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Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy
Environmental Pollution xxx (xxxx) xxx
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy* Carl Van Colen*, Brecht Vanhove, Anna Diem, Tom Moens Ghent University, Biology Department, Marine Biology Research Group, Krijgslaan 281 e S8, B 9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2019 Received in revised form 14 October 2019 Accepted 22 October 2019 Available online xxx
Litter is omnipresent in the ocean where it can be ingested by marine biota. Although ingestion of microplastics (MPs) is abundantly reported, insights into how MP can influence predator-prey interactions currently limits our understanding of the ecological impact of MPs. Here we demonstrate trophic transfer of MPs from zooplankton to benthic filter feeders, through consumption of contaminated prey (i.e. prey with ingested MP). However, predation rates of contaminated prey were significantly lower as compared to predation rates of prey that had no MPs ingested. As filter feeder clearance rates were not affected by consumption of MPs, the lower predation rates of contaminated prey appear to be primarily explained by disruption in zooplankton swimming behaviour that reduces their filtration risk. This is the first study that shows how MPs can change predator-prey interactions that are involved in the coupling between the pelagic and seabed habitat. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Microplastics Embryogenesis Trophic transfer Predator-prey interactions Larviphagy
1. Introduction Its light weight, durability and relatively low production cost make plastic omnipresent in the Anthropocene. In this ‘Plastic Age’ (Avio et al., 2017), around 8 million tonnes of plastic debris are transported from land to the ocean each year (Jambeck et al., 2015), accounting for 73% of the global marine debris (Bergmann et al., 2015). Microplastics (plastics smaller than 5 mm (GESAMP, 2016), further referred to as MPs) are considered to dominate ocean plastic pollution across the marine realm where MPs are present throughout the water column and in the seabed (Thompson et al., 2004; Choy et al., 2019). Plastics can be taken up directly or indirectly via ingestion of contaminated prey, with direct ingestion being either deliberate when plastic items are mistaken for prey, or accidental when plastics are passively consumed, e.g. by filter feeding (Ryan, 2019). Empirical evidence of indirect ingestion of contaminated prey via filter feeding is however limited despite that MPs are readily ingested by zooplankton (see Botterell et al., 2019 for a review) and that ingestion of zooplankton e such as pelagic shellfish larvae e by e.g. adult benthic filter feeders through non-selective feeding on
* This paper has been recommended for acceptance by Eddy Y. Zeng. * Corresponding author. E-mail address: [email protected] (C. Van Colen).
phytoplankton (i.e. larviphagy) is a common pathway in marine food webs (Troost et al., 2008a and references therein). The consumption of contaminated zooplankton by benthic filter feeders thus represents a possible trophic pathway of MPs between the pelagic and benthic environment. Ingestion of MPs can alter the behaviour of planktonic organisms, e.g. immobilization in Daphnia magna (Rehse et al., 2016) and reduction in swimming velocity of Cyprinodon variegatus larvae (Choy et al., 2019). MPs can therefore possibly alter predator-prey interactions, depending on how specific MP properties, such as buoyancy and shape, disrupt prey swimming abilities which for pelagic shellfish larvae are known to determine the risk of being preyed upon by benthic filter feeders (Troost et al., 2008a). Finally, altered predator filtration rates in response to MP exposure (Wegner et al., 2012) can also affect the ingestion of prey contaminated with MPs. Since predator-prey interactions can have cascading ecosystem effects, insights into whether and how these interactions are affected by MP is essential to enhance our understanding of the ecological impact of MP (Galloway et al., 2017, Windsor et al., 2018). Here we experimentally test whether and how the uptake of MP by zooplankton can alter the rate at which zooplankton is preyed upon by adult benthic filter feeders. We used two endobenthic bivalves as model species: the filter feeding common cockle Cerastoderma edule and the deposit feeding Baltic tellin Limecola balthica. Both species are dominant members of benthic
https://doi.org/10.1016/j.envpol.2019.113479 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479
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C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx
communities in coastal soft sediments in North West Europe, where they can represent up to 75% of the macrofaunal biomass (Van Colen et al., 2008). We hypothesized that MP ingestion would alter predator e prey interactions due to changes in prey behaviour and in filtration rates of predators.
start and end of the experiment, respectively, and standardised these per dry weight of the predator (in g) that was determined after drying at 60 C for two days (Ong et al., 2017). As larvae were not homogenously distributed in the grazing chambers (see results), predation rates on larvae were quantified as:
2. Material and methods
ðC0 Ct Þ=C0
2.1. Biota collection and experimental design First, we analysed whether and how embryonal exposure to MP would affect hatching success of L. balthica embryos. Adult clams were collected in March 2018 at the Paulina polder tidal flat in the Westerschelde estuary, the Netherlands (512102400 N, 3 420 5100 E). Clams were induced to spawn following a heat shock, and embryos were reared at 15 C in the laboratory (for more details: see Van Colen et al. (2009)). After fertilisation, embryos were collected pooled per 1000, and exposed to 6 different concentrations of green fluorescent polystyrene spheres with a diameter of 4.8 mm (density of 1.05 g.cm3, Fluoro-Max, Thermo Scientific): 0, 1, 10, 100, 1000, and 10,000 particles mL1. Embryos and plastics were kept suspended in 0.2-mm filtered UV sterilised seawater (salinity 32 PSU, density ¼ 1.02 g.cm3) by continuous rotation of 250-ml glass culture flasks at 3 rotations per minute. L. balthica larvae start to feed on phytoplankton about 3e4 days after fertilisation when embryos metamorphose into veliger larvae (pers. obs. C. Van Colen). No phytoplankton was added to the cultures to avoid the possible influence of MP ingestion on feeding (Lizarraga et al., 2017). After 4 days of incubation, development was stopped by adding a buffered formaldehyde solution (final concentration in the sample ¼ 1%). Subsequently, hatching success was determined as the percentage of larvae that developed a D-shaped shell in each replicate culture (n ¼ 5) and the average number of ingested MPs per individual was determined from 30 randomly selected hatchlings per replicate culture using a fluorescence microscope (Zeiss Axioskop, Jena, Germany). For the predation and behavior experiment, adult C. edule and L. balthica were sampled in March 2019 at the Paulina polder tidal flat in the Westerschelde estuary, the Netherlands (512102400 N, 3 420 5100 E). Adult L. balthica were induced to spawn and embryos were reared following the same procedure as for the hatching experiment. Subsequently, 4-day old veliger larvae were exposed to a concentration of 100,000 MP mL1 (4.8 mm polystyrene microspheres, see above) for 40 min on a roller table (3 rpm), ensuring uptake in >90% of the larvae with an average MP concentration of 9.0 ± 0.9 (SE) particles larva1 (n ¼ 30). A control batch of larvae was treated in the same way but without addition of MP. Larvae with (i.e. exposed organisms) and without (i.e. control organisms) MP are further referred to as MP larvae and C larvae, respectively. In the predation experiment, 250 larvae of both batches were added to grazing chambers (n ¼ 5 per treatment) that were filled with 800 ml of 0.2-mm filtered seawater (32 PSU, density ¼ 1.02 g.cm3) of 15 C. Subsequently, algae (Isochrysis galbana at 40,000 cells ml1) and one C. edule individual (28.5 ± 1.5 (SE) mm) were added per chamber, and predation rates of algae and larvae were simultaneously quantified during one-hour incubations. Algal concentrations were quantified with a Beckman Multisizer coulter counter; they followed an exponential decline during pilot tests with 10-min sampling intervals. We calculated algal clearance rates as:
ðV = tÞ lnðC0 = Ct Þ where V is the volume of the grazing chamber (L), t is the time (h) spent filtering, and C0 and Ct are the concentrations of algae at the
where C0 and Ct are the concentrations of larvae at the start and end of the experiment, respectively. A similar set-up was used in the behavior experiment, where MP larvae and C larvae of L. balthica were added at a density of ~3 larvae ml1 in the grazing chambers with and without C. edule (n ¼ 5 per treatment, 25.7 ± 1.5 (SE) mm). Subsequently, the vertical distribution of larvae was determined from 45-ml samples collected at 0.5, 5, and 9.5 cm above the bottom of the grazing chamber (total height 12 cm) where the predator was positioned. Larvae from all experiments were stored in a neutralised 1% formaldehyde e seawater solution awaiting counting under a stereomicroscope at 50 magnification. 2.2. Statistical data analysis Differences in hatching success between MP exposure treatments were analysed using one-way analysis of variance (ANOVA). Furthermore, a paired t-test was applied to investigate the difference in algal clearance rates and larval predation rates (i.e. % of larvae consumed) by C. edule exposed to C and MP larvae. The differences in vertical distribution between C and MP larvae, measured as the number of larvae present per height relative to the total number of larvae present at the three sampling heights (i.e. relative abundance), were analysed using two-way ANOVA with MP treatment (C or MP) and height in the water column (bottom, middle, top) as fixed factors. Percentage data (i.e. hatching success, predation rate, and relative vertical distribution) were arcsin squareroot transformed prior to analysis and the homoscedasticity of variances was confirmed using Levene tests. All statistical data analyses were conducted in Statistica 5.5 (Statsoft, Inc). 3. Results and discussion Hatching success of L. balthica varied between 68.5 and 86.2% but did not differ between treatments (one-way ANOVA: p ¼ 0.422, df5,24). Uptake of MP was only observed from an exposure concentration of 100 particles ml1 onwards, with both ingestion success (i.e. the percentage of individuals that ingested particles) and the number of ingested particles per individual increasing linearly with particle concentration (Fig. 1). Similar results were observed for development and uptake of 3-mm polystyrene (PS) beads (at 50e10,000 particles ml1) by 2-day old Mytilus galloprovincialis embryos (Capolupo et al., 2018). However, effects may vary depending on MP size, shape and type, and on morpho-functional differences in development between species (e.g. Balbi et al., 2017; Cole and Galloway, 2015; Kaposi et al., 2014; Wathsala et al., 2018). At the end of the predation experiment, free-swimming larvae still contained on average 2.5 MP ± 0.7 SE (n ¼ 30). Consequently, the consumption of contaminated L. balthica larvae by adult C. edule individuals during the course of the experiment demonstrates trophic transfer of MP from zooplankton to benthic filter feeders. Similar findings were reported by Set€ al€ a, 2014 for transfer of MP in planktonic copepods to mysid shrimps within an experimental pelagic food web. Nevertheless, the ingestion of MP by C. edule did not influence C. edule clearance rates (t-test: p ¼ 0.752, df1,8) (Fig. 2). In contrast, predation rates on larvae differed significantly between treatments (t-test: p ¼ 0.004, df1,8), with ~30% less
Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479
C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx
consumption of MP larvae as compared to C larvae (Fig. 2). Cockles did not produce pseudofaeces during the experiment, confirming that filtered larvae were ingested and not rejected. In treatments without predators, the vertical distribution of larvae differed significantly between MP larvae and C larvae (twoway ANOVA: MP x height, p ¼ 0.004, df2,24), with C larvae concentrating near the bottom whereas MP larvae were homogenously distributed in the water column (Fig. 3). In the treatments with predators, both MP larvae and C larvae concentrated in the upper water layer suggesting that larvae cannot escape predation once trapped by inhalant suction currents, corroborating results for
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oyster and mussel larvae (Troost et al., 2008b). Because ingestion of MP contaminated larvae did not affect C. edule clearance rates we hypothesize that the lower predation on MP larvae can be explained by effects of MPs on swimming behaviour that move the larvae away from the inhalant flow field near the predator (i.e. bottom of grazing chamber) where C larvae were found to concentrate. Such behavioural response could possibly have resulted from an obstruction of the gastric system that could have reduced the feeding capacity of the larvae as was found in pelagic copepods (Cole et al., 2013, 2015). Consequently, MP larvae might swim continuously to increase food intake
Fig. 1. Hatching (blue) and ingestion (orange) success (primary y-axis), and number of ingested MP per L. balthica individual (secondary y-axis) as a function of MP exposure (mean ± SE; n ¼ 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Algal clearance rate (primary y axis) and larval consumption rates (secondary y-axis) by predators (adult C. edule) in the presence and absence of MP in the prey (L. balthica veliger larvae).
Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479
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C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx
Fig. 3. Vertical distribution of L. balthica veliger larvae with (solid line) and without (dashed line) MP ingested in the presence and absence of a filter feeding predator (adult C. edule). Vertical distribution is presented as the relative fraction of larvae found in the three sampling heights.
(Jonsson et al., 1991), explaining their homogeneous distribution throughout the water column. In contrast, the aggregated distribution of C larvae at the bottom of the water column is expected when larvae perform normal intermittent swimming behaviour characterized by rapid sinking when larvae close their valves and stop feeding (Troost et al., 2008a). Other studies have, however, demonstrated that daphnids with ingested MPs can temporarily immobilize (Rehse et al., 2016) and no effects of MP exposure on feeding was found for oyster larvae (Cole and Galloway, 2015). Reponses might thus differ between species and/or the properties of the MPs. Additional research is thus needed to elucidate eventual additional mechanistic explanations of the observed reduction in predation rate and to further corroborate our hypothesis. By exposing embryos to MPs in suspension, we augmented the ecological relevance of our work in comparison to still-water toxicity tests using well-plates. However, the particularly difficult identification process for the smaller MPs in field samples hampers a reliable estimation of small-sized MP in situ concentrations (Windsor et al., 2018), possibly limiting the relevance of applied MP concentrations in laboratory experiments. Nevertheless, while virtually unknown, these concentrations are supposedly high (e.g. Enders et al., 2015) and will undoubtedly increase in the future due to fragmentation of larger plastics that are currently present in the ocean (Galloway et al., 2017). Finally, filtration risk of larvae will strongly depend on environmental conditions and feeding currents produced by the predator (Troost et al., 2009). Nevertheless, this study is e to the best of our knowledge - the first to provide empirical insight into how microplastics can change predator-prey interactions, such as larviphagy.
Acknowledgments This work was performed in the framework of the EU project PLASTOX (JPIO Oceans) and funded by The Belgian Federal Science Policy (BELSPO, contract number BR/154/A1/PLASTOX). The research leading to results presented in this publication was carried out with infrastructure funded by EMBRC Belgium - FWO project GOH3817N. The authors want to thank three anonymous reviewers for their comments and suggestions to improve the manuscript. We thank Arne Denolf and Eezin Ong for assistance in the experiments and the collection of bivalves in the field.
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C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx Chemosphere 153, 91e99. Ryan, P.G., 2019. Ingestion of plastics by marine organisms. In: Takada, H., Karapanagioti, H.K. (Eds.), Hazardous Chemicals Associated with Plastics in the Marine Environment, vol. 2019. Hdb Env Chem, pp. 235e266, 78. €la €, O., et al., 2014. Ingestion and transfer of microplastics in the planktonic food Seta web. Environ. Pollut. 185, 77e83. Thompson, R.C., Olsen, A., Mitchell, R.P., Davis, A., Rowland, S.J., John, A.W.G., McConigie, D., Russel, A.E., 2004. Lost at sea: where is all the plastic? Science 304, 383. Troost, K., Kammermans, P., Wolff, W.J., 2008a. Larviphagy in native bivalves and an introduced oyster. J. Sea Res. 60, 157e163. Troost, K., Stamhuis, E.J., van Duren, L.A., Wolff, W.J., 2009. Feeding current characteristics of three morphologically different bivalve suspension feeders, Crassostrea gigas, Mytilus edulis and Cerastoderma edule, in relation to food competition. Mar. Biol. 156, 355e372. Troost, K., Veldhuizen, R., Stamhuis, E.J., Wolff, W.J., 2008b. Can bivalve veligers
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escape feeding currents of adult bivalves? J. Exp. Mar. Biol. Ecol. 358, 185e196. Van Colen, C., Lenoir, J., De Backer, A., Vanelslander, B., Vincx, M., Degraer, S., Ysebaert, T., 2009. Settlement of Macoma balthica larvae in response to benthic diatom films. Mar. Biol. 156 (10), 2161e2171. Van Colen, C., Montserrat, F., Vincx, M., Herman, P.M.J., Ysebaert, T., Degraer, S., 2008. Macrobenthic recovery from Hypoxia in an estuarine tidal mudflat. Mar. Ecol. Prog. Ser. 372, 31e42. Wathsala, R.H.G.R., Franzellitti, S., Scaglione, M., Fabbri, E., 2018. Styrene impairs normal embryo development in the Mediterranean mussel (Mytilus galloprovincialis). Aquat. Toxicol. 201, 58e65. Wegner, A., Besseling, E., Foekema, E.M., Kamermans, P., Koelmans, A.A., 2012. Effects of nanopolystyrene on the feeding behavior of the Blue mussel (Mytilus edulis L.). Environ. Toxicol. Chem. 31, 2490e2497. Windsor, F.M., Durance, I., Horton, A.A., Thompson, R.C., Tyler, C.R., Ormerod, S.J., 2018. A catchment-scale perspective of plastic pollution. Glob. Chang. Biol. 25, 1207e1221.
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Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy* Carl Van Colen*, Brecht Vanhove, Anna Diem, Tom Moens Ghent University, Biology Department, Marine Biology Research Group, Krijgslaan 281 e S8, B 9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2019 Received in revised form 14 October 2019 Accepted 22 October 2019 Available online xxx
Litter is omnipresent in the ocean where it can be ingested by marine biota. Although ingestion of microplastics (MPs) is abundantly reported, insights into how MP can influence predator-prey interactions currently limits our understanding of the ecological impact of MPs. Here we demonstrate trophic transfer of MPs from zooplankton to benthic filter feeders, through consumption of contaminated prey (i.e. prey with ingested MP). However, predation rates of contaminated prey were significantly lower as compared to predation rates of prey that had no MPs ingested. As filter feeder clearance rates were not affected by consumption of MPs, the lower predation rates of contaminated prey appear to be primarily explained by disruption in zooplankton swimming behaviour that reduces their filtration risk. This is the first study that shows how MPs can change predator-prey interactions that are involved in the coupling between the pelagic and seabed habitat. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Microplastics Embryogenesis Trophic transfer Predator-prey interactions Larviphagy
1. Introduction Its light weight, durability and relatively low production cost make plastic omnipresent in the Anthropocene. In this ‘Plastic Age’ (Avio et al., 2017), around 8 million tonnes of plastic debris are transported from land to the ocean each year (Jambeck et al., 2015), accounting for 73% of the global marine debris (Bergmann et al., 2015). Microplastics (plastics smaller than 5 mm (GESAMP, 2016), further referred to as MPs) are considered to dominate ocean plastic pollution across the marine realm where MPs are present throughout the water column and in the seabed (Thompson et al., 2004; Choy et al., 2019). Plastics can be taken up directly or indirectly via ingestion of contaminated prey, with direct ingestion being either deliberate when plastic items are mistaken for prey, or accidental when plastics are passively consumed, e.g. by filter feeding (Ryan, 2019). Empirical evidence of indirect ingestion of contaminated prey via filter feeding is however limited despite that MPs are readily ingested by zooplankton (see Botterell et al., 2019 for a review) and that ingestion of zooplankton e such as pelagic shellfish larvae e by e.g. adult benthic filter feeders through non-selective feeding on
* This paper has been recommended for acceptance by Eddy Y. Zeng. * Corresponding author. E-mail address: [email protected] (C. Van Colen).
phytoplankton (i.e. larviphagy) is a common pathway in marine food webs (Troost et al., 2008a and references therein). The consumption of contaminated zooplankton by benthic filter feeders thus represents a possible trophic pathway of MPs between the pelagic and benthic environment. Ingestion of MPs can alter the behaviour of planktonic organisms, e.g. immobilization in Daphnia magna (Rehse et al., 2016) and reduction in swimming velocity of Cyprinodon variegatus larvae (Choy et al., 2019). MPs can therefore possibly alter predator-prey interactions, depending on how specific MP properties, such as buoyancy and shape, disrupt prey swimming abilities which for pelagic shellfish larvae are known to determine the risk of being preyed upon by benthic filter feeders (Troost et al., 2008a). Finally, altered predator filtration rates in response to MP exposure (Wegner et al., 2012) can also affect the ingestion of prey contaminated with MPs. Since predator-prey interactions can have cascading ecosystem effects, insights into whether and how these interactions are affected by MP is essential to enhance our understanding of the ecological impact of MP (Galloway et al., 2017, Windsor et al., 2018). Here we experimentally test whether and how the uptake of MP by zooplankton can alter the rate at which zooplankton is preyed upon by adult benthic filter feeders. We used two endobenthic bivalves as model species: the filter feeding common cockle Cerastoderma edule and the deposit feeding Baltic tellin Limecola balthica. Both species are dominant members of benthic
https://doi.org/10.1016/j.envpol.2019.113479 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479
2
C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx
communities in coastal soft sediments in North West Europe, where they can represent up to 75% of the macrofaunal biomass (Van Colen et al., 2008). We hypothesized that MP ingestion would alter predator e prey interactions due to changes in prey behaviour and in filtration rates of predators.
start and end of the experiment, respectively, and standardised these per dry weight of the predator (in g) that was determined after drying at 60 C for two days (Ong et al., 2017). As larvae were not homogenously distributed in the grazing chambers (see results), predation rates on larvae were quantified as:
2. Material and methods
ðC0 Ct Þ=C0
2.1. Biota collection and experimental design First, we analysed whether and how embryonal exposure to MP would affect hatching success of L. balthica embryos. Adult clams were collected in March 2018 at the Paulina polder tidal flat in the Westerschelde estuary, the Netherlands (512102400 N, 3 420 5100 E). Clams were induced to spawn following a heat shock, and embryos were reared at 15 C in the laboratory (for more details: see Van Colen et al. (2009)). After fertilisation, embryos were collected pooled per 1000, and exposed to 6 different concentrations of green fluorescent polystyrene spheres with a diameter of 4.8 mm (density of 1.05 g.cm3, Fluoro-Max, Thermo Scientific): 0, 1, 10, 100, 1000, and 10,000 particles mL1. Embryos and plastics were kept suspended in 0.2-mm filtered UV sterilised seawater (salinity 32 PSU, density ¼ 1.02 g.cm3) by continuous rotation of 250-ml glass culture flasks at 3 rotations per minute. L. balthica larvae start to feed on phytoplankton about 3e4 days after fertilisation when embryos metamorphose into veliger larvae (pers. obs. C. Van Colen). No phytoplankton was added to the cultures to avoid the possible influence of MP ingestion on feeding (Lizarraga et al., 2017). After 4 days of incubation, development was stopped by adding a buffered formaldehyde solution (final concentration in the sample ¼ 1%). Subsequently, hatching success was determined as the percentage of larvae that developed a D-shaped shell in each replicate culture (n ¼ 5) and the average number of ingested MPs per individual was determined from 30 randomly selected hatchlings per replicate culture using a fluorescence microscope (Zeiss Axioskop, Jena, Germany). For the predation and behavior experiment, adult C. edule and L. balthica were sampled in March 2019 at the Paulina polder tidal flat in the Westerschelde estuary, the Netherlands (512102400 N, 3 420 5100 E). Adult L. balthica were induced to spawn and embryos were reared following the same procedure as for the hatching experiment. Subsequently, 4-day old veliger larvae were exposed to a concentration of 100,000 MP mL1 (4.8 mm polystyrene microspheres, see above) for 40 min on a roller table (3 rpm), ensuring uptake in >90% of the larvae with an average MP concentration of 9.0 ± 0.9 (SE) particles larva1 (n ¼ 30). A control batch of larvae was treated in the same way but without addition of MP. Larvae with (i.e. exposed organisms) and without (i.e. control organisms) MP are further referred to as MP larvae and C larvae, respectively. In the predation experiment, 250 larvae of both batches were added to grazing chambers (n ¼ 5 per treatment) that were filled with 800 ml of 0.2-mm filtered seawater (32 PSU, density ¼ 1.02 g.cm3) of 15 C. Subsequently, algae (Isochrysis galbana at 40,000 cells ml1) and one C. edule individual (28.5 ± 1.5 (SE) mm) were added per chamber, and predation rates of algae and larvae were simultaneously quantified during one-hour incubations. Algal concentrations were quantified with a Beckman Multisizer coulter counter; they followed an exponential decline during pilot tests with 10-min sampling intervals. We calculated algal clearance rates as:
ðV = tÞ lnðC0 = Ct Þ where V is the volume of the grazing chamber (L), t is the time (h) spent filtering, and C0 and Ct are the concentrations of algae at the
where C0 and Ct are the concentrations of larvae at the start and end of the experiment, respectively. A similar set-up was used in the behavior experiment, where MP larvae and C larvae of L. balthica were added at a density of ~3 larvae ml1 in the grazing chambers with and without C. edule (n ¼ 5 per treatment, 25.7 ± 1.5 (SE) mm). Subsequently, the vertical distribution of larvae was determined from 45-ml samples collected at 0.5, 5, and 9.5 cm above the bottom of the grazing chamber (total height 12 cm) where the predator was positioned. Larvae from all experiments were stored in a neutralised 1% formaldehyde e seawater solution awaiting counting under a stereomicroscope at 50 magnification. 2.2. Statistical data analysis Differences in hatching success between MP exposure treatments were analysed using one-way analysis of variance (ANOVA). Furthermore, a paired t-test was applied to investigate the difference in algal clearance rates and larval predation rates (i.e. % of larvae consumed) by C. edule exposed to C and MP larvae. The differences in vertical distribution between C and MP larvae, measured as the number of larvae present per height relative to the total number of larvae present at the three sampling heights (i.e. relative abundance), were analysed using two-way ANOVA with MP treatment (C or MP) and height in the water column (bottom, middle, top) as fixed factors. Percentage data (i.e. hatching success, predation rate, and relative vertical distribution) were arcsin squareroot transformed prior to analysis and the homoscedasticity of variances was confirmed using Levene tests. All statistical data analyses were conducted in Statistica 5.5 (Statsoft, Inc). 3. Results and discussion Hatching success of L. balthica varied between 68.5 and 86.2% but did not differ between treatments (one-way ANOVA: p ¼ 0.422, df5,24). Uptake of MP was only observed from an exposure concentration of 100 particles ml1 onwards, with both ingestion success (i.e. the percentage of individuals that ingested particles) and the number of ingested particles per individual increasing linearly with particle concentration (Fig. 1). Similar results were observed for development and uptake of 3-mm polystyrene (PS) beads (at 50e10,000 particles ml1) by 2-day old Mytilus galloprovincialis embryos (Capolupo et al., 2018). However, effects may vary depending on MP size, shape and type, and on morpho-functional differences in development between species (e.g. Balbi et al., 2017; Cole and Galloway, 2015; Kaposi et al., 2014; Wathsala et al., 2018). At the end of the predation experiment, free-swimming larvae still contained on average 2.5 MP ± 0.7 SE (n ¼ 30). Consequently, the consumption of contaminated L. balthica larvae by adult C. edule individuals during the course of the experiment demonstrates trophic transfer of MP from zooplankton to benthic filter feeders. Similar findings were reported by Set€ al€ a, 2014 for transfer of MP in planktonic copepods to mysid shrimps within an experimental pelagic food web. Nevertheless, the ingestion of MP by C. edule did not influence C. edule clearance rates (t-test: p ¼ 0.752, df1,8) (Fig. 2). In contrast, predation rates on larvae differed significantly between treatments (t-test: p ¼ 0.004, df1,8), with ~30% less
Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479
C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx
consumption of MP larvae as compared to C larvae (Fig. 2). Cockles did not produce pseudofaeces during the experiment, confirming that filtered larvae were ingested and not rejected. In treatments without predators, the vertical distribution of larvae differed significantly between MP larvae and C larvae (twoway ANOVA: MP x height, p ¼ 0.004, df2,24), with C larvae concentrating near the bottom whereas MP larvae were homogenously distributed in the water column (Fig. 3). In the treatments with predators, both MP larvae and C larvae concentrated in the upper water layer suggesting that larvae cannot escape predation once trapped by inhalant suction currents, corroborating results for
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oyster and mussel larvae (Troost et al., 2008b). Because ingestion of MP contaminated larvae did not affect C. edule clearance rates we hypothesize that the lower predation on MP larvae can be explained by effects of MPs on swimming behaviour that move the larvae away from the inhalant flow field near the predator (i.e. bottom of grazing chamber) where C larvae were found to concentrate. Such behavioural response could possibly have resulted from an obstruction of the gastric system that could have reduced the feeding capacity of the larvae as was found in pelagic copepods (Cole et al., 2013, 2015). Consequently, MP larvae might swim continuously to increase food intake
Fig. 1. Hatching (blue) and ingestion (orange) success (primary y-axis), and number of ingested MP per L. balthica individual (secondary y-axis) as a function of MP exposure (mean ± SE; n ¼ 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Algal clearance rate (primary y axis) and larval consumption rates (secondary y-axis) by predators (adult C. edule) in the presence and absence of MP in the prey (L. balthica veliger larvae).
Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479
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C. Van Colen et al. / Environmental Pollution xxx (xxxx) xxx
Fig. 3. Vertical distribution of L. balthica veliger larvae with (solid line) and without (dashed line) MP ingested in the presence and absence of a filter feeding predator (adult C. edule). Vertical distribution is presented as the relative fraction of larvae found in the three sampling heights.
(Jonsson et al., 1991), explaining their homogeneous distribution throughout the water column. In contrast, the aggregated distribution of C larvae at the bottom of the water column is expected when larvae perform normal intermittent swimming behaviour characterized by rapid sinking when larvae close their valves and stop feeding (Troost et al., 2008a). Other studies have, however, demonstrated that daphnids with ingested MPs can temporarily immobilize (Rehse et al., 2016) and no effects of MP exposure on feeding was found for oyster larvae (Cole and Galloway, 2015). Reponses might thus differ between species and/or the properties of the MPs. Additional research is thus needed to elucidate eventual additional mechanistic explanations of the observed reduction in predation rate and to further corroborate our hypothesis. By exposing embryos to MPs in suspension, we augmented the ecological relevance of our work in comparison to still-water toxicity tests using well-plates. However, the particularly difficult identification process for the smaller MPs in field samples hampers a reliable estimation of small-sized MP in situ concentrations (Windsor et al., 2018), possibly limiting the relevance of applied MP concentrations in laboratory experiments. Nevertheless, while virtually unknown, these concentrations are supposedly high (e.g. Enders et al., 2015) and will undoubtedly increase in the future due to fragmentation of larger plastics that are currently present in the ocean (Galloway et al., 2017). Finally, filtration risk of larvae will strongly depend on environmental conditions and feeding currents produced by the predator (Troost et al., 2009). Nevertheless, this study is e to the best of our knowledge - the first to provide empirical insight into how microplastics can change predator-prey interactions, such as larviphagy.
Acknowledgments This work was performed in the framework of the EU project PLASTOX (JPIO Oceans) and funded by The Belgian Federal Science Policy (BELSPO, contract number BR/154/A1/PLASTOX). The research leading to results presented in this publication was carried out with infrastructure funded by EMBRC Belgium - FWO project GOH3817N. The authors want to thank three anonymous reviewers for their comments and suggestions to improve the manuscript. We thank Arne Denolf and Eezin Ong for assistance in the experiments and the collection of bivalves in the field.
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Please cite this article as: Van Colen, C et al., Does microplastic ingestion by zooplankton affect predator-prey interactions? An experimental study on larviphagy, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113479