Environ Sci Pollut Res DOI 10.1007/s11356-015-4119-1

RESEARCH ARTICLE

Acute and chronic toxicity of emerging contaminants, alone or in combination, in Chlorella vulgaris and Daphnia magna María Victoria Pablos & Pilar García-Hortigüela & Carlos Fernández

Received: 5 June 2014 / Accepted: 11 January 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract This work presents the toxicity results of different compounds classified as emerging contaminants on primary producers and primary consumers in the aquatic compartment. The objectives were to (1) obtain acute and chronic toxicity results for algae and Daphnia magna using standardised or currently used tests, (2) study the relationship between the effects on the impaired feeding rate for daphnia and the effects of reproduction and (3) examine the responses on daphnia and algae after binary combinations of environmentally relevant compounds and perfluorooctane sulfonate (PFOS). Toxicity data on personal care products (PCPs), not reported in the scientific literature up to now, are presented. The results confirmed that the Daphnia feeding bioassay can be a sensitive, ecologically relevant endpoint to detect sublethal effects and could complement the information obtained with the reproduction test on Daphnia. The results also suggested that the concomitant occurrence of PFOS and other emerging contaminants in the aquatic compartment could affect the toxicity of some compounds according to their lipophilicity. Keywords Emerging contaminants . Daphnia magna . Chlorella vulgaris . Combined toxicity . Parabens . UV filters . Fragrances . PFOS Introduction The term “emerging contaminants” describes a variety of chemicals that are currently used and are released into the environment. They are of special concern because of their Responsible editor: Henner Hollert M. V. Pablos (*) : P. García-Hortigüela : C. Fernández Laboratory for Ecotoxicology, Department of Environment, Spanish National Institute for Agricultural and Food Research and Technology (INIA), Crta. A Coruña km 7,5, 28040 Madrid, Spain e-mail: [email protected]

widespread occurrence and potentially toxic effects (Wong 2006). This group encompasses, among others, pharmaceuticals, personal care products (PCPs) (fragrances, preservatives and UV filters), perfluorinated compounds (PFCs) and flame retardants, such as polybrominated diphenyl ethers (PBDEs). They derive from domestic and industrial uses, and their presence in municipal, agricultural and industrial wastewaters is well-documented (Lee et al. 2005; Eljarrat et al. 2008; Kumar and Xagoraraki 2010). PCPs are among the most frequently detected compounds in surface waters (Peck 2006). In the last few years, the number of ecotoxicity studies conducted on the most frequently detected PCPs in waters, which have assessed both acute and sublethal effects, has significantly grown (Brausch and Rand 2011; Yamamoto et al. 2011; Kaiser et al. 2012). Nonetheless, information about their toxicity is not so extensive if compared with other chemicals, i.e. pharmaceuticals, especially for primary producers (algae) and primary consumers (aquatic invertebrates). The environmental risk assessment of these compounds entails evaluating acute (lethal) effects and, in some cases, studying of long-term effects (reproduction). However, sublethal endpoints generally precede lethal responses and are manifested at lower exposure levels (Gerhardt 1996; McWilliam and Baird 2002). Thus, continuous occurrence of PCPs at low concentrations in the aquatic compartment suggests that subtle ecological effects can be underestimated if standardised assays are used (Barata et al. 2008). To study sublethal effects of ecological relevance, one alternative proposed in recent years has been the Daphnia magna feeding assay (Barata et al. 2008). Feeding impairment has been related to other relevant effects, such as growth and reproduction (Barata and Baird 2000), and results in population responses (Barata et al. 2008). The present work studies the relationship between chronic endpoints and feeding impairment in order to determine if the effects on reproduction can be explained by a general toxic stress response.

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Aquatic organisms are invariably exposed to contaminant mixtures, and additive, synergistic or antagonistic toxic effects are produced (Barata et al. 2006; Schnell et al. 2009; Rodea-Palomares et al. 2012). The coexistence of different emerging contaminants in the aquatic compartment has demonstrated interactions between compounds that could influence both uptake and toxicity. By way of example, Liu et al. (2009) studied the interaction between PFOS and pentachlorophenol, atrazine and diuron on the growth of Scenedesmus obliquus. Their results demonstrated that PFOS can affect the cell uptake and toxicity of these compounds differently by increasing or reducing the toxicity of these compounds according to their hydrophobicity. Other works have also evaluated these interactions have also done the same (Rodea-Palomares et al. 2012). Thus, studying the effects of the interaction between PFOS and other emerging contaminants frequently presented in water on algae and D. magna is clearly justified. The goal of this study was to generate ecotoxicological information about the toxicity of PCPs on aquatic organisms. Specifically the objectives were to (1) obtain acute and chronic toxicity results for Chlorella vulgaris and D. magna using standardised or currently used tests, (2) study the relationship between the effects on the impaired feeding rate for daphnia and the effects of reproduction and (3) examine the responses on D. magna and C. vulgaris after binary combinations of environmentally relevant compounds and PFOS.

(González-Doncel et al. 2014), methylparaben (San Segundo et al. 2013), fluoxetine (Martini et al. 2010) and PFOS (unpublished data). Table 1 shows the nominal concentrations used in the alga and daphnia tests. D. magna feeding bioassay The test was conducted according to Barata et al. (2008). Briefly, groups of five neonates were exposed to serial concentrations of PCP substances in a final volume of 15 ml of reconstituted water in the presence of C. vulgaris cells used as food at a concentration of 5×105 cells/ml. The test was conducted in the dark. Four replicates per concentration were used, in addition to a control medium (with no substance, but with Chlorella). A control blank (without daphnids) was used in order to ensure that the initial algal concentration did not significantly increase during the exposure period. The feeding rate of daphnids was measured by determining the change of cell densities after 24 h in accordance with Barata and Baird (2000). Cell density was estimated from the absorbance measurements taken at 650 nm (Gutiérrez et al. 2009). The no observed effect concentration (NOEC) was estimated using an ANOVA with a Dunnett test when data were normally distributed or using a non-parametric test (Kruskal-Wallis test) for those cases with non-normally distributed data. SPSS 13.0® package software was used for the statistical analysis. D. magna reproduction test

Material and methods Test substances and tested concentrations Preservatives methylparaben (MP) and propylparaben (PP) were provided by Sigma-Aldrich. UV filter substances ethylhexyl methoxycinnamate (EHMC) and octocrylene (OC) were purchased from Merck. Benzophenone-3 (BP3) and 3-(4-methyl-benzylidene) camphor (4-MBC) were obtained from Sigma-Aldrich. Polycyclic musks AHTN (Tonalide®) and HHCB (Galaxolide®) were acquired from LGC Standards. Perfluorooctane sulfonate (PFOS) was provided by Sigma-Aldrich and fluoxetine (FL) was purchased from Interchem. Concentrations were selected according to previous toxicity data or to pre-screening test data. To reach nominal concentrations, several compounds were solubilised with the help of organic solvents (DMSO, acetone). The solvent concentration never exceeded 100 mg/l. Analytical determinations of the test compounds were not conducted. However, previous studies have demonstrated the stability of some of these compounds in different media; this is the case of propylparaben

A semi-static reproduction test with D. magna was performed according to OECD guideline 211 (OECD 2008). Juveniles (less than 24 h old) were placed in 30-ml beakers with 15 ml of reconstituted water as a test medium. Ten replicates with one daphnid per replicate were used and five serial concentrations were tested. The test medium was replaced every 3 days. After each change, daphnids were fed with a suspension of C. vulgaris at a concentration of 4.5×105 cells/ml. The number of living offspring per parent animal was recorded and neonates were removed from test beakers daily. When differences in animal size were detected, daphnids where photographed using a stereomicroscope (Olympus SZX12) with a digital camera (Olympus Camedia C-5060 Camera). Body length was measured with Image Pro-Plus image analysis software (Media Cybernetics, Sylver Sprong, MD, USA), and any statistically significant differences in relation to the control were evaluated. If the samples passed the Kolmogorov-Smirnov test for normality and the Levene’s test for homogeneity of variances, an ANOVA was conducted with Dunnett’s multiple comparison test; otherwise, the non-parametric Kruskal-Wallis test

16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 in 1 % DMSO

25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 in 0.5 % acetone

1, 0.5, 0.25, 0.125, 0.062, 0.031, 0.015, 0.007 in 0.5 % acetone

12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09 in 0.5 % acetone

n.t

n.t

0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 mg/l FL with 0, 0.1, 0.5 or 1 mg/l PFOS 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156 mg/l HHCB with 0, 0.1, 0.5 or 1 mg/l PFOS n.t

Methylparaben Propylparaben OC

BP3

EHMC

4-MBC

HHCB

AHTN

FL + PFOS

n.t not tested

EHMC + PFOS

HHCB + PFOS

Chlorella vulgaris growth inhibition test

Substance

2.4, 1.2, 0.6, 0.3, 0.15 and 0.075 mg/l EHMC with 0, 0.1, 0.5 or 1 mg/l PFOS

6.25, 3.12, 1.56, 0.78, 0.39, 0.195 in 0.01 % Cremophor 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19 in 0.05 % DMSO 5, 2.5, 1.25, 0.625, 0.3125, 0.156 mg/l FL with 0, 0.1, 0.5 or 1 mg/l PFOS 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 mg/l HHCB with 0, 0.1, 0.5 or 1 mg/l PFOS

n.t

n.t

n.t

n.t n.t n.t

Daphnia magna acute immobilisation test

Nominal test concentrations of emerging contaminants (in mg/l)

Table 1

n.t

n.t

3, 1.5, 0.75, 0.375, 0.187 in 0.003 % Cremophor 3, 1.5, 0.75, 0.375, 0.187 in 0.003 % DMSO n.t

0.12, 0.06, 0.03, 0.015, 0.0075 in 0.012 % acetone

0.12, 0.06, 0.03, 0.015, 0.0075 in 0.1 % acetone

0.8, 0.4, 0.2, 0.1, 0.05 in 0.02 % acetone

10, 5, 2.5, 1.25 4, 2, 1, 0.5, 0.25 0.4, 0.2, 0.1, 0.05, 0.025 in 0.01 % DMSO

Daphnia magna feeding assay

n.t

n.t

n.t

n.t

12, 6, 3, 1.5, 0.75 4, 2, 1, 0.5, 0.25 0.05, 0.025, 0.0125, 0.00625, 0.00312 in 0.01 % DMSO 0.2, 0.1, 0.05, 0.025, 0.0125 in 0.02 % acetone 0.015, 0.0075, 0.00375, 0.0019, 0.00093 in 0.1 % acetone 0.06, 0.03, 0.015, 0.0075, 0.00375 in 0.006 % acetone n.t

Daphnia magna reproduction test

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was used. The NOEC and the lowest observed effect concentration (LOEC) were estimated. D. magna acute immobilisation test This assay was conducted in compliance with OECD guideline 202 (OECD 2004) but with a small test medium volume. The test was done to estimate the acute effects of combining of different PCPs together with PFOS. Ten juveniles (less than 24 h old) were exposed in duplicate in a final volume of 15 ml of reconstituted water to serial concentrations of the PCP substances combined with different fixed concentrations of PFOS (0, 100, 500 or 1000 μg/l). Toxicity tests were run in a thermostatised chamber (20±1 °C) in a 16-h light/8-h dark photoperiod. The control medium was carried out in duplicate. Those daphnids unable to swim for 15 s after gentle stirring were considered immobile, and the percentages of immobilisation relating to either the control medium or solvent control were used to estimate EC50 with log-logistic concentrationeffect curves by Statgraphics Plus 5.0 software package. C. vulgaris growth inhibition test This is a modified test of the standardised algae growth inhibition test (OECD 2011) for low volumes and the protocol is described elsewhere (Ramos et al. 1996; Pablos et al. 1999; Carbonell et al. 2010). Briefly, the test was run in 96-well microplates using the Bold Basal Medium (BBM) as a nutrient source and an initial algal concentration of 10×104 cells/ ml. Test conditions were constant light with an intensity of 8000 lx and a constant temperature (20±2 °C) maintained for 72 h. Plates were shaken in an orbital shaker (Ika Labortechnik) to maintain algae suspensions homogeneous.

Eight or nine serial concentrations with a factor of 2 were tested with six replicates per sample; whenever necessary, a solvent control was incorporated. Algal growth was estimated by measuring absorbance (450 nm) and fluorescence (430 Ex/ 680 Em) in a multiwell plate reader (TECAN-Genius spectrofluorometer). Percentages of growth increase or reduction in relation to the control were calculated to estimate IC50, using log-logistic concentration-effect curves, and Statgraphics Plus 5.0 software package was applied for this fit.

Results and discussion D. magna feeding bioassay Table 2 provides the results of the feeding inhibition assays after a sublethal exposure to different PCPs. Parabens and the fragrance HHCB had the mildest effects on algae feeding inhibition. However, the UV filters, particularly OC and 4MBC, displayed the greatest sensitivities for feeding inhibition with NOEC values of 50 and 60 μg/l, respectively. The feeding assay has been proposed to be a sensitive, robust and ecologically relevant endpoint to detect sublethal effects of substances and toxic effluents either directly or in postexposure assays (Allen et al. 1995; Barata et al. 2007, 2008; Damasió et al. 2008). Several studies with different pollutants have demonstrated that this bioassay is more sensitive than standardised acute assays (Allen et al. 1995; Barata and Baird 2000; McWilliam and Baird 2002; Barata et al. 2008), although in some cases, i.e. pesticides, sensitivity has been reported to be compound-specific (Barata et al. 2008). The results provided by such assays also help to complement information on chronic responses since it is well-known

Table 2 Summary of NOECs for feeding, reproduction and length (in mg/l) of emerging contaminants tested on Daphna magna versus other bibliographic toxicity results Substance

NOEC 24 hfeeding

Propylparaben

≥4

NOEC 21 dayslengtha

Bibliographic data

Reference

0.25

≥0.5

LOEC 7 daysreproduction =6 LOEC 7 daysgrowth =0.4 LOEC 7 daysreproduction =1.5 LOEC 7 daysgrowth =6 n.f. NOECreproduction =0.5

Dobbins et al. 2009

NOEC 21 daysreproduction

Methylparaben

2.5

0.75

No effect

OC BP3

0.05 0.4

≥0.05 ≥0.2

No effect No effect

EHMC

≥0.12

≥0.015

No effect

NOECreproduction =0.04

Sieratowicz et al 2011

4MBC

0.06

0.015b

No effect

NOECreproduction =0.1

Sieratowicz et al 2011

NOECreproduction =0.02

Fent et al 2010

HHCB AHTN

1.5 0.187

n.t n.t

n.t n.t

NOECreproduction =0.11 NOECreproduction =0.196

Balk and Ford 1999 Balk and Ford 1999

a

Effects on parental size related to control were observed only for propylparaben

b

At higher concentrations, induction in the reproduction rate was observed

n.f. not found, n.t not tested

Dobbins et al. 2009 – Sieratowicz et al. 2011

Environ Sci Pollut Res Table 3 Summary of EC50s and CI 95 % (in mg/l) of binary combination of emerging contaminants and PFOS tested on Daphna magna acute immobilisation test Substance

EC50s 0 μg/l PFOS

100 μg/l PFOS

500 μg/l PFOS

1000 μg/l PFOS

HHCB

3.33 (3.07–3.56)

1.715 (1.62–1.80)

0.867 (0.7–1)

0.853 (0.64–1)

Fl EHMC

2.517 (n.c) 0.1512 (0.1511–0.1512)

2.58 (2.58–2.58) 0.283 (n.c)

4.913 (4.910–4.915) 0.0824 (0.0823–0.0826)

4.887 (4.887–4.887) 0.088 (0.075–0.098)

n.c not calculable, due to the high jump in the immobilisation effect

that effects on feeding are linked with reproduction and growth (Barata and Baird 2000). Some studies have evaluated if the short-term energy budget changes provoked by pollutants correlate with long-term effects on reproduction. De Coen and Janssen (1997) compared the effects on not only the metabolic balance using the cellular energy allocation (CEA) methodology but also the effects on reproduction on D. magna after exposure to HgCl2 and lindane. Their results suggested that the effects obtained by the short-term CEA assay (96 h) were comparable with those obtained with the long-term population-level endpoint (intrinsic rate of natural increase, or rm). Although we did not study the metabolic balance of the organisms, the impairment of the feeding rate on Daphnia due to toxic stress can be considered a measure of energy consumption. Thus, a relationship between the feeding inhibition assay and the reproduction test could be studied. D. magna reproduction test The reproduction tests were conducted in order to generate information that was not available at the time we did our literature review, or which has been not generated until now; in other cases, the results found during the literature review were obtained by following non-standard methodologies. The outcomes of the D. magna reproduction tests (Table 2) reflected that the bibliographic results generally fell within the same range as our data; for example, in EHMC, our results came close to those found by Sieratowicz et al. (2011), where NOEC for 21 days for reproduction was 0.04 mg/l. In other cases, such as methylparaben and propylparaben, the LOEC for 7 days obtained by Dobbins et al. (2009) was in line with our NOEC for 21 days. The exception was UV filter 4-MBC, where our NOEC for 21-day reproduction was 15 μg/l, estimated based on the increase in the number of offspring at the highest concentrations tested (30 and 60 μg/l). However, other studies have demonstrated different effects for this compound. In the work of Sieratowicz et al. (2011), the NOEC for 21 days was 100 μg/l, based on a decrease in the length of the parental animals, although the number of released neonates was not

significantly affected. On the other hand, the study of Fent et al. (2010) showed a negative effect on reproduction at 50 μg/l, estimating thus a NOEC 21 days for reproduction of 20 μg/l. But the exposure of 4-MBC to other invertebrates has also enhanced reproduction, which also occurred in our study. Schmitt et al. (2008) found that a number of unshelled embryos of mudsnails increased for a spiked sediment exposure with 4-MBC and 3-BC, although they reported that the same exposure produced a decrease in the reproduction of the blackworm Lumbriculus variegatus. Some studies have suggested a potential endocrine disruption of UV filters (Schlumpf et al. 2001; Kunz et al. 2006; Schmitt et al. 2008; Brausch and Rand 2011), but in these cases, inhibition of reproduction is the most expected response for an endocrine disruptor. However, different reproduction responses after chronic exposure have been reported for other pollutants, such as nonylphenol and fluoxetine. For these compounds, some experiments have produced enhanced offspring production for Daphnia rather than inhibition (LeBlanc, et al. 2000; Brooks et al. 2003; Flaherty and Dodson 2005), and this contradictory effect can be explained as a stage-dependent or a food-dependent effect (Campos et al. 2012). Previous authors have also demonstrated the strong influence of maternal nutritional status on the chronic response of D. magna to pesticide exposure (Pieters and Liess 2006). Therefore, this hypothesis could explain the different response patterns shown for 4-MBC in different bioassays. Taking into account both feeding and reproduction results jointly, we can hypothesise that for parabens EHMC and HHCB, feeding inhibition did not influence reproduction success in Daphnia because the concentrations that affected feeding were higher than those that affected reproduction. For BP3, the literature review indicated that no effects on reproduction occurred for this compound. So feeding inhibition at more than 400 μg/l of BP3 could produce a sublethal effect, which is not reflected on reproduction. Finally, the feeding inhibition assay results for OC, AHTN and 4-MBC could explain how the effects on reproduction might be produced by general toxic stress caused by pollutants. In the particular case of 4-MBC, and as described above, the increase in the number of offspring produced at 30 and 60 μg/l could be

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related to feeding inhibition (NOEC 60 μg/l), as could the NOEC of 100 μg/l, based on the reduced parent size, which has been found in the literature (Sieratowicz et al. 2011). The Daphnia reproduction test also revealed, in the particular case of propylparaben, differences in the parental size between concentrations, but not statistically significant related to control. Thus, the NOEClength was estimated ≥0.5 mg/l (Table 2); in all other cases, no differences in the parental size were observed. D. magna acute immobilisation test The acute immobilisation test on D. magna was conducted for estimating the binary toxicity of some PCPs and pharmaceuticals with PFOS simultaneously. Table 3 shows the effects of different concentrations of PFOS on the toxicity of three emerging contaminants, HHCB, fluoxetine and EHMC, selected for the broad lipophilicity range, frequent occurrence in the aquatic compartment and toxicity to aquatic organisms. The results revealed that the toxicity of lipophilic compounds (high Kow), such as EHMC and HHCB, increased with the presence of PFOS, unlike compounds with a lower Kow, i.e. fluoxetine, where the presence of high concentrations of PFOS reduced toxicity, but this toxicity was not modified by the lowest PFOS concentration tested (100 μg/l). Our results corroborated previous theories which considered that PFOS, with surfactant properties, could modify membrane cellular properties by increasing the bioavility of pollutants with high lipophilicity. Hu et al. (2003) showed that the toxicity of

tetrachlorodibenzo-p-dioxin and estradiol caused by PFOS increased; Liu et al. (2008) demonstrated an increase in algal cell permeability, and they later suggested that PFOS could affect algal cell uptake and toxicity of various compounds differently (Liu et al. 2009). Rodea-Palomares et al. (2012) showed similar results for cyanobacterium bioluminescent toxicity test; in this case, PFOS displayed synergistic interactions with the organic compounds 2,4-D, furazolidone and mitomycin C. The authors explained this fact by means of the potential role of PFOS which enhanced the accessibility and cell uptake of co-existing hydrophobic compounds (RodeaPalomares et al. 2012). In addition, the concomitant presence of PFOS with other compounds in the aquatic compartment could favour the water solubility of other lipophilic compounds by increasing their bioavility and, thus, their toxicity. C. vulgaris growth inhibition test Table 4 summarises the results of the emerging contaminants tested in the C. vulgaris growth inhibition test in our laboratory. The results are presented together with the data found in the literature, obtained using different species of unicellular algae. The data confirmed that, in the majority of cases, PCPs presented low toxicity for algae, and toxic values generally fell within the same range in all the algae species tested, except for BP3 exposure, where the results for C. vulgaris showed a slightly less sensitive response than the other species tested. This table also presents the results on the toxicity of C. vulgaris by binary exposures of PFOS and of other

Table 4 Summary of IC50, IC10, NOEC 72 h and 95 % confidence intervals (in mg/l) data for emerging contaminants tested on Chlorella vulgaris growth inhibition test versus other bibliographic toxicity results Substance

IC50 (CI 95 %)

IC10 (CI 95 %)

Propylparaben

6.8 (4.2–11)

n.c

Methylparaben

≥16

n.c

n.c

≥16

3.12 3.12

Bibliographic data

Reference

IC50 72 h=15

Yamamoto et al. 2011

IC50 72 h=36 NOEC72h=7.4

Yamamoto et al. 2011

IC50 72 h=91

Yamamoto et al. 2011

IC50 72 h=80 NOEC72h=21

Yamamoto et al. 2011

n.f IC50 =0.96 (0.9–1.02) IC10 =0.61 (0.45–0.81)

– Sieratowicz et al. 2011

OC BP3

20.3 (18.5–22.4) 22.4 (20.1–25.5)

EHMC

≥1

n.c

≥1

IC10 =0.07 (0.01–0.56)

Sieratowicz et al. 2011

4MBC

≥12.5

n.c

≥12.5

Sieratowicz et al. 2011

Fl + PFOS HHCB + PFOS

No effect No effect

– –

IC50 =7.66 (3.51–16.7) IC10 =0.81 (0.09–7.49) – –

n.f. not found, n.t not tested, n.c not calculable

0.4 (1.6) 2.2 (4.69)

NOEC

No effect No effect

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emerging pollutants. In this case, no effects of any of the exposed pollutants were observed on toxicity, unlike the effects that PFOS provoked on the toxicity of different compounds in the study of Liu et al. (2009). However in this case, the effects noted for the uptake or growth inhibition of different pollutants were observed at higher concentrations of PFOS (up to 40 mg/l) compared with our study, where the maximum concentration used was 1 mg/l.

Acknowledgments This work has been funded by Spanish Project CTM-2013-44986-R.

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