504,

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p = 0.001, with Holm-Sidak’s post-hoc, p = 0.390). Both samples have low TN and TOC values
(Table 2), and they could be used, for example, to dilute Sample 2 or Sample 3 before using

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them as media for biomass production. Sample 5 exhibits similar growth pattern and cell density
for all the percentages tested up to 80% compared with the control sample. A 1-way ANOVA
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comparison performed on the data presented in Figure 3b demonstrates clearly that there are not
significant differences in the growth dynamics of the populations for all amendments of Sample
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5 (d.f = 5, F = 1.740, p = 0.176). Microscopic examination showed the presence of a large

number of healthy microalgae cells in this sample (Figure 4a). Note the 4-cells colonial form,
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which is characteristic of this genus of green algae. Figure 4b shows the development of green
color over time resulting from algae growth. It should be noted that the control and the 80%
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wastewater amendment have similar color. According to the test results, this sample could be
used to dilute other samples with higher toxicity in order to achieve a non-toxic blend for algae
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growth.
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Finally, the specific growth rate constants were also calculated for all the samples and are
presented in Table 3. As shown, µ for the controls and the samples remained similar when the
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samples did not show toxicity at any of the amendment levels performed (i.e. Sample 5, 8 and
10) while they could not be determined when samples were highly toxic and, in consequence, S.
obliquus could not grow (Sample 1, 6 and 7). On the other hand, µ decreased when inhibition
became a factor such as in Sample 2, 3, 4, and 9, where the reduction of the growth rates were
different at different amendment levels according to the composition of each sample and as
previously explained in detail. In all the cases, the lack or grade of toxicity for the amendment

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levels of each sample assessed derived similar conclusions either by analyzing the cell density or
the specific growth rates.

3.2.Evaluation of a Potential Mixture on the Growth of S. obliquus

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Based on the characterization and algae toxicity test results, a blend was prepared from different
untreated wastewater streams, mixed in selected proportions, and then evaluated as an algae

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growth medium following the experimental protocol described in Table 1. The composition of
the mixture was as follows: 55% of Sample 5, 40% of Sample 9 and 5% of Sample 3 diluted

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1,000 times in Sample 8. The mixture corresponds to 122 mg/L of TN, 900 mg/L of TOC, 28
mg/L of NTO and 24 mg/L of RDX. Results from the algae growth experiments for the blend are

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shown in Figure 5. S. obliquus exhibited normal growth characterisics in the untreated
wastewater mixture without inhibition for all the percentages tested between 20% and 80% (1-
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way ANOVA, d.f = 4, F = 1.918, p = 0.160). The average observed growth rates for this strain
(SD in parenthesis) were 0.66 d-1 (0.01), 0.65 d-1 (0.01), 0.66 d-1 (0.01), 0.65 d-1 (0.01), 0.62 d-1
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(0.01) for control, 20%, 40%, 60 % and 80% (v/v) amendments, respectively. Conversely, the
90% amendment of wastewater mixture, which did not contain any other macro and micro
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nutrient augmentation, did not show any appreciable algae growth. Therefore, it is concluded that
relevant macro and micronutrients such as phosphates, magnesium and trace metals must be
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supplied in order to support algae growth.

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4. Conclusions
The feasibility of growing algae by taking advantage of nutrient rich, untreated energetic-laden
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wastewater has been successfully demonstrated in this work. Algae toxicity of the different
wastewater streams to S. obliquus was assessed by plotting the algae cell concentration profiles
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for different amendment levels and calculating the specific growth rates. The results showed that
the majority of the untreated wastewater samples tested (seven out of ten) had low or no toxicity
to S. obliquus, suggesting their potential use as a medium to grow algae. The remaining three
samples showed high algae toxicity due to the type and concentration of EC present, namely
DNAN, NQ and NTO. Consequently, the latter samples require further treatment prior to their
utilization for algae growth.

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After characterization and algae toxicity screening of the samples, a mixture from various
untreated wastewater streams was prepared and tested for growth of the microalgae S. obliquus.
The results exhibited growth patterns similar to those in the control, with no toxicity or growth
inhibition observed even in the presence of 24 mg/L of RDX and 28 mg/L of NTO when
phosphorous, magnesium and trace metals were added to the wastewater mixture.

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This study demonstrates that energetic-laden wastewater from munitions production facilities is

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amenable to water and nutrient recovery (mainly nitrogen) by microalgae cultivation and can be
used as a feedstock alternative for cost-effective biofuel production while providing pretreatment

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of the wastewater for the attainment of regulatory compliance prior to discharge. The
reclamation of macronutrients and reutilization of wastewater streams to produce valuable bio-

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stocks align with sustainable and efficient production practices and may open a path to the
reduction of the energy footprint and generation of waste at munitions production facilities.
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Acknowledgements
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This work is supported by the Consortium for Energy, Environment and Demilitarization
(CEED) contract number SINIT-15-0013. Authors would like to thank Dr. Terracciano and Dr.
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Su for their analytical help, Dr. Liang for facilitating the access to the microplate reader, and Dr.
Attygalle and the Center for Mass Spectromey (Stevens Institute of Technology) for MS scan
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analysis.
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References
Burton, D.T., Turley, S.D., Peters, G.T.,1994. The toxicity of hexahydro-1,3,5-trinitro-
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1,3,5-triazine (RDX) to the freshwater green alga Selenastrum capricornutum. Water, Air and
Soil Pollut. 76, 449-457. https://doi.org/10.1007/BF00482717
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Chen, W., Zhang, Q., Dai, S., 2009. Effects of nitrate on intracellular nitrite and growth
of Microcystis aeruginosa. J. Appl. Phycol. 21, 701–706. https://doi.org/10.1007/s10811-009-
9405-1
Chul-Woong Cho, Thi Phuong Thuy Pham, Sok Kim, Young-Ran Kim, You-Chul Jeon,
Yeoung-Sang Yun., 2009. Toxicity assessment of common organic solvents using a biosensor

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based on algal photosynthetic activity measurement. J. Appl. Phycol. 21(6):683-689.

http://dx.doi.org/10.1007/s10811-009-9401-5
Dodard, S. G., Sarrazin, M., Hawari, J., Paquet, L., Ampleman, G.,Thiboutot, S.,
Sunahara, G.I., 2013. Ecotoxicological assessment of a high energetic and insensitive munitions
compound: 2,4-Dinitroanisole (DNAN). J. Hazard. Mater. 262, 143–150.

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http://dx.doi.org/10.1016/j.jhazmat.2013.08.043.
Geider, R., La Roche, J., 2002. Redfield revisited: variability of C: N: P in marine

RI
microalgae and its biochemical basis. Eur. J. Phycol. 37, 1-17.
https://doi.org/10.1017/S0967026201003456

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Hadnagy, E., Mai, A., Smolinski, B., Braida, W., Koutsospyros, A., 2018.
Characterization of Mg-based bimetal treatment of insensitive munition 2,4-dinitroanisole.

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Environ Sci Pollut Res, https://doi.org/10.1007/s11356-018-2493-1.
Haley V, Kuperman RG, Checkai, R.T., 2009. Aquatic toxicity of 3-nitro-1,2,4-triazol-5-
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one. ECBC-TR-726. US Army Research, Development and Engineering Command, Edgewood
Chemical Biological Center, Aberdeen Proving Ground, MD.
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Ho, S.-H., Chen, W.-M., Chang, J.-S., 2010. Scenedesmus obliquus CNW-N as a
potential candidate for CO2 mitigation and biodiesel production. Bioresour. Technol. 101, 8725–
D

8730. https://doi.org/10.1016/j.biortech.2010.06.112.
Kitcher, E., Braida, W., Koutsospyros, A., Pavlov, J., Su, T.L., 2017. Characteristics and
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products of the reductive degradation of 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole

(DNAN) in a Fe-Cu bimetal system. Environ. Sci. Pollut. Res. 24, 2744-2753.
EP

https://doi.org/10.1007/s11356-016-8053-7.
Koutsospyros, A., Pavlov, J., Fawcett, J., Strickland, D., Smolinski, B., Braida, W., 2012.
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Degradation of high energetic and insensitive munitions compounds by Fe/Cu bimetal reduction.
J. Hazard. Mater. 219-220, 75-81. https://doi.org/10.1016/j.jhazmat.2012.03.048
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Monteiro, C.M., Fonseca, S. C., Castro, P.M.L, Malcata, F.X., 2011. Toxicity of
cadmium and zinc on two microalgae, Scenedesmus obliquus and Desmodesmus pleiomorphus,
from Northern Portugal. J. Appl. Phycol. 23, 97-103.
Mukhi, S., Pan, X., Cobb, G.P., Patiño, R., 2005. Toxicity of hexahydro 1,3,5-trinitro-
1,3,5-triazine to larval zebrafish (Danio rerio). Chemosphere 61, 178-185.
http://dx.doi.org/10.1016/j.chemosphere.2005.02.067

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Organization for Economic Co-operation and Development (OECD) Guidelines for the
testing of chemicals 201. July 2011.
Perez-Garcia, O., Froylan M.E., Escalante, F.M.E, de-Bashan, L.E., Bashan, Y., 2011.
Heterotrophic cultures of microalgae: Metabolism and potential products. Water Res. 45, 11-36.
http://dx.doi.org/10.1016/j.watres.2010.08.037

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Quiroz Arita, C.E., Peebles, C., Bradley, H.B., 2015. Scalability of combining
microalgae-based biofuels with wastewater facilities: A review. Algal Res. 9, 160-169.

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http://dx.doi.org/10.1016/j.algal.2015.03.001
Reddy, G., Song, J., Kirby, P., Lent E. M., Crouse, L.C., Johnson, M.S., 2011.

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Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one
(NTO). Mutation Res. 719, 35-40. https://doi.org/ 10.1016/j.mrgentox.2010.11.004

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Reyimu, Z. and Özçimen, D., 2017. Batch cultivation of marine microalgae
Nannochloropsis oculata and Tetraselmis suecica in treated municipal wastewater toward
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bioethanol production. J. Clean. Prod. 150, 40-46. https://doi.org/10.1016/j.jclepro.2017.02.189
Stanley, J.K., Lotufo, G.R., Biedenbach, J.M., Chapelli, P., Gust, K., 2015.Toxicity of the
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conventional energetics TNT and RDX relative to new insensitive munitions constituents DNAN
and NTO in Rana Pipiens tadpoles. Environ. Toxicol. Chem. 34 (4), 873–879.
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https://doi.org/10.1002/etc.2890
Tang, D., Han, W., Li, P., Miao, X., Zhong. J., 2011. CO2 biofixation and fatty acid
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composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2

levels. Bioresour Technol. 102 (3), 3071-3076. https://doi.org/10.1016/j.biortech.2010.10.047
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Taziki, M., Ahmadzadeh, H., Murry, M.A., 2015.Growth of Chlorella vulgaris in High
Concentrations of Nitrate and Nitrite for Wastewater Treatment. Curr. Biotechnol. 4, 000-000.
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https://doi.org/10.2174/2211550104666150930204835
U.S Environmental Protection Agency Method 200.7, Revision 4.4: Determination of
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Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma-Atomic
Emission Spectrometry.
U.S Environmental Protection Agency Method 3015A. Microwave assisted acid
digestion of aqueous samples and extracts.
U.S Environmental Protection Agency Method 8330B. Nitroaromatics, nitramines, and
nitrates esters by high performance liquid chromatography (HPLC).

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Xin, L., Hong-ying, H., Jia, Y., 2010. Lipid accumulation and nutrient removal properties
of a newly isolated freshwater microalga, Scenedesmus sp. LX1, growing in secondary effluent.
New Biotechnol. 27 (1), 59-63. https://doi.org/10.1016/j.nbt.2009.11.006
Xing, X., Zhao, F., Ma, S., Xu, K., Xiao, L., Gao, T. A., Hu, R., 2012. Specific Heat
Capacity, Thermal Behavior, and Thermal Hazard of 2,4-Dinitroanisole. Propellants Explos.

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Pyrotech. 37, 179-182. https://doi.org/10.1002/prep.201000077.
Yan, H., Pan, G., 2002. Toxicity and bioaccumulation of copper in three green microalgal

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species. Chemosphere 49 (5), 471-476.
Yang, Z., Pavlov, J., Attygalle, A.B., 2012. Quantification and remote detection of nitro

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explosives by helium plasma ionization mass spectrometry (HePI-MS) on a modified
atmospheric pressure source designed for electrospray ionization. J. Mass Spectrom. 47 (7), 845-

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Tables

Table 1. Algae Toxicity Testing Protocol.

% of Test Substance 90%* 80% 60% 40% 20% 10% 0% Blank

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(Control)

Volume of Test Substance (µL) 2700 2400 1800 1200 600 300 0 0

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Volume of 1X Medium or DI 0 0 600 1200 1800 2100 2400 2700
water (µL)

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Volume of 10X Medium (µL) 0 300 300 300 300 300 300 300

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Volume of Cell Suspension (µL) 300 300
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Final Volume for each well (uL) 3000 3000 3000 3000 3000 3000 3000 3000
*This percentage was only tested in the experiments of growth with the blend of untreated
wastewaters selected.
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Table 2. Characterization of Wastewater Streams Obtained From a Munitions Facility.

Waste pH TN * N-NH3 * N-NO3- N-NO2- TP * TOC MS scans Energetic

streams (mg/L) (mg/L) (mg/L) (mg/L)) (mg/L) (mg/L) compounds

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(ppm)

1 0.66 (0.01) 10200 B.D.L 9923 (3973) 429 (100) 0.01 40 (4) NQ 9556 NQ

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2 0.67 (0.01) 6910 B.D.L. 28700 (1158) 0 B.D.L (0.004) NO3- , 4-anisaldehyde, MeOH -

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NO3-, NH3, DMF, acetone,
3 5.12 (0.01) 287000 82400 119220 (20682) 29525(1449) B.D.L 192 (25) dimethylacetamide -

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4 30216 RDX, Solex, HMX, acetone 76 RDX, 1 NQ

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3.95 (0.01) 295 14.20 26 (3) 123 (18) 0.04 (1800)

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5 6.75 (0.01) 33.70 0.53 5.74 (0.14) 27 (4) 0.04 57 (2) RDX, DNAN, acetone 10 RDX, 1 NQ

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6 NTO, NQ 8234 NTO, 415
12.15 (0.01) 4870 19.80 1671 (726) 4991 (284) B.D.L 569 (13) NQ

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7 7.08 (0.01) 47.00 B.D.L. 1.33 (0.03) 34 (3) B.D.L 194 (10) DNP, 5 RDX

8 4.61 (0.01) 16.30 28.30 B.D.L 54 (8) 0.04 174.9 (0.4) N.A N.A
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NTO, NQ, RDX, MeOH, acetone 46 RDX, 70

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9 6.59 (0.01) 221 12.70 B.D.L. 413 (5) 0.12 2132 (2) NTO

10 7.67 (0.01) 4.23 B.D.L. 0.69 (0.02) 36.58 (0.77) 0.04 4.89 (0.36) N.A N.A
Results expressed as mean of triplicates with SD in parenthesis. B.D.L: below detection limit, N.A: not analyzed, * Hach kits (no SD). RDX: 1,3,5-trinitroperhydro-1,3,5-triazine;
DNAN: 1-methoxy-2,4-dinitrobenzene; NTO: 3-nitro-1,2,4-triasol-5-one; NQ: 1-nitroguanidine; HMX: 1,3,5,7-Tetranitro-1,3,5,7-tetrazocane; DNP: 2,4-dinitrophenol, DMF:
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Table 3. Algae Toxicity Levels of the Wastewater Streams Evaluated.

Samples Specific Growth Rates, day-1 (SD) Toxicity likely due to Toxicity level Required action
before use for
0% 10% 20% 40% 60%
algae
growth/discharge
1 0.63 NQ Toxic Treatment EC
(0.02)
2-2x 0.52 0.52 0.50 0.46 0.36 NO3- Non- toxic ≤ 20% None if diluted 2x

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(0.03) (0.03) (0.03) (0.04) (0.03)
3-1000x 0.67 0.65 0.61 0.42 0.43 NO3- and NH3 Non-toxic ≤ 20% None if diluted
(0.01) (0.01) (0.01) (0.03) (0.03) 1000x
4 0.60 0.61 0.59 0.59 0.44 acetone, RDX Non-toxic < 60% None below 40%

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(0.01) (0.01) (0.02) (0.06) (0.05)
5 0.68 0.66 0.66 0.67 0.65* - Non-toxic ≤ 80% None
(0.01) (0.01) (0.02) (0.01) (0.02)
6 0.45 0.42 NTO, NQ Toxic > 10% Treatment EC

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(0.01) (0.02)
7 0.57 DNAN, DNP Toxic Treatment EC
(0.01)
8 0.64 0.64 0.61 0.61 0.59 - Non-toxic ≤ 60% None
(0.01) (0.01) (0.01) (0.01) (0.01)

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9 0.62 0.63 0.63 0.62 0.49 Acetone, ECs Non-toxic < 60% None below 40%
(0.02) (0.02) (0.02) (0.02) (0.08)
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10 0.60 0.60 0.61 0.62 0.60 - Non-toxic ≤ 60% None
(0.01) (0.01) (0.01) (0.02) (0.01)
RDX: 1,3,5-trinitroperhydro-1,3,5-triazine; DNAN: 1-methoxy-2,4-dinitrobenzene; DNP: 2,4-dinitrophenol; NTO: 3-nitro-1,2,4-
triasol-5-one; NQ; 1-nitroguanidine; ECs: energetic compounds. *80%: 0.66 (0.01)
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Figures captions

1. Growth curves of S. obliquus in samples with high nitrogen concentrations a) Sample

2, b) Sample 2 diluted twice, c) Sample 3 diluted 500x and, d) Sample 3 diluted 1,000x.
2. Growth curves of S. obliquus in samples with high concentrations of energetic

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compounds a) Sample 1, b) Sample 6, and c) Sample 7.
3. Growth curve of S. obliquus in samples with low or no toxicity a) Sample 4, b) Sample

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5 and, c) Sample 8.
4. Image of S. obliquus growing under normal conditions in Sample 5 a) at augmentation

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of 20X and b) color development under naked eye for day 0 (left) and day 6 (right).
5. Growth curve of S. obliquus in a blend of untreated wastewaters.

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Figure 1.

3.0e+7 3.0e+7
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Cell Density (cells/mL)

Cell Density (cells/mL)

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Figure 2.

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Figure 3.

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Figure 5.

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Supplementary Information
Table 1S. Concentrations (mg/L) of Total Metals in Wastewater Samples.
Sample

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N° Al Cd Co Cr Cu Fe Mn Mo Ni Zn Pb As
1 2.8 BDL BDL 0.25 39.65 6.95 0.1 BDL 0.25 22.15 BDL 0.12
2 1.05 BDL BDL 0.10 39.65 4.30 0.05 BDL 0.3 23.5 BDL 0.32

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3 2.00 BDL 0.10 12.13 14.57 18.47 1.13 0.10 6.07 7.83 0.07 0.37
4 2.60 BDL BDL 0.10 40.75 13.65 0.10 0.05 0.55 22.80 0.09 0.71

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5 1.50 BDL BDL 0.10 40.25 5.80 0.00 BDL 0.20 22.35 0.10 0.23
6 4.15 BDL BDL 0.20 42.40 13.10 0.15 BDL 0.30 24.15 0.05 1.12
BDL BDL BDL

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7 0.93 0.10 12.87 2.17 0.00 0.10 7.33 0.06 0.10
8 2.33 BDL BDL 0.10 39.80 7.63 0.10 BDL 0.20 22.43 BDL 0.03

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9 1.20 BDL BDL 0.20 40.03 4.00 0.00 BDL 0.20 23.23 0.09 0.03
10 0.87 BDL BDL 0.13 12.97 2.57 0.03 BDL 0.17 7.70 0.18 BDL

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Results expressed as mean of triplicates with SD of 0.01. BDL: below detection limit (10 ppb).

Table 2S. Concentrations (mg/L) of Soluble Metals in Wastewater Samples.

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N° Al Cd Co Cr Cu Fe Mn Mo Ni Zn Pb
1 0.11 N.A N.A 0.1 0.08 1.35 0.05 N.A 0.08 0.28 N.A
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2 0.02 N.A N.A 0.03 0.01 0.27 0.01 N.A 0.03 0.07 N.A
3 0.10 N.A 0 14.60 1.80 14.80 1.03 0.7 7.00 0.50 0
4 0.30 N.A N.A 0.00 0.00 0.30 0.00 0 0.00 0.10 0
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6 0.00 N.A N.A 0.01 0.00 0.00 0.01 N.A 0.02 0.01 0
7 0.03 N.A N.A 0.00 0.00 0.00 0.00 N.A 0.00 0.01 0
8 0.01 N.A N.A 0.00 0.01 0.00 0.00 N.A 0.00 0.04 N.A
9 0.01 N.A N.A 0.01 0.01 0.00 0.00 N.A 0.00 0.00 0
10 0.05 N.A N.A 0.00 0.00 0.00 0.00 N.A 0.00 0.00 0.15
Results expressed as mean of triplicates with SD of 0.01. N.A: not analyzed.

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Table 3S. Concentrations of Nitrates in Sample 2 and Control.

Percentage (%) of Sample #2 60 % 40 % 20 % 10 % Control
(from medium)
NO3- (mg/L) in sample 17220 11480 5740 2870 1971
-
NO3 (mg/L) in sample diluted 2 times 8610 5740 2870 1435 1971

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Highlights

• Algae toxicity assessments of wastewaters from a munition production facility.

• The majority of untreated wastewaters analyzed were not toxic to Scenedesmus obliquus.
• A blend of untreated wastewaters was formulated to recover nutrients for algae growth.

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• A proof of concept for valorizing wastewaters from a munition production facility.

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