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Effects of elutriates from contaminated coastal sediments on different life cycle phases of planktonic diatoms
Marine Environmental Research 155 (2020) 104890
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Effects of elutriates from contaminated coastal sediments on different life cycle phases of planktonic diatoms A. Pelusi , F. Rotolo , A. Gallo , M.I. Ferrante , M. Montresor * Stazione Zoologica Anton Dohrn, Villa Comunale 1, 80121, Napoli, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Diatoms Plankton Sexual reproduction Spores Elutriates Sediment pollution PAHs Heavy metals
We assessed the effects of elutriates from sediments collected at three stations in the polluted Bay of BagnoliCoroglio along the Campania coast (Tyrrhenian Sea, Italy) using three planktonic diatoms regularly occurring in the area, Pseudo-nitzschia multistriata, P. arenysensis and Chaetoceros socialis. Specifically, we tested the pro duction of sexual stages in the heterothallic Pseudo-nitzschia species with the hypothesis that pollutants could impair sexual reproduction. We also tested the seeding capacity of spores of C. socialis after up to six months of storage in elutriates, assuming that pollutants could affect the capability of resting stages to germinate. Elutriate from station 56, with the highest concentrations of pollutants, impaired growth, sexual reproduction and spore germination. Elutriates from stations 25 and 84 caused moderate enhancement of growth and sexual repro duction in Pseudo-nitzschia as compared with control conditions, and also had intermediate effect on spore seeding capacity.
1. Introduction Marine sediments are the repository for a broad range of contami nants of anthropogenic origin (Merhaby et al., 2019). They are also the habitat of benthic organisms and of organisms that include benthic stages in their life cycle, such as resting eggs of crustaceans and cysts of unicellular eukaryotes. Assessing the toxicity of sediments is very chal lenging due to the diversity of pollutants, the different levels of bioavailability and the interaction with the physical and chemical characteristics of the environment. Contaminants directly affect benthic organisms, but the resuspension of sediments due to natural and anthropogenic processes, such as storms or dredging, can mobilize pollutants in the water column, thus potentially impacting a larger fraction of the marine community (Roberts, 2012). A broad range of tests spanning from acute toxicity bioassays, biomarker experiments and bioaccumulation assays have been implemented using bacteria, micro algae, planktonic and benthic meiofauna organisms of different size and life cycle complexity (Simpson et al., 2016). To test the toxicity of marine sediments in aqueous phase, sediments are first re-suspended in seawater so to release the adsorbed pollutants; the resulting water is the elutriate. A standard testing protocol, the ‘Alga growth inhibition test ISO 10253’, is normally applied to investigate the effects of a single substance, of water samples or sediment elutriates on
the growth of selected species, which include the marine diatoms Phaeodactylum tricornutum and Skeletonema sp. (ISO, 2016), and the green alga Dunaliella tertiolecta (ISO 10253, 2016). Cell growth is nor mally tested at 24 h interval over 3 days, and growth inhibition is considered as the main chronic endpoint. Phaeodactylum tricornutum is a model marine diatom originally selected for the easiness of cultivation and for the small genome size (Bowler et al., 2008), but it is rarely abundant in marine ecosystems and its ecological relevance is limited. For the present work, we have selected diatom species belonging to the genera Pseudo-nitzschia and Chaetoceros, commonly found in the marine environment, and we have tested the effect of sediment elutriates on specific phases of their life cycle, which can impact on population dynamics and community composition. Chaetoceros socialis is a widely distributed species (Harrison et al., 2015) that has been chosen because of its ability to form resting stages, called spores. Spores are surrounded by very thick siliceous valves and are produced following two mitotic divisions of a vegetative cell (Pelusi et al., 2019). Spores sink to the sea bottom where they form ‘seed beds’ €rnstro €m et al., 2011) and be that can remain dormant for a long time (Ha therefore exposed to pollutants present in the sediments. Pseudo-nitzschia is also a globally distributed genus of planktonic diatoms, important members of the phytoplankton communities in
* Corresponding author. E-mail address: [email protected] (M. Montresor). https://doi.org/10.1016/j.marenvres.2020.104890 Received 31 October 2019; Received in revised form 21 January 2020; Accepted 21 January 2020 Available online 22 January 2020 0141-1136/© 2020 Elsevier Ltd. All rights reserved.
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whereas lower values (1.84 μg L 1 and 0.13 μg L 1) were recorded in elutriates E25 and E84, respectively. Polychlorinated biphenyls were below the limit of detection (<0.05 μg L 1). Elutriates from the top 50 cm of three sediment cores (25, 56 and 84) were prepared according to the (US EPA, 1991) standard protocol. Briefly, the homogenized sedi ment was combined with filtered natural seawater, collected from a reference site, in a 1:4 solid-to-liquid volumetric ratio. The mixture was stirred at 300 rpm for 1 h at 22 � C on an orbital shaker followed by 1 h settling. Then, the aqueous fraction was siphoned off without disturbing the settled material and centrifuged at 5,100 g for 20 min at 4 � C. The supernatant was collected as elutriate and stored at 20 � C. Before starting the experiments, the needed volume of elutriate was covered with aluminium foil and thawed. Before use, salinity measurements were taken to make sure that the values were in the same range of those of sea water.
coastal and oceanic waters, including the Campania region (Ruggiero et al., 2015). Many species in this genus produce domoic acid, a neurotoxin responsible for Amnesic Shellfish Poisoning (Bates et al., 2018). The two Pseudo-nitzschia species have been chosen because they have a controllable life cycle including a sexual phase. The occurrence of sexual reproduction is essential for the persistence of the diatom popu lation because this is the phase in which large-sized cells are produced, thus compensating the progressive reduction of cell size that occurs in diatoms when they grow, i.e. divide mitotically (Montresor et al., 2016). Sexual reproduction is induced by mixing two strains of opposite mating type (MT) and sexual stages (gametes, zygotes, auxospores and initial cells) appear with a known timing in laboratory conditions (Scalco et al., 2016). Vegetative diatom cells are surrounded by a siliceous frustule, while gametes, zygotes and auxospores are deprived of the siliceous cell wall and thus potentially more susceptible to the effect of pollutants. The Tyrrhenian coast in Italy is impacted by several anthropic per �, 2014), and one of the most turbations (Tornero & Ribera d’Alcala affected sites is the Bay of Bagnoli-Coroglio (Gulf of Pozzuoli), which hosted a large steel factory that greatly contributed to the pollution of the nearby marine environment (Romano et al., 2004). The ABBaCo project, launched in 2017, is aimed at assessing the quality of water and sediments of the Bay of Bagnoli-Coroglio and at implementing envi ronmental remediation and restoration strategies (Morroni et al., 2020, this issue). In this study, we have assessed the effect of elutriates prepared from sediment samples collected in the Bay of Bagnoli-Coroglio within the ABBaCo project on different phases of the life cycle of three planktonic diatoms naturally occurring in the area: the pennate diatoms Pseudonitzschia multistriata and P. arenysensis, and the centric diatom Chaeto � et al., 2004; Margiotta et al., 2020, this ceros socialis (Ribera d’Alcala issue). Three elutriates were selected differing in the concentration of heavy metals and polycyclic aromatic hydrocarbons (PAHs) (Carotenuto et al., 2020, this issue). The rationale of our study was to consider not only the direct impact of contaminated elutriates on diatom growth but also on sexual stages, which represent the most delicate phase of the life cycle, and on resting spores that are protected by a thick cell wall but – being stored in marine sediments - can be negatively affected by pollutants.
2.2. Growth inhibition tests
2. Material and methods
A protocol for the two Pseudo-nitzschia species was adapted from the ISO standardized protocol for the toxicity tests with the diatom Phaeo dactylum tricornutum (ISO, 2016). The tests were performed in 6-wells culture plates (Corning, Missouri City, USA). For each strain five con ditions were tested in duplicate: 1) control with f/2 growth medium (4.5 mL); 2) control with sterilized seawater (SSW; 4 mL); 3) elutriate E25 (4 mL); 4) elutriate E56 (4 mL); 5) elutriate E84 (4 mL). SSW and the three elutriates were amended with 0.5 mL of f/2 medium so to rule out possible nutrient deficiency during the experiment. The seawater used for the preparation of the f/2 medium and for all the experimental conditions was sampled at LTER-MC station on 24/05/2019 and it had a salinity of 36. After having thoroughly mixed the cultures, the cellular abundance of the two strains was assessed in duplicate using a Malassez counting chamber with a Zeiss Axiophot microscope (ZEISS, Oberkochen, Ger many). Cultures of the two species were diluted with f/2 growth medium in a 15 mL plastic tube (Corning Missouri City, USA) to reach a con centration of 105 cells⋅mL 1. A volume of 0.5 mL of this culture was added to each well to reach a final abundance of 104 cells⋅mL 1. The culture plates were incubated in the culture chamber and cell abundance was estimated after 3 days with two replicate counts, after fixing the content of the plates with Lugol.
2.1. Strains, cultivation conditions, and preparation of elutriates
2.3. Sexual reproduction inhibition tests
Pseudo-nitzschia multistriata strains LV168 (MTþ) and RA8 (MT–), and Pseudo-nitzschia arenysensis strains SV4 (MTþ) and 18 (MT–) derive from single initial cells manually isolated from laboratory crosses of strains collected at the Long Term Ecological Research station Mar eChiara (LTER-MC), in the Gulf of Naples (Tyrrhenian Sea, Mediterra nean Sea). Strains were grown in f/2 culture medium (Guillard, 1975) in 25 cm2 sterile tissue culture flasks, in a culture chamber at 18 � C, photoperiod 12:12 h light:dark, irradiance � 100 μmol photons m 2 s 1. A single short chain of Chaetoceros socialis strain APC12 was isolated from the germination of spores collected from sediments at the LTER station MareChiara in the Gulf of Naples on September 2015. The strain was preserved as dormant spores in the dark at a temperature of 7 � C (Pelusi et al., 2019). An aliquot of spores was germinated to obtain a culture of vegetative cells one month before the beginning of the experiment. The culture was kept at the same conditions illustrated above. We used elutriates obtained from superficial sediments collected at three stations – 56, 25 and 84 - in the Bay of Bagnoli, representing different levels of pollution (high, medium, and low, respectively) for PAHs (Carotenuto et al., 2020, this issue). Elutriate E84 contained the highest concentration of heavy metals (12.8 mg L 1), whereas lower concentrations were detected in E25 and E56 (~5 mg L 1). The highest concentration of PAHs (8.62 μg L 1) was recorded in elutriate E56,
For each strain of the two Pseudo-nitzschia species a flask was pre pared with an abundance of 2*105 cells⋅mL 1. Strains of the two species were below the cell size threshold for sex that, for both species is comprised between 50 and 55 μm (D’Alelio et al., 2009); P. arenysensis reported as P. delicatissima 1 in (Amato et al., 2005). Two 6-well culture plates for each species were prepared with the same treatments and controls as above and inoculated with 0.25 mL of culture from each mating type; the total abundance of algal cells in each well was 2*104 cells⋅mL 1. The plates were incubated in the culture chamber and counts were performed on day 3 after fixing the content of the plates with Lugol. From each well, 1 mL was collected after gentle mixing, placed into a Sedgewick-Rafter count chamber, and gametes, zygotes, auxospores and initial cells present in the whole chamber were counted using a Zeiss Axiophot microscope. At least 400 vegetative cells were counted in random fields. 2.4. Seeding capacity of spores A culture of Chaetoceros socialis in exponential growth phase was inoculated in 200 mL of nitrogen-limited (23 μM of nitrates) f/2 medium for one week to obtain the formation of spores (Pelusi et al., 2019). The culture was then split in four 45 mL subsamples. Three samples were 2
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centrifuged for 15 min at 1321 g and re-suspended in the three elutriates obtained from cores 25, 56 and 84; a control was prepared with the spores in the culture medium in which they were produced. An aliquot from each suspension was counted using a Sedgwick-Rafter chamber on a Zeiss Axiophot microscope at 400x magnification. The treatments and control suspensions were dispensed in Eppendorf vials. Vials were sealed with parafilm, wrapped in aluminum foil and stored at 7 � C in a black box. One Eppendorf from each stock was used to test spore germination every month for six consecutive months. The same amount of spores, to reach a final abundance of 2,000 spores mL 1 for each treatment and the control, was inoculated in triplicate glass tubes containing 10 mL of f/2 medium; one blank containing only f/2 medium was also prepared. The fluorescence (relative fluorescence units) was measured at the same time for four consecutive days with a Turner Designs fluorometer (model 10-005R. Turner Designs, INC. Sunnyvale, CA, USA).
zygote developed into an auxospore (Fig. 1). This life stage is deprived of the rigid siliceous frustule and can thus elongate and host the formation of the large initial cell (Scalco et al., 2016). Quite surprisingly, in both P. multistriata (Fig. 2A, C) and P. arenysensis (Fig. 2B, D) the highest absolute and relative abundances of sexual stages were observed in the treatment with elutriates of E25 and E84, while lower values were recorded in elutriate E56 and in the controls. When comparing the total percentage of sexual stages obtained in the reference condition, i.e. the f/2 medium in which the strains are cultivated, differences were detected especially for P. multistriata, where all treatments showed significant differences (Supplementary Table 1), either positive (elutriates 25 and 84) or negative (elutriate 56). Differ ences between the f/2 control and SSW, which only differ in the con centration of nutrients, were not significant. The same comparison with P. arenysensis showed that differences between the reference f/2 con dition and the treatments were not significant, although in the same direction, suggesting a different sensitivity for the sexual stages of the two species. The lower abundance of sexual stages recorded for P. multistriata in these latter conditions could also be explained by the fact that in these conditions the MT þ strain had much lower growth rates (Table 1) and thus the ratios of the two mating types was unbalanced. The aim of this pilot experiment was not to depict the fine dynamics of sexual reproduction but to test if gametes and auxospores could be produced in elutriates obtained from sediments with different concen trations of pollutants. Cytotoxicity of PAHs of atmospheric origin was detected in bioassays carried out with human cell lines (Bai and Zhang, 2017; Niu et al., 2017), thus suggesting that these compounds can negatively affect cells that are not surrounded by a thick cell wall, as diatoms sexual stages. Sexual reproduction of Pseudo-nitzschia species has been seldom recorded in the natural environment, possibly due to the fact that it is a very ephemeral event lasting only a few days. A massive sex event, involving P. cf. delicatissima and P. cf. calliantha, was recorded in surface waters at the LTER-MC station (Sarno et al., 2010) and a second one, involving P. australis and P. pungens, was detected in the surf zone along the Washington state coast (Holtermann et al., 2010). It is thus possible that sexual events can occur also relatively close to sediments and therefore be negatively affected in case of high concentration of resuspended pollutants. The results of our experiments show that sexual stages were apparently not negatively affected by elutriates of E25 and E84, with low and medium concentration of PAH, respectively, but with concentrations of mercury (0.74 � 0.19 and 0.84 � 0.2 μgL 1 respectively) exceeding the Environmental Quality Stan dards (Carotenuto et al., 2020, this issue). Elutriates obtained from polluted sediments include a broad range of compounds whose specific or combined effect on this key phase of the life cycle deserves further investigations. Pollutants can affect the viability of unicellular micro algae directly but can also act only on a specific, more sensitive, stage of their life cycle thus potentially affect in a negative or even positive way the dynamics of their populations.
2.5. Statistical analyses Welch’s tests on percentages of sexual stages over the total cell number for P. multistrianta and P. arenysensis were performed using GraphPad Prism 6. Nominal alpha level was kept at 0.05 throughout by � � applying the Holm-Síd ak correction. 3. Results and discussion 3.1. Growth and sexual reproduction inhibition in Pseudo-nitzschia Growth rates of monocultures of P. multistriata and P. arenysensis estimated over a 3-days interval showed variability both between the two species and the experimental conditions (Table 1). Growth rates of P. multistriata were unexpectedly higher in the elutriates as compared to the two controls, with the exception of the MT þ strain that did not grow in elutriate E56, which had the highest concentration of PAHs (Car otenuto et al., 2020, this issue). P. arenysensis grew at similar rates in the standard f/2 medium and in elutriates E25 and E84, while lower growth rates were measured in elutriate E56 and in the SSW treatment. A recent study showed that sediment elutriates contaminated by heavy metals, ranging from 0.03 for copper to 217.1 μg L 1 for molybdenum, enhanced growth of natural phytoplankton measured in immersed mi crocosms (Ben Othman et al., 2017). The Authors suggested that nutri ents, and/or the presence of essential trace metals, can counteract the possible harmful effects of contaminants. Moreover, the study showed that a shift in the phytoplankton community occurred due to the addi tion of toxic elutriates, leading to a proliferation of potentially toxic species, including Pseudo-nitzschia spp, possibly because of their higher tolerance for heavy metals. All of this suggests that that tests only assessing growth properties could be insufficient to capture the potential harmful effects of contaminants. In our study, elutriates were tested also on Pseudo-nitzschia cultures undergoing sexual reproduction. Within the 3 days in which strains of opposite mating type were co-cultured, gametangial cells, i.e., the cells that undergo meiosis and produce two round gametes, got in contact. Syngamy occurred between gametes of opposite mating type and the
3.2. The seeding capacity of Chaetoceros socialis spores The effect of elutriates on the seeding capacity of spores was tested monitoring growth of cultures obtained from the germination of spores stored for up to 6 months in elutriates obtained from the three sediment samples. Considering that spores were incubated in the same culture medium and at the same environmental conditions, differences in the growth curves should be related to differences in the inoculum size, i.e. to the number of spores capable to germinate. The results of our experiment showed that differences in seeding capacity were related to the length of the storage period in the different treatments. After one month of storage, the growth curves of the controls and the treatments were almost identical. In the following incubation tests, growth re sponses were always higher in control conditions and progressively decreased in elutriates E84, E25 and E56. The spores stored for 6 months
Table 1 Growth rates (divisions⋅day 1) of each mating types of P. multistriata and P. arenysensis in mono-culture after three days of incubation; Data shown as average � SD (n ¼ 2). P. multistriata E56 E25 E84 f/2 SSW
P. arenysensis
LV 168 (MTþ)
RA8 (MT–)
SV4 (MTþ)
18 (MT–)
0.03 � 0.04 1.20 � 0.08 1.30 � 0.07 0.17 � 0.16 0.09 � 0.20
1.06 � 1.32 � 1.29 � 0.90 � 0.29 �
0.49 � 1.08 � 1.32 � 1.34 � 0.49 �
0.45 � 1.03 � 1.45 � 1.24 � 0.99 �
0.06 0.01 0.08 0.04 0.12
0.06 0.01 0.04 0.09 0.04
0.01 0.07 0.04 0.05 0.04
3
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Fig. 1. Light micrographs of sexual stages of P. multistriata. A. Gametangia with two gametes (arrows) and two developing zygotes (arrowheads). B. Elongated auxospore (thick arrow). C. Initial cell of species-specific maximum size (thick arrow). Scale bars: 20 μm.
Fig. 2. Abundance (cells⋅ml 1; A, B) and percentage (C, D) of gametes þ zygotes, auxospores and initial cells in co-cultures of opposite mating types of P. multistriata and P. arenysensis undergoing sexual reproduction in the three elutriates (E56, E25 and E84) and controls (f/2 and SSW). Data shown as average � SD (n ¼ 2).
in elutriate E56 did not germinate at all (Fig. 3). Diatom spores are surrounded by a heavily silicified frustule that can be interpreted as a defensive trait. Spores can in fact germinate after the passage through copepod gut (Kuwata and Tsuda, 2005) and they can remain viable in the sediments for decades (Lewis et al., 1999). These stages represent a pool of diversity of populations produced in subse quent years that can be brought back to vegetative growth upon
germination and thus fuel blooms in subsequent years (Jones and Len non, 2010). The results of our experiments show that heavily polluted sediments can negatively impact the preservation of diatom resting stages, impairing their seeding capacity, with potential consequences for the dynamics of planktonic populations. Benthic resting stages are produced by several unicellular eukaryotes (Ellegaard and Ribeiro, 2018) but also by crustaceans (Holm et al., 2018) and other 4
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Fig. 3. Growth curves of cultures obtained from the germination of C. socialis spores that were stored for different time intervals in the three elutriates (E56, E25 and E84) and control (f/2). Growth was monitored measuring fluorescence (RFU: relative fluorescent units); data are shown as average values � SD (n ¼ 3).
multicellular organisms, but the resistance of benthic resting stages to long term storage in areas with high PAHs and/or heavy metals is completely unknown. Studies carried out in the same study area revealed detrimental effects of pollution on the meiofauna and the prokaryotic benthic assemblages- (Gambi et al., 2020, this issue; Tan gherlini et al., 2020, this issue), suggesting that the impact of contam inants may be high also on the benthic dormant stages of the meroplanktonic organisms.
elutriates, E25 and E84, was instead more variable confirming species-specific sensitivity to different pollutants (Gallo et al., 2020, this issue). This pilot study was a first exploration of the opportunity to support/integrate standard ecotoxicology tests with additional experi ments targeting key species and key features of their life cycle to achieve a better understanding of the potential impact of pollutants on the phytoplankton community. Declaration of competing interest
4. Conclusions
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
This study represented an attempt to test the effect of contaminated sediments on species naturally occurring in the given polluted envi ronment and on distinct key stages of their life cycle. The data obtained for the two Pseudo-nitzschia species and C. socialis, indicating that elutriate E56 was the most toxic in all conditions tested and also affected diatom sexual reproduction and spore seeding capacity, are in line with the results obtained in the ISO 10253 test for Skeletonema sp. and Phaeodactylum tricornutum (Gallo et al., 2020, this issue), for which elutriate E56 strongly inhibited growth. The response to the other two
CRediT authorship contribution statement A. Pelusi: Investigation, Data curation, Writing - original draft, Visualization. F. Rotolo: Investigation, Data curation, Visualization, Writing - original draft. A. Gallo: Resources. M.I. Ferrante: Conceptu alization, Writing - review & editing, Funding acquisition, Supervision. 5
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Marine Environmental Research 155 (2020) 104890
M. Montresor: Conceptualization, Writing - review & editing, Funding acquisition, Supervision.
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Acknowledgments This study was supported by the project ABBaCo funded by the Italian Ministry for Education, University and Research (grant number C62F16000170001). The authors thank Tomas Vega Fernandez for helpful discussions. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.marenvres.2020.104890. References Amato, A., Orsini, L., D’Alelio, D., Montresor, M., 2005. Life cycle, size reduction patterns, and ultrastructure of the pennate planktonic diatom Pseudo-nitzschia delicatissima (Bacillariophyceae). J. Phycol. 41, 542–556. Bai, H., Zhang, H., 2017. Characteristics, sources, and cytotoxicity of atmospheric polycyclic aromatic hydrocarbons in urban roadside areas of Hangzhou, China. J. Environ. Sci. Health Part A 52, 303–312. Bates, S.S., Hubbard, K.A., Lundholm, N., Montresor, M., Leaw, C.P., 2018. Pseudonitzschia, Nitzschia, and domoic acid: new research since 2011. Harmful Algae 79, 3–43. Ben Othman, H., Pringault, O., Louati, H., Sakka Hlaili, A., Leboulanger, C., 2017. Impact of contaminated sediment elutriate on coastal phytoplankton community (Thau lagoon, Mediterranean Sea, France). J. Exp. Mar. Biol. Ecol. 486, 1–12. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., Maheswari, U., Martens, C., Maumus, F., Otillar, R.P., Rayko, E., Salamov, A., Vandepoele, K., Beszteri, B., Gruber, A., Heijde, M., Katinka, M., Mock, T., Valentin, K., Verret, F., Berges, J.A., Brownlee, C., Cadoret, J.-P., Chiovitti, A., Choi, C.J., Coesel, S., De Martino, A., Detter, J.C., Durkin, C., Falciatore, A., Fournet, J., Haruta, M., Huysman, M.J.J., Jenkins, B.D., Jiroutova, K., Jorgensen, R.E., Joubert, Y., Kaplan, A., Kr€ oger, N., Kroth, P.G., La Roche, J., Lindquist, E., Lommer, M., Martin–J�ez�equel, V., Lopez, P.J., Lucas, S., Mangogna, M., McGinnis, K., Medlin, L. K., Montsant, A., Marie-Pierre, Oudot–Le Secq, M.-P., Napoli, C., Obornik, M., Schnitzler Parker, M., Petit, J.-L., Porcel, B.M., Poulsen, N., Robison, M., Rychlewski, L., Rynearson, T.A., Schmutz, J., Shapiro, H., Siaut, M., Stanley, M., Sussman, M.R., Taylor, A.R., Vardi, A., von Dassow, P., Vyverman, W., Willis, A., Wyrwicz, L.S., Rokhsar, D.S., Weissenbach, J., Armbrust, E.V., Green, B.R., Van de Peer, Y., Grigoriev, I.V., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456, 239–244. Carotenuto, Y., Vitiello, V., Gallo, A., Libralato, G., Trifuoggi, M., Toscanesi, M., Lofrano, G., Esposito, F., Buttino, I., 2020. Assessment of the relative sensitivity of the copepods Acartia tonsa and Acartia clausi exposed to sediment-derived elutriates from the Bagnoli-Coroglio industrial area. Mar. Environ. Res. (this issue). D’Alelio, D., Amato, A., Luedeking, A., Montresor, M., 2009. Sexual and vegetative phases in the planktonic diatom Pseudo-nitzschia multistriata. Harmful Algae 8, 225–232. Ellegaard, M., Ribeiro, S., 2018. The long-term persistence of phytoplankton resting stages in aquatic ’seed banks. Biol. Rev. 93, 166–183. Gallo, A., Guida, M., Armiento, G., Siciliano, A., Mormile, N., Carraturo, F., Pellegrini, D., Morroni, L., Tosti, E., Ferrante, M.I., Montresor, M., Molisso, F., Sacchi, M., Danovaro, R., Libralato, G., 2020. Species-specific sensitivity of three microalgae to sediment elutriates. Mar. Environ. Res. (this issue). Gambi, C., Dell’Anno, A., Corinaldesi, C., Lo Martire, M., Musco, L., Da Ros, Z., Armiento, G., Danovaro, R., 2020. Impact of historical contamination on meiofaunal assemblages: the case study of the Bagnoli-Coroglio Bay (Southern Tyrrhenian Sea). Mar. Environ. Res. (this issue). Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum Press, New York, pp. 29–60. H€ arnstr€ om, K., Ellegaard, M., Andersen, T.J., Godhe, A., 2011. Hundred years of genetic structure in a sediment revived diatom population. Proc. Natl. Acad. Sci. Unit. States Am. 108, 4252–4257.
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Effects of elutriates from contaminated coastal sediments on different life cycle phases of planktonic diatoms A. Pelusi , F. Rotolo , A. Gallo , M.I. Ferrante , M. Montresor * Stazione Zoologica Anton Dohrn, Villa Comunale 1, 80121, Napoli, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Diatoms Plankton Sexual reproduction Spores Elutriates Sediment pollution PAHs Heavy metals
We assessed the effects of elutriates from sediments collected at three stations in the polluted Bay of BagnoliCoroglio along the Campania coast (Tyrrhenian Sea, Italy) using three planktonic diatoms regularly occurring in the area, Pseudo-nitzschia multistriata, P. arenysensis and Chaetoceros socialis. Specifically, we tested the pro duction of sexual stages in the heterothallic Pseudo-nitzschia species with the hypothesis that pollutants could impair sexual reproduction. We also tested the seeding capacity of spores of C. socialis after up to six months of storage in elutriates, assuming that pollutants could affect the capability of resting stages to germinate. Elutriate from station 56, with the highest concentrations of pollutants, impaired growth, sexual reproduction and spore germination. Elutriates from stations 25 and 84 caused moderate enhancement of growth and sexual repro duction in Pseudo-nitzschia as compared with control conditions, and also had intermediate effect on spore seeding capacity.
1. Introduction Marine sediments are the repository for a broad range of contami nants of anthropogenic origin (Merhaby et al., 2019). They are also the habitat of benthic organisms and of organisms that include benthic stages in their life cycle, such as resting eggs of crustaceans and cysts of unicellular eukaryotes. Assessing the toxicity of sediments is very chal lenging due to the diversity of pollutants, the different levels of bioavailability and the interaction with the physical and chemical characteristics of the environment. Contaminants directly affect benthic organisms, but the resuspension of sediments due to natural and anthropogenic processes, such as storms or dredging, can mobilize pollutants in the water column, thus potentially impacting a larger fraction of the marine community (Roberts, 2012). A broad range of tests spanning from acute toxicity bioassays, biomarker experiments and bioaccumulation assays have been implemented using bacteria, micro algae, planktonic and benthic meiofauna organisms of different size and life cycle complexity (Simpson et al., 2016). To test the toxicity of marine sediments in aqueous phase, sediments are first re-suspended in seawater so to release the adsorbed pollutants; the resulting water is the elutriate. A standard testing protocol, the ‘Alga growth inhibition test ISO 10253’, is normally applied to investigate the effects of a single substance, of water samples or sediment elutriates on
the growth of selected species, which include the marine diatoms Phaeodactylum tricornutum and Skeletonema sp. (ISO, 2016), and the green alga Dunaliella tertiolecta (ISO 10253, 2016). Cell growth is nor mally tested at 24 h interval over 3 days, and growth inhibition is considered as the main chronic endpoint. Phaeodactylum tricornutum is a model marine diatom originally selected for the easiness of cultivation and for the small genome size (Bowler et al., 2008), but it is rarely abundant in marine ecosystems and its ecological relevance is limited. For the present work, we have selected diatom species belonging to the genera Pseudo-nitzschia and Chaetoceros, commonly found in the marine environment, and we have tested the effect of sediment elutriates on specific phases of their life cycle, which can impact on population dynamics and community composition. Chaetoceros socialis is a widely distributed species (Harrison et al., 2015) that has been chosen because of its ability to form resting stages, called spores. Spores are surrounded by very thick siliceous valves and are produced following two mitotic divisions of a vegetative cell (Pelusi et al., 2019). Spores sink to the sea bottom where they form ‘seed beds’ €rnstro €m et al., 2011) and be that can remain dormant for a long time (Ha therefore exposed to pollutants present in the sediments. Pseudo-nitzschia is also a globally distributed genus of planktonic diatoms, important members of the phytoplankton communities in
* Corresponding author. E-mail address: [email protected] (M. Montresor). https://doi.org/10.1016/j.marenvres.2020.104890 Received 31 October 2019; Received in revised form 21 January 2020; Accepted 21 January 2020 Available online 22 January 2020 0141-1136/© 2020 Elsevier Ltd. All rights reserved.
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whereas lower values (1.84 μg L 1 and 0.13 μg L 1) were recorded in elutriates E25 and E84, respectively. Polychlorinated biphenyls were below the limit of detection (<0.05 μg L 1). Elutriates from the top 50 cm of three sediment cores (25, 56 and 84) were prepared according to the (US EPA, 1991) standard protocol. Briefly, the homogenized sedi ment was combined with filtered natural seawater, collected from a reference site, in a 1:4 solid-to-liquid volumetric ratio. The mixture was stirred at 300 rpm for 1 h at 22 � C on an orbital shaker followed by 1 h settling. Then, the aqueous fraction was siphoned off without disturbing the settled material and centrifuged at 5,100 g for 20 min at 4 � C. The supernatant was collected as elutriate and stored at 20 � C. Before starting the experiments, the needed volume of elutriate was covered with aluminium foil and thawed. Before use, salinity measurements were taken to make sure that the values were in the same range of those of sea water.
coastal and oceanic waters, including the Campania region (Ruggiero et al., 2015). Many species in this genus produce domoic acid, a neurotoxin responsible for Amnesic Shellfish Poisoning (Bates et al., 2018). The two Pseudo-nitzschia species have been chosen because they have a controllable life cycle including a sexual phase. The occurrence of sexual reproduction is essential for the persistence of the diatom popu lation because this is the phase in which large-sized cells are produced, thus compensating the progressive reduction of cell size that occurs in diatoms when they grow, i.e. divide mitotically (Montresor et al., 2016). Sexual reproduction is induced by mixing two strains of opposite mating type (MT) and sexual stages (gametes, zygotes, auxospores and initial cells) appear with a known timing in laboratory conditions (Scalco et al., 2016). Vegetative diatom cells are surrounded by a siliceous frustule, while gametes, zygotes and auxospores are deprived of the siliceous cell wall and thus potentially more susceptible to the effect of pollutants. The Tyrrhenian coast in Italy is impacted by several anthropic per �, 2014), and one of the most turbations (Tornero & Ribera d’Alcala affected sites is the Bay of Bagnoli-Coroglio (Gulf of Pozzuoli), which hosted a large steel factory that greatly contributed to the pollution of the nearby marine environment (Romano et al., 2004). The ABBaCo project, launched in 2017, is aimed at assessing the quality of water and sediments of the Bay of Bagnoli-Coroglio and at implementing envi ronmental remediation and restoration strategies (Morroni et al., 2020, this issue). In this study, we have assessed the effect of elutriates prepared from sediment samples collected in the Bay of Bagnoli-Coroglio within the ABBaCo project on different phases of the life cycle of three planktonic diatoms naturally occurring in the area: the pennate diatoms Pseudonitzschia multistriata and P. arenysensis, and the centric diatom Chaeto � et al., 2004; Margiotta et al., 2020, this ceros socialis (Ribera d’Alcala issue). Three elutriates were selected differing in the concentration of heavy metals and polycyclic aromatic hydrocarbons (PAHs) (Carotenuto et al., 2020, this issue). The rationale of our study was to consider not only the direct impact of contaminated elutriates on diatom growth but also on sexual stages, which represent the most delicate phase of the life cycle, and on resting spores that are protected by a thick cell wall but – being stored in marine sediments - can be negatively affected by pollutants.
2.2. Growth inhibition tests
2. Material and methods
A protocol for the two Pseudo-nitzschia species was adapted from the ISO standardized protocol for the toxicity tests with the diatom Phaeo dactylum tricornutum (ISO, 2016). The tests were performed in 6-wells culture plates (Corning, Missouri City, USA). For each strain five con ditions were tested in duplicate: 1) control with f/2 growth medium (4.5 mL); 2) control with sterilized seawater (SSW; 4 mL); 3) elutriate E25 (4 mL); 4) elutriate E56 (4 mL); 5) elutriate E84 (4 mL). SSW and the three elutriates were amended with 0.5 mL of f/2 medium so to rule out possible nutrient deficiency during the experiment. The seawater used for the preparation of the f/2 medium and for all the experimental conditions was sampled at LTER-MC station on 24/05/2019 and it had a salinity of 36. After having thoroughly mixed the cultures, the cellular abundance of the two strains was assessed in duplicate using a Malassez counting chamber with a Zeiss Axiophot microscope (ZEISS, Oberkochen, Ger many). Cultures of the two species were diluted with f/2 growth medium in a 15 mL plastic tube (Corning Missouri City, USA) to reach a con centration of 105 cells⋅mL 1. A volume of 0.5 mL of this culture was added to each well to reach a final abundance of 104 cells⋅mL 1. The culture plates were incubated in the culture chamber and cell abundance was estimated after 3 days with two replicate counts, after fixing the content of the plates with Lugol.
2.1. Strains, cultivation conditions, and preparation of elutriates
2.3. Sexual reproduction inhibition tests
Pseudo-nitzschia multistriata strains LV168 (MTþ) and RA8 (MT–), and Pseudo-nitzschia arenysensis strains SV4 (MTþ) and 18 (MT–) derive from single initial cells manually isolated from laboratory crosses of strains collected at the Long Term Ecological Research station Mar eChiara (LTER-MC), in the Gulf of Naples (Tyrrhenian Sea, Mediterra nean Sea). Strains were grown in f/2 culture medium (Guillard, 1975) in 25 cm2 sterile tissue culture flasks, in a culture chamber at 18 � C, photoperiod 12:12 h light:dark, irradiance � 100 μmol photons m 2 s 1. A single short chain of Chaetoceros socialis strain APC12 was isolated from the germination of spores collected from sediments at the LTER station MareChiara in the Gulf of Naples on September 2015. The strain was preserved as dormant spores in the dark at a temperature of 7 � C (Pelusi et al., 2019). An aliquot of spores was germinated to obtain a culture of vegetative cells one month before the beginning of the experiment. The culture was kept at the same conditions illustrated above. We used elutriates obtained from superficial sediments collected at three stations – 56, 25 and 84 - in the Bay of Bagnoli, representing different levels of pollution (high, medium, and low, respectively) for PAHs (Carotenuto et al., 2020, this issue). Elutriate E84 contained the highest concentration of heavy metals (12.8 mg L 1), whereas lower concentrations were detected in E25 and E56 (~5 mg L 1). The highest concentration of PAHs (8.62 μg L 1) was recorded in elutriate E56,
For each strain of the two Pseudo-nitzschia species a flask was pre pared with an abundance of 2*105 cells⋅mL 1. Strains of the two species were below the cell size threshold for sex that, for both species is comprised between 50 and 55 μm (D’Alelio et al., 2009); P. arenysensis reported as P. delicatissima 1 in (Amato et al., 2005). Two 6-well culture plates for each species were prepared with the same treatments and controls as above and inoculated with 0.25 mL of culture from each mating type; the total abundance of algal cells in each well was 2*104 cells⋅mL 1. The plates were incubated in the culture chamber and counts were performed on day 3 after fixing the content of the plates with Lugol. From each well, 1 mL was collected after gentle mixing, placed into a Sedgewick-Rafter count chamber, and gametes, zygotes, auxospores and initial cells present in the whole chamber were counted using a Zeiss Axiophot microscope. At least 400 vegetative cells were counted in random fields. 2.4. Seeding capacity of spores A culture of Chaetoceros socialis in exponential growth phase was inoculated in 200 mL of nitrogen-limited (23 μM of nitrates) f/2 medium for one week to obtain the formation of spores (Pelusi et al., 2019). The culture was then split in four 45 mL subsamples. Three samples were 2
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centrifuged for 15 min at 1321 g and re-suspended in the three elutriates obtained from cores 25, 56 and 84; a control was prepared with the spores in the culture medium in which they were produced. An aliquot from each suspension was counted using a Sedgwick-Rafter chamber on a Zeiss Axiophot microscope at 400x magnification. The treatments and control suspensions were dispensed in Eppendorf vials. Vials were sealed with parafilm, wrapped in aluminum foil and stored at 7 � C in a black box. One Eppendorf from each stock was used to test spore germination every month for six consecutive months. The same amount of spores, to reach a final abundance of 2,000 spores mL 1 for each treatment and the control, was inoculated in triplicate glass tubes containing 10 mL of f/2 medium; one blank containing only f/2 medium was also prepared. The fluorescence (relative fluorescence units) was measured at the same time for four consecutive days with a Turner Designs fluorometer (model 10-005R. Turner Designs, INC. Sunnyvale, CA, USA).
zygote developed into an auxospore (Fig. 1). This life stage is deprived of the rigid siliceous frustule and can thus elongate and host the formation of the large initial cell (Scalco et al., 2016). Quite surprisingly, in both P. multistriata (Fig. 2A, C) and P. arenysensis (Fig. 2B, D) the highest absolute and relative abundances of sexual stages were observed in the treatment with elutriates of E25 and E84, while lower values were recorded in elutriate E56 and in the controls. When comparing the total percentage of sexual stages obtained in the reference condition, i.e. the f/2 medium in which the strains are cultivated, differences were detected especially for P. multistriata, where all treatments showed significant differences (Supplementary Table 1), either positive (elutriates 25 and 84) or negative (elutriate 56). Differ ences between the f/2 control and SSW, which only differ in the con centration of nutrients, were not significant. The same comparison with P. arenysensis showed that differences between the reference f/2 con dition and the treatments were not significant, although in the same direction, suggesting a different sensitivity for the sexual stages of the two species. The lower abundance of sexual stages recorded for P. multistriata in these latter conditions could also be explained by the fact that in these conditions the MT þ strain had much lower growth rates (Table 1) and thus the ratios of the two mating types was unbalanced. The aim of this pilot experiment was not to depict the fine dynamics of sexual reproduction but to test if gametes and auxospores could be produced in elutriates obtained from sediments with different concen trations of pollutants. Cytotoxicity of PAHs of atmospheric origin was detected in bioassays carried out with human cell lines (Bai and Zhang, 2017; Niu et al., 2017), thus suggesting that these compounds can negatively affect cells that are not surrounded by a thick cell wall, as diatoms sexual stages. Sexual reproduction of Pseudo-nitzschia species has been seldom recorded in the natural environment, possibly due to the fact that it is a very ephemeral event lasting only a few days. A massive sex event, involving P. cf. delicatissima and P. cf. calliantha, was recorded in surface waters at the LTER-MC station (Sarno et al., 2010) and a second one, involving P. australis and P. pungens, was detected in the surf zone along the Washington state coast (Holtermann et al., 2010). It is thus possible that sexual events can occur also relatively close to sediments and therefore be negatively affected in case of high concentration of resuspended pollutants. The results of our experiments show that sexual stages were apparently not negatively affected by elutriates of E25 and E84, with low and medium concentration of PAH, respectively, but with concentrations of mercury (0.74 � 0.19 and 0.84 � 0.2 μgL 1 respectively) exceeding the Environmental Quality Stan dards (Carotenuto et al., 2020, this issue). Elutriates obtained from polluted sediments include a broad range of compounds whose specific or combined effect on this key phase of the life cycle deserves further investigations. Pollutants can affect the viability of unicellular micro algae directly but can also act only on a specific, more sensitive, stage of their life cycle thus potentially affect in a negative or even positive way the dynamics of their populations.
2.5. Statistical analyses Welch’s tests on percentages of sexual stages over the total cell number for P. multistrianta and P. arenysensis were performed using GraphPad Prism 6. Nominal alpha level was kept at 0.05 throughout by � � applying the Holm-Síd ak correction. 3. Results and discussion 3.1. Growth and sexual reproduction inhibition in Pseudo-nitzschia Growth rates of monocultures of P. multistriata and P. arenysensis estimated over a 3-days interval showed variability both between the two species and the experimental conditions (Table 1). Growth rates of P. multistriata were unexpectedly higher in the elutriates as compared to the two controls, with the exception of the MT þ strain that did not grow in elutriate E56, which had the highest concentration of PAHs (Car otenuto et al., 2020, this issue). P. arenysensis grew at similar rates in the standard f/2 medium and in elutriates E25 and E84, while lower growth rates were measured in elutriate E56 and in the SSW treatment. A recent study showed that sediment elutriates contaminated by heavy metals, ranging from 0.03 for copper to 217.1 μg L 1 for molybdenum, enhanced growth of natural phytoplankton measured in immersed mi crocosms (Ben Othman et al., 2017). The Authors suggested that nutri ents, and/or the presence of essential trace metals, can counteract the possible harmful effects of contaminants. Moreover, the study showed that a shift in the phytoplankton community occurred due to the addi tion of toxic elutriates, leading to a proliferation of potentially toxic species, including Pseudo-nitzschia spp, possibly because of their higher tolerance for heavy metals. All of this suggests that that tests only assessing growth properties could be insufficient to capture the potential harmful effects of contaminants. In our study, elutriates were tested also on Pseudo-nitzschia cultures undergoing sexual reproduction. Within the 3 days in which strains of opposite mating type were co-cultured, gametangial cells, i.e., the cells that undergo meiosis and produce two round gametes, got in contact. Syngamy occurred between gametes of opposite mating type and the
3.2. The seeding capacity of Chaetoceros socialis spores The effect of elutriates on the seeding capacity of spores was tested monitoring growth of cultures obtained from the germination of spores stored for up to 6 months in elutriates obtained from the three sediment samples. Considering that spores were incubated in the same culture medium and at the same environmental conditions, differences in the growth curves should be related to differences in the inoculum size, i.e. to the number of spores capable to germinate. The results of our experiment showed that differences in seeding capacity were related to the length of the storage period in the different treatments. After one month of storage, the growth curves of the controls and the treatments were almost identical. In the following incubation tests, growth re sponses were always higher in control conditions and progressively decreased in elutriates E84, E25 and E56. The spores stored for 6 months
Table 1 Growth rates (divisions⋅day 1) of each mating types of P. multistriata and P. arenysensis in mono-culture after three days of incubation; Data shown as average � SD (n ¼ 2). P. multistriata E56 E25 E84 f/2 SSW
P. arenysensis
LV 168 (MTþ)
RA8 (MT–)
SV4 (MTþ)
18 (MT–)
0.03 � 0.04 1.20 � 0.08 1.30 � 0.07 0.17 � 0.16 0.09 � 0.20
1.06 � 1.32 � 1.29 � 0.90 � 0.29 �
0.49 � 1.08 � 1.32 � 1.34 � 0.49 �
0.45 � 1.03 � 1.45 � 1.24 � 0.99 �
0.06 0.01 0.08 0.04 0.12
0.06 0.01 0.04 0.09 0.04
0.01 0.07 0.04 0.05 0.04
3
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Fig. 1. Light micrographs of sexual stages of P. multistriata. A. Gametangia with two gametes (arrows) and two developing zygotes (arrowheads). B. Elongated auxospore (thick arrow). C. Initial cell of species-specific maximum size (thick arrow). Scale bars: 20 μm.
Fig. 2. Abundance (cells⋅ml 1; A, B) and percentage (C, D) of gametes þ zygotes, auxospores and initial cells in co-cultures of opposite mating types of P. multistriata and P. arenysensis undergoing sexual reproduction in the three elutriates (E56, E25 and E84) and controls (f/2 and SSW). Data shown as average � SD (n ¼ 2).
in elutriate E56 did not germinate at all (Fig. 3). Diatom spores are surrounded by a heavily silicified frustule that can be interpreted as a defensive trait. Spores can in fact germinate after the passage through copepod gut (Kuwata and Tsuda, 2005) and they can remain viable in the sediments for decades (Lewis et al., 1999). These stages represent a pool of diversity of populations produced in subse quent years that can be brought back to vegetative growth upon
germination and thus fuel blooms in subsequent years (Jones and Len non, 2010). The results of our experiments show that heavily polluted sediments can negatively impact the preservation of diatom resting stages, impairing their seeding capacity, with potential consequences for the dynamics of planktonic populations. Benthic resting stages are produced by several unicellular eukaryotes (Ellegaard and Ribeiro, 2018) but also by crustaceans (Holm et al., 2018) and other 4
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Fig. 3. Growth curves of cultures obtained from the germination of C. socialis spores that were stored for different time intervals in the three elutriates (E56, E25 and E84) and control (f/2). Growth was monitored measuring fluorescence (RFU: relative fluorescent units); data are shown as average values � SD (n ¼ 3).
multicellular organisms, but the resistance of benthic resting stages to long term storage in areas with high PAHs and/or heavy metals is completely unknown. Studies carried out in the same study area revealed detrimental effects of pollution on the meiofauna and the prokaryotic benthic assemblages- (Gambi et al., 2020, this issue; Tan gherlini et al., 2020, this issue), suggesting that the impact of contam inants may be high also on the benthic dormant stages of the meroplanktonic organisms.
elutriates, E25 and E84, was instead more variable confirming species-specific sensitivity to different pollutants (Gallo et al., 2020, this issue). This pilot study was a first exploration of the opportunity to support/integrate standard ecotoxicology tests with additional experi ments targeting key species and key features of their life cycle to achieve a better understanding of the potential impact of pollutants on the phytoplankton community. Declaration of competing interest
4. Conclusions
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
This study represented an attempt to test the effect of contaminated sediments on species naturally occurring in the given polluted envi ronment and on distinct key stages of their life cycle. The data obtained for the two Pseudo-nitzschia species and C. socialis, indicating that elutriate E56 was the most toxic in all conditions tested and also affected diatom sexual reproduction and spore seeding capacity, are in line with the results obtained in the ISO 10253 test for Skeletonema sp. and Phaeodactylum tricornutum (Gallo et al., 2020, this issue), for which elutriate E56 strongly inhibited growth. The response to the other two
CRediT authorship contribution statement A. Pelusi: Investigation, Data curation, Writing - original draft, Visualization. F. Rotolo: Investigation, Data curation, Visualization, Writing - original draft. A. Gallo: Resources. M.I. Ferrante: Conceptu alization, Writing - review & editing, Funding acquisition, Supervision. 5
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Marine Environmental Research 155 (2020) 104890
M. Montresor: Conceptualization, Writing - review & editing, Funding acquisition, Supervision.
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