Introduction

A disruption in communities of microalgae can generate serious problems for the system productivity, imbalance in food webs and, in consequence, impairment of ecosystem functioning given the role played by these organisms as primary producers. Planktonic microalgae have been employed in environmental risk assessment [1] and recommended as test organisms in international guidelines [2-4] because of their ecological relevance, feasibility for cultivation and sensitivity to different contaminants [1]. More recently, following the same trend, the interest in using benthic diatoms has increased appreciably [5], and estuarine species have attracted particular interest [6-11]. The estuarine microphytobenthos community is mainly composed of sediment-inhabiting benthic diatoms, which are fundamental for estuarine systems, as they are, to a large extent, responsible for the input of energy into the system, stabilization of the sediment through the production of polysaccharides, and constitute a major food source for many species, mainly deposit-feeders [12,13]. Due to their important role in estuarine systems, the use of microphytobenthos in environmental risk assessment has been strongly encouraged [14]. However, in practice, the use of benthic diatoms in toxicity testing has been limited to estimations of population growth (e.g. see references in Araújo et al. [14]). Being motile organisms, diatoms exhibit measurable behavioral responses associated with their displacement [15]; these responses may be of interest for ecotoxicological testing but have been neglected in this context.

The intriguing, sophisticated and complex horizontal displacement mechanism of benthic diatoms has been explored over many years [16]. Besides this typical displacement, benthic diatoms are also able to perform vertical displacement in the sediment in response to environmental gradients of light, nutrients, salinity, and temperature [17-19]. Although, in general, studies about diatom displacement have not focused specifically on habitat disruption scenarios, the displacement velocity of the benthic diatom Craticula cuspidata has been shown to be a good indicator of the toxicity of freshwater sediment elutriates [17]. Therefore, it is possible to hypothesize that motility reactions could occur as an active response to avoid contamination. Recently, avoidance by swimming to escape less favorable contaminated environments has received special attention, justified by the ecological relevance of the results with different invertebrate and vertebrate species [20-23]. Such a response may be very significant for the immediate decline of a population, as it can be observed in the short-term at sub-lethal contamination levels, with consequences ecologically similar to the death of the population [23,24]. For microalgae, the most common approach to assess contaminant effects on their displacement ability does not take into account a potential avoidance response, but rather the use of parameters related to their innate motility (e.g. percentage of swimming cells, velocity and distance), such as the Erlanger flagellate test with the freshwater green alga Euglena gracilis [25]. The displacement of microalgae by swimming was recently documented in the planktonic marine golden brown algae Heterosigma akashiwo, but in this case to escape predation [26]. Nevertheless, given that, in the case of benthic diatoms, active displacement is not expected to enable cells to move long distances; self-propelled (active) avoidance of contamination would not be an effective response to the detected contamination. Therefore, drift, characterized by the displacement of the organisms by means of the water flow [27], may be anticipated as an alternative mechanism for microalgae to move sufficiently long distances.

Drift has been widely recorded in freshwater macroinvertebrates in response to predation, population density, life-cycle stage, food availability and quality, water flow/discharge, photoperiod, and water chemistry [27,28]. Additionally, this type of displacement has also been verified for organisms exposed to contaminants, especially agrochemicals [29-31]. To date, drift has not been recorded for benthic diatoms, but changes from horizontal to vertical movement patterns were verified in the benthic diatom Cylindrotheca closterium when exposed to extreme salinity and nutrient conditions [15]. These authors observed that, under optimal conditions, the typical movement presented by that species was gliding (their innate horizontal pattern); however, under dramatic hypo-saline conditions, changes from gliding to pivot and pirouette movements (vertical patterns) were recorded. This movement by cells (interpreted as an attempt to detach themselves from sediment) could therefore be an indication of their potential for transportation. Given the low active mobility of C. closterium (velocity up to 840 µm min^-1 [32]), its ability to change the movement pattern from horizontal to vertical under stressful conditions (e.g. salinity) [15], as well as the evidence that the spatial distribution of estuarine benthic diatoms can be strongly salinity-driven [33], we hypothesized that C. closterium may use drift as an avoidance response to escape less favorable environments.

The main goal of the present study, therefore, was to verify the drift response of C. closterium to avoid potentially stressful (decreased) salinity conditions. Classical population growth inhibition assays were carried out together with drift assays to verify how C. closterium population growth is inhibited by decreases in salinity; and, on this basis, both growth and drift responses have been combined to estimate a toxicity parameter that is more relevant in ecological terms – the immediate population decline. Salinity was used as stress factor as important changes in salinity values in estuaries due to, for instance, extreme weather events (intense drought or rainfall) can cause disruption in the habitats and biological communities [34].

Materials and Methods

Ethics statement: for water sampling in Río San Pedro, specific permission is not required because this is a public beach zone; the field studies did not involve endangered or protected species.

Test system for drift assays

The test system used in the drift assays was formed by three interconnected compartments (transparent polypropylene plastic flasks; 35 mL each) and a recirculation reservoir (1 L capacity, but filled with 500 mL), forming a closed flow system (Figure 1). The first and second compartments (respectively at the closed end and in the middle) were both designed to reduce water turbulence and prevent chaotic drift; the first was used as water entry chamber and the second to smooth the flow and make it more laminar. The third compartment (at the open end to allow the water to flow out) was designed as the exposure chamber, i.e., the compartment containing the microalgae cells. The water flow was maintained at 440 ± 13 mL min^-1 by a pump placed in the recirculation reservoir. The pump was wrapped in a mesh of 5 µm to block the reintroduction of the drifted microalgae into the system.

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Figure 1. Scheme of the three-compartment assay chamber to evaluate drift by the benthic estuarine microalgae Cylindroteca closterium.

https://doi.org/10.1371/journal.pone.0081073.g001

Results

Growth assay

Significant differences in growth inhibition were found among salinity values (one-way ANOVA: F_6,14 = 61.993, p < 0.0001), but a significant inhibition in growth relative to the control was found only from salinities 24 to 0 (Dunnett´s test: p < 0.05). Whereas in the control treatment the cell density increased 8.6 times during the assay, cell growth was non-existent at salinities as low as 12, with final cell densities equal to the initial. At the two lowest salinity values (5 and 0) the final cell number was lower than the initial cell number. Taking the cell density in the control treatment as maximum growth (100%), the growth inhibition percentage for different salinities tested ranged between 2.2 and 113% (Figure 2). For salinities equal to or lower than 19 the growth inhibition was higher than 50%. The 72-h IGS₅₀ salinity value was 19 (95% CI: ±4).

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Figure 2. Mean percentage of growth inhibition (n = 3), total drift (n = 4) and immediate population decline of the benthic microalgae Cylindrotheca closterium exposed to different salinities.

Data of immediate decline represent the sum of 12-h drift percentages and 72-h growth inhibition percentages of the remaining cells.

Error bars indicate + 1 standard deviation. Statistical differences in responses relative to the control are indicated by * (p<0.05) and ** (p<0.01) according to the Dunnett’s multiple comparison test.

https://doi.org/10.1371/journal.pone.0081073.g002

Discussion

The ability of the estuarine benthic diatom C. closterium to employ drifting to avoid stressful conditions caused by a decrease in salinity has been studied. Although effects on population growth have previously been the most frequently-used endpoint in toxicity testing, drift may also have important effects on the population dynamics of benthic diatoms, as a mechanism for avoiding a less favorable area and, consequently, colonizing other areas or, afterwards, the same area upon habitat recovery. In addition, both responses acting simultaneously can accelerate effects on the population decline, although drift has more immediate consequences. At salinity 24, for instance, the C. closterium population had its growth inhibited by 33% (IGS₃₃), while at this salinity the estimated drift was around 36%, slightly higher than control. Therefore, a decrease measured in population growth should be estimated taking into account ca. 64% of the non-drifter population. On the other hand, with a reduction in salinity to 19, growth was inhibited by 60% and drift was around 50%. This most extreme stress scenario highlights the significant effect that drift can have on the maintenance of the population of benthic diatoms, and the potential effects of drift on local population decline. The drift results obtained in the present study were to some extent expected, since diatoms are organisms very capable of responding to environmental physicochemical alterations, and they can use complex mechanisms to execute their response [37]. Among their behavioral changes, movement-related parameters have been widely studied [17-19,38,39].

In particular, movement responses to stressful environmental factors can be the result of an adaptation that allows cells to inhabit highly variable environments [17]. Sauer et al. [19] showed that vertical migratory movements of diatom assemblages were influenced by light and salinity. Also, light- and grain size-dependent vertical motility was observed in C. closterium [40]. Vertical migration behavior was also demonstrated in function of the nutrient levels in subsurface sediments [18]. McLachlan et al. [39] did not find a phototactic response in C. closterium, whereas that response was observed in the estuarine benthic microalgae Navicula perminuta. Temperature has also been shown to be a factor able to change cell speed in horizontal movements of diatoms, which was reduced at 40 °C [41]. Particularly in estuarine systems, sediment-inhabiting diatoms are propelled to move constantly due to the dynamics of these systems and, in this context, salinity therefore has a strong influence on the displacement of benthic diatoms, as a factor controlling migration [19,42,43].

According to Apoya-Horton et al. [15], movement modalities, referred to as pirouettes and pivots, exhibited by C. closterium when exposed to suboptimal conditions may be mechanisms adopted by individual cells to detach themselves from sediment and thus avoid longer exposure to those conditions. Those movements could indicate that cells are demonstrating a propensity to perform drifting. Although this interpretation cannot be considered certain, the ability of C. closterium to drift (rather than to remain attached to a grain of sediment) under stressful conditions is obvious.

There is additional value in studying the way drift is used as measurable avoidance response by benthic diatoms, since such an approach does not focus exclusively on variation in the type or velocity of the cell’s activity/movement as its response to a stressful environment. This response represents displacement activity that is both reliable and very relevant for studying the effects of environmental disruption on the spatial distribution of benthic diatom populations. In an ecotoxicological context, data about the impairment of microalgae motility is scarce [44]. The latter authors showed that the motility of the marine microalgae Isochrysis galbana and Tetraselmis chui is a rapid and sensitive endpoint for assessing metals toxicity. Motility in the benthic diatom Craticula cuspidata was also reduced when exposed to potentially toxic elutriates [17]. Measurement of the motility and orientation of the flagellate green microalgae Euglena gracilis was shown to be effective for assessing exposure to toxic compounds [25]. Measuring the distance travelled by free-swimming microalgae may be a more accurate indicator of active avoidance; and recently it was shown that the microalga H. Akashiwo presented active avoidance behavior when exposed to predators [26]. Although vertical movement response typically studied in benthic diatoms can contribute to a better understanding of temporal and spatial patterns of diatom distribution [41], in the scenario of a large spatial contamination gradient, this type of response may not achieve effective avoidance; therefore the horizontal displacement achieved by means of drift could be an alternative response by which benthic diatoms move away from contamination by sufficiently long distances.

It can be difficult to discriminate separately the effects of salinity from those of other environmental disruptors (e.g. contaminants). Our results indicate that C. closterium cells can grow at salinity values lower than 35-37, i.e., at values close to 30, at which growth was optimal. However at salinities of 24 and less, growth will be inhibited, and simultaneously, local populations can be further reduced as a result of drift and not necessarily due to direct toxic effects. Although being a largely neglected perspective in ecotoxicity studies, contaminants can act as habitat disruptors, affecting population dynamics, community structure and ecosystem functioning with no effect whatsoever at the individual level of biological organization [45,46]. Thus, although determining the main factors that give rise to drift by microphytobenthos is an important finding, it is equally relevant to understand and quantify the effects that this displacement mechanism can generate on natural population and the consequences for ecosystem structure and functioning.

Conclusions

Drift response by the estuarine benthic diatom microalgae C. closterium was detected in response to hypo-saline stress. Although the inhibition of population growth as a standard response was as sensitive as drift, the combination of both responses for estimating the real salinity effect on the immediate population decline showed that the drift response of C. closterium should not be neglected under such stressful conditions.

Acknowledgments

Assistance with the graphics of Figure 1 was provided by Nadia Romero.