- Email: [email protected]
Effects of microplastics on juveniles of the common goby (Pomatoschistus microps): Confusion with prey, reduction of the predatory performance and efficiency, and possible influence of developmental conditions
Environmental Pollution 196 (2015) 359e362
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Short communication
Effects of microplastics on juveniles of the common goby (Pomatoschistus microps): Confusion with prey, reduction of the predatory performance and efficiency, and possible influence of developmental conditions a, b, Luís G. Luís a, b, Lúcia Guilhermino a, b, * Luís Carlos de Sa a ICBAS e Institute of Biomedical Sciences of Abel Salazar, University of Porto, Department of Population Studies, Laboratory of Ecotoxicology, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal b CIIMAR/CIMAR-LA e Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Research Group on Ecotoxicology, Stress Ecology and Environmental Health, Rua dos Bragas, 209, 4050-123 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 April 2014 Received in revised form 12 July 2014 Accepted 30 October 2014 Available online
Microplastics (MP) are ubiquitous contaminants able to cause adverse effects on organisms. Three hypotheses were tested here: early Pomatoschistus microps juveniles can ingest MP; the presence of MP may reduce fish predatory performance and efficiency; developmental conditions may influence the prey selection capability of fish. Predatory bioassays were carried out with juveniles from two estuaries with differences in environmental conditions: Minho (M-est) and Lima (L-est) Rivers (NW Iberian coast). Polyethylene MP spheres (3 types) alone and in combination with Artemia nauplii were offered as prey. All the MP types were ingested, suggesting confusion with food. Under simultaneous exposure to MP and Artemia, L-est fish showed a significant reduction of the predatory performance (65%) and efficiency (up to 50%), while M-est fish did not, suggesting that developmental conditions may influence the prey selection capability of fish. The MP-induced reduction of food intake may decrease individual and population fitness. © 2014 Published by Elsevier Ltd.
Keywords: Pomatochistus microps Early 0þ juveniles Predatory behaviour Microplastics
1. Introduction As the result of the intensive use of plastics by our society over decades and the long environmental life and high dispersion that these pollutants in general have (Andrady, 2011; Cole et al., 2011; Zarfl and Matthies, 2010) plastic debris have been accumulating into the environment. A large part of them is believed to be small plastic particles known as microplastics (MP) having ubiquitous occurrence. In the marine environment, where they have been particularly investigated, ocean gyres, industrial and/or densely populated estuarine and coastal areas are considered hotspots of MP concentration (Cole et al., 2011; Wright et al., 2013). MP are ingested by organisms (Besseling et al., 2013; Cole et al., €la € et al., 2014), including fish (Boerger et al., 2010; Lusher 2013; Seta et al., 2013; Possatto et al., 2011). Among other potential effects, the * Corresponding author. ICBAS e Institute of Biomedical Sciences of Abel Salazar, University of Porto, Department of Population Studies, Laboratory of Ecotoxicology, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. ), [email protected] E-mail addresses: [email protected] (L.C. de Sa (L.G. Luís), [email protected] (L. Guilhermino). http://dx.doi.org/10.1016/j.envpol.2014.10.026 0269-7491/© 2014 Published by Elsevier Ltd.
ingestion of MP instead of food may lead to growth delay, decrease of the reproductive output, and mortality increase (e.g. due to starvation), with negative effects on the population fitness. Moreover, MP can be transferred from lower to higher food web levels €la € et al., 2014). In the marine biota, MP have been shown to (Seta cause physical and chemical adverse effects, to interfere with the toxicity of other environmental contaminants, and to transfer chemicals to organisms (Antunes et al., 2013; Oliveira et al., 2013; Rochman et al., 2014; Wright et al., 2013). Three hypotheses were tested here: (i) early 0þ age group Pomatoschistus microps juveniles can ingest MP; (ii) the presence of MP simultaneously to real prey may decrease the fish predatory performance and efficiency; and (iii) the environmental conditions during the onthogenic development may influence the capability of juveniles to discriminate real prey from MP. P. microps was selected as model species mainly because it is an important intermediary ~o et al., 2006; Mehner, predator in several European estuaries (Leita 1992; Monteiro et al., 2007), including some located in urban and industrial regions where MP concentrations are expected to be high.
360
et al. / Environmental Pollution 196 (2015) 359e362 L.C. de Sa
2. Material and methods
2.4. Statistical analysis
2.1. Microplastics
Data were tested for variance homogeneity and normality of distribution and data transformations were made when necessary. Percentages were arcsine transformed, using the Bartlett's correction (Zar, 1996). The percentage of overall preys ingested by fish from distinct estuaries was compared using a two-way Analysis of Variance (ANOVA) with interaction (main factors: treatments and fish provenience - estuary). The Student's t test was used to compare M-est and L-est fish inside treatments, and their length and weight. A one-way ANOVA was carried out with data from each estuary separately, followed by the Tukey's test to compare fish performance towards different prey types. SPSS software, version 20, was used. The significant level was 0.05.
Polyethylene microspheres (PMP) were from Cospheric - Innovations in Microtechnology (USA). All PMP were spherical (>90%), opaque and had 420e500 mm diameter; white PMP (WPMP): 1.2 g/cc density; black PMP (B-PMP): paramagnetic, 1.15 g/cc density; red PMP (R-PMP): 0.98 g/cc density (manufacturer indications). 2.2. Fish collection and acclimatization Juveniles were collected (spring 2013) in the estuaries of Minho (M-est) and Lima (L-est) Rivers (NW Iberian coast) that have several ~es et al., 2012). differences in environmental conditions (Guimara Fish were transported to the laboratory and acclimatized for 2 weeks (Oliveira et al., 2013). Briefly, conditions were: 14 h light/10 h dark photoperiod, artificial salt water 18 g/l salinity (ASW), 21 ± 1 C, continuous water aeration and filtration, 98 l glass aquaria (200 fish each). 2.3. Bioassay After the acclimation, 126 fish (63 from each estuary) with about 1.5e2 cm long were selected. Bioassay conditions were similar to those above indicated, with some changes: food supply was stopped 24 h before the bioassay starting; randomly selected fish were put individually in glass beakers containing 500 ml of ASW where they were maintained for 96 h (static conditions, with aeration, no food). Water parameters were measured at 0 h and at each 24 h. After 96 h, a predatory behaviour assay was done. Seven prey types (treatments, with 9 fish from each estuary per treatment) were: 30 24 h old Artemia franciscana nauplii (Art) alone (controls); 30 W-PMP, R-PMP or B-PMP alone; 15 Art in combination with 15 W-PMP, B-PMP or R-PMP. Briefly, each fish was transferred to a prey-exposure chamber containing 300 ml of ASW. After 5 min, 30 preys were introduced into the test chamber (A), and the total number of preys ingested by the fish in a 3 min time interval was recorded. The fish was removed and the number of preys remaining in the prey-exposure chamber was counted by visual observation (B). The number of ingested prey was calculated (A - B). Fish predatory performance was determined as the percentage of the overall ingested preys relatively to the total number of preys offered (30) (IP/A*100). Fish predatory efficiency was calculated as the percentage of Art ingested relatively to the total number of preys ingested. The relative percentage of PMP ingested was calculated relatively to the total number of preys ingested. The hypotheses were tested by investigating if: fish ingested PMP (first hypothesis); the predatory performance and efficiency were lower under simultaneous exposure to Art and PMP relatively to control groups (second hypothesis); comparing the predatory performance and efficiency of M-est and L-est fish (third hypothesis). White, red and black PMP were used because colour may be important in the perception of particles as prey by fish: W-PMP because the main natural prey of early P. microps juveniles (zooplankton) are whitish; R-PMP because some of their potential benthic preys may have reddish colours; B-PMP because they have lower colour similarity to juveniles natural preys. In preliminary assays, 30 Art was found to be ad libitum food for a 3 min predation time; the ingestion of 420e500 mm PMP by 24 h old Art was never observed.
3. Results and discussion 3.1. General conditions No fish mortality occurred. Water pH and temperature variations were lower than 1 pH unity and 1 C, respectively; water dissolved oxygen was higher than 8 mg/l. Mean and standard deviation of total length and weight were: 2.017 ± 0.194 cm and 0.087 ± 0.025 g, respectively, for M-est fish; 1.938 ± 0.214 cm and 0.088 ± 0.028 g, respectively, for L-est fish. No significant differences in length (N ¼ 63; t ¼ 0.939; d.f. ¼ 124, p > 0.05) or weight (N ¼ 63; t ¼ 0.039; d.f. ¼ 124, p > 0.05) were found. 3.2. MP ingestion and confusion with real prey Fish ingested all PMP types, either under individual or combined exposures (Figs. 1e2), thus corroborating our first hypothesis. These results are in good agreement with data from previous studies reporting the ingestion of small plastic debris by other fish
Fig. 1. Percentage of the total amount of prey (Artemia nauplii þ microplastics) ingested by juveniles of the common goby (Pomatoschistus microps) from the estuaries of Minho (M-est) and Lima Rivers (L-est) when exposed to a total number of 30 preys in the water for 3 min. The values indicate the mean of 9 fish per treatment with the corresponding standard error bars. Art e Artemia nauplii (control group); W e exposure to white polyethylene micro-plastic spheres (PMP) alone; B e exposure to black PMP alone; R e exposure to red PMP alone; Art þ W e combined exposure to Artemia nauplii and white PMP; Art þ B e combined exposure to Artemia nauplii and black PMP; Art þ R e combined exposure to Artemia nauplii and red PMP; different letters (A and B e Minho estuary; a and b e Lima estuary) indicate statistical significant differences relatively to the respective control group as indicated by the Tukey multicomparisons test (p 0.05). The predatory performance of M-est and L-est fish exposed to each treatment was compared by the Student's t-test and significant differences (p < 0.05) between fish from distinct estuaries were only found under Art þ BPMP and Art þ R-PMP exposures.
et al. / Environmental Pollution 196 (2015) 359e362 L.C. de Sa
361
Table 1 Results of the two-way ANOVA on the overall percentage of preys ingested by fish from the estuaries of the Minho and Lima Rivers under exposure to different treatments (Artemia nauplii only, white microplastics alone, black microplastics alone, red microplastics alone, Artemia nauplii þ white microplastics, Artemia nauplii þ black microplastics; Artemia nauplii þ red micro-plastics). Comparison
F
P
Type of preys offered (treatments) Origin of the fish (estuaries) Interaction
F(6,112) ¼ 19.908 F(1,112) ¼ 6.963 F(6,112) ¼ 1.183
p < 0.05 p < 0.05 p < 0.05
performance and efficiency of early P. microps juveniles, depending on the MP type and fish condition.
Fig. 2. Relative percentages of Artemia nauplii and microplastics ingested by juveniles of the common goby (Pomatoschistus microps) from the estuaries of Minho and Lima Rivers in combined exposures. The values indicate the mean of 9 fish per treatment with the corresponding standard error bar. Art þ W e combined exposure to Artemia nauplii and white PMP; Art þ B e combined exposure to Artemia nauplii and black PMP; Art þ R e combined exposure to Artemia nauplii and red PMP.
species (Boerger et al., 2010; Dantas et al., 2012; Lusher et al., 2013; Possato et al., 2011). Significant differences among treatments, between fish from distinct estuaries and a significant interaction between the two factors were found (Table 1). Relatively to the respective control feed with Art only, fish showed a significant reduction (M-est: F(6,56) ¼ 6.563, p < 0.05; L-est F(6,56) ¼ 16.492, p < 0.05) of the predatory performance when exposed to PMP alone (Fig. 1). Moreover, in all the combined treatments, M-est fish ingested considerably more Art than PMP (Fig. 2). Thus, fish showed prey selectivity, preferring Art over PMP. They were able to select Art among PMP when both were present in the water. Thus, they can discriminate food from MP, despite making some mistakes. Early P. microps juveniles are visual predators feeding mainly on zooplankton (Mehner, 1992). Art were alive and moved while PMP did not. Thus, Art motion may have helped fish to discriminate it from PMP. In the wild, MP are likely to be in movement too as the result of water currents and other factors, potentially increasing the difficulty of selecting food among MP. In combined exposures, L-est fish ingested more W-PMP than BPMP or R-PMP (Fig. 2) suggesting that they confuse W-PMP with food more than the other PMP. W-PMP are more similar to the whitish colour of early P. microps natural preys than the other PMP. Thus, white MP may have a high likelihood of being ingested by early P. microps due to their colour and visibility, as suggest for other plastic types and fish species (Carpenter et al., 1972; Boerger et al., 2010).
3.3. The presence of MP simultaneously to prey reduces fish predatory performance and efficiency In combined exposures, M-est fish were effective in selecting Art from MP during the 3 min measurement period, ingesting only a small proportion (z10%) of PMP (Fig. 2). Thus, they seem to confuse PMP with preys only in a small extent, and the presence of PMP did not reduce significantly their predatory performance (Fig. 1). On the contrary, under combined exposures, L-est fish predatory performance was significantly reduced in the presence of B-PMP or RPMP, and their predatory efficiency was reduced in the presence of all PMP types (Figs. 1e2). Overall, the above findings indicate that the presence of MP in water may decrease the predatory
3.4. The environmental conditions during early developmental phases may influence the capability of juveniles to discriminate real prey from MP Under combined treatments, L-est fish ingested less Art and more MP than M-est fish, particularly W-PMP (Figs. 1e2). This can be due to a lower capability of L-est fish to discriminate PMP from the real prey or because they were less able in capturing Art during its attempt to escape from the predator than M-est fish. Because no significant differences in the predatory performance between fish from distinct estuaries were found under exposure to Art alone (Fig. 1), the second possibility can be excluded. Therefore, L-est fish showed a reduced capability in discriminating PMP from the real prey relatively to M-est animals. Fish from the two estuaries were collected in the same week, they have no significant differences in weight and size, and acclimation and experimental conditions were the same. Thus, the differences in the predatory performance and efficiency found were likely due to the influence of distinct environmental conditions during previous developmental phases. Mest and L-est have several differences, including in their levels of ~es et al., 2012). Previous studies with older pollution (Guimara P. microps wild juveniles indicated a decreased health status of L-est fish relatively to those from the M-est, likely caused by higher levels of background pollution in the L-est, among other possible factors ~es et al., 2012). Despite the difsuch as prey availability (Guimara ferences in feeding ecology between early and latter P. microps ~o et al., 2006), these studies support juveniles (Mehner, 1992; Leita our hypothesis that onthogenic developmental conditions may influence the capability of fish to discriminate real prey from MP. Acknowledgements We thank Prof. Natividade Vieira for providing parental Artemia, and Dr. Miguel Oliveira for discussions. This study was funded by the Portuguese Foundation for Science and Technology (FCT) with national and European Regional Development Fund (ERDF) funds through the Operational Competitiveness Programme under the projects “SIGNAL” (PTDC/AAC-AMB/110331/2009; FCOMP-01-0124FEDER-013876) and PEst-C/MAR/LA0015/2013. References Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62, 1596e1605. Antunes, J.C., Frias, J.G.L., Micaelo, A.C., Sobral, P., 2013. Resin pellets from beaches of the Portuguese coast and adsorbed persistent organic pollutants. Estuar. Coast. Shelf Sci. 130, 62e69. Besseling, E., Wegner, A., Foekema, E.M., van den Heuvel-Greve, M.J., Koelmans, A., 2013. Effects of microplastic on fitness and PCB bioaccumulation by the lugworm Arenicola marina (L.). Environ. Sci. Technol. 47, 593e600. Boerger, C.M., Lattin, G.L., Moore, S.L., Moore, C.J., 2010. Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre. Mar. Pollut. Bull. 60, 2275e2278.
362
et al. / Environmental Pollution 196 (2015) 359e362 L.C. de Sa
Carpenter, E.J., Smith, K.L., 1972. Plastics on the Sargasso sea surface. Science 175, 1240e1241. Cole, M., Lindeque, P., Halsband, C., Galloway, T.S., 2011. Microplastics as contaminants in the marine environment: a review. Mar. Pollut. Bull. 62, 2588e2597. Cole, M., Lindeque, P., Fileman, E., Halsband, C., Goodhead, R., Moger, J., Galloway, T.S., 2013. Microplastic ingestion by zooplankton. Environ. Sci. Technol. 47, 6646e6655. Dantas, D.V., Barletta, M., Ferreira da Costa, M., 2012. The seasonal and spatial patterns of ingestion of polyfilament nylon fragments by estuarine drums (sciaenidae). Environ. Sci. Pollut. Res. 19, 600e606. ~es, L., Medina, M.H., Guilhermino, L., 2012. Health status of Pomatoschistus Guimara microps populations in relation to pollution and natural stressors: implications for ecological risk assessment. Biomarkers 17, 62e77. Leit~ ao, R., Martinho, F., Neto, J.M., Cabral, H., Marques, J.C., Pardal, M.A., 2006. Feeding ecology, population structure and distribution of Pomatoschistus microps (Krøyer, 1838) and Pomatoschistus minutus (Pallas, 1770) in a temperate estuary, Portugal. Estuar. Coast. Shelf Sci. 66, 231e239. Lusher, A.L., McHugh, M., Thompson, R.C., 2013. Occurrence of microplastics in the gastrointestinal tract of pelagic and demersal fish from the English Channel. Mar. Pollut. Bull. 67, 94e99. Mehner, T., 1992. Diet spectra of Pomatoschistus microps (Krøyer) and Pomatoschistus minutus (Pallas) (teleostei, gobiidae) during first weeks after hatching. Zool. Anz. 229, 13e20.
Monteiro, M., Quintaneiro, C., Nogueira, A.J.A., Morgado, F., Soares, A.M.V.M., Guilhermino, L., 2007. Impact of chemical exposure on the fish Pomatoschistus micropsis Krøyer (1838) in estuaries of the Portuguese Northwest coast. Chemosphere 66, 514e522. Oliveira, M., Ribeiro, A., Hylland, K., Guilhermino, L., 2013. Single and combined effects of microplastics and pyrene on juveniles (0þ group) of the common goby Pomatoschistus microps (teleostei: gobiidae). Ecol. Indic. 34, 641e647. Possatto, F.E., Barletta, M., Costa, M.F., Ivar do Sul, J.A., Dantas, D.V., 2011. Plastic debris ingestion by marine catfish: an unexpected fisheries impact. Mar. Pollut. Bull. 62, 1098e1102. Rochman, C.M., Hentschel, B.T., The, S.J., 2014. Long-Term sorption of metals is similar among plastic types: implications for plastic debris in aquatic environments. PLoS ONE 9, e85433. http://dx.doi.org/10.1371/ journal.pone.0085433y1. €l€ Seta a, O., Fleming-Lehtinen, V., Lehtiniemi, M., 2014. Ingestion and transfer of microplastics in the planktonic food web. Environ. Pollut. 185, 77e83. Wright, S.L., Thompson, R.C., Galloway, T.S., 2013. The physical impacts of microplastics on marine organisms: a review. Environ. Pollut. 178, 483e492. Zar, J.H., 1996. Biostatistical Analysis. Prentice Hall, United Kingdom. Zarfl, C., Matthies, M., 2010. Are marine plastic particles transport vectors for organic pollutants to the Arctic? Mar. Pollut. Bull. 60, 1810e1814.
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Short communication
Effects of microplastics on juveniles of the common goby (Pomatoschistus microps): Confusion with prey, reduction of the predatory performance and efficiency, and possible influence of developmental conditions a, b, Luís G. Luís a, b, Lúcia Guilhermino a, b, * Luís Carlos de Sa a ICBAS e Institute of Biomedical Sciences of Abel Salazar, University of Porto, Department of Population Studies, Laboratory of Ecotoxicology, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal b CIIMAR/CIMAR-LA e Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Research Group on Ecotoxicology, Stress Ecology and Environmental Health, Rua dos Bragas, 209, 4050-123 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 April 2014 Received in revised form 12 July 2014 Accepted 30 October 2014 Available online
Microplastics (MP) are ubiquitous contaminants able to cause adverse effects on organisms. Three hypotheses were tested here: early Pomatoschistus microps juveniles can ingest MP; the presence of MP may reduce fish predatory performance and efficiency; developmental conditions may influence the prey selection capability of fish. Predatory bioassays were carried out with juveniles from two estuaries with differences in environmental conditions: Minho (M-est) and Lima (L-est) Rivers (NW Iberian coast). Polyethylene MP spheres (3 types) alone and in combination with Artemia nauplii were offered as prey. All the MP types were ingested, suggesting confusion with food. Under simultaneous exposure to MP and Artemia, L-est fish showed a significant reduction of the predatory performance (65%) and efficiency (up to 50%), while M-est fish did not, suggesting that developmental conditions may influence the prey selection capability of fish. The MP-induced reduction of food intake may decrease individual and population fitness. © 2014 Published by Elsevier Ltd.
Keywords: Pomatochistus microps Early 0þ juveniles Predatory behaviour Microplastics
1. Introduction As the result of the intensive use of plastics by our society over decades and the long environmental life and high dispersion that these pollutants in general have (Andrady, 2011; Cole et al., 2011; Zarfl and Matthies, 2010) plastic debris have been accumulating into the environment. A large part of them is believed to be small plastic particles known as microplastics (MP) having ubiquitous occurrence. In the marine environment, where they have been particularly investigated, ocean gyres, industrial and/or densely populated estuarine and coastal areas are considered hotspots of MP concentration (Cole et al., 2011; Wright et al., 2013). MP are ingested by organisms (Besseling et al., 2013; Cole et al., €la € et al., 2014), including fish (Boerger et al., 2010; Lusher 2013; Seta et al., 2013; Possatto et al., 2011). Among other potential effects, the * Corresponding author. ICBAS e Institute of Biomedical Sciences of Abel Salazar, University of Porto, Department of Population Studies, Laboratory of Ecotoxicology, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. ), [email protected] E-mail addresses: [email protected] (L.C. de Sa (L.G. Luís), [email protected] (L. Guilhermino). http://dx.doi.org/10.1016/j.envpol.2014.10.026 0269-7491/© 2014 Published by Elsevier Ltd.
ingestion of MP instead of food may lead to growth delay, decrease of the reproductive output, and mortality increase (e.g. due to starvation), with negative effects on the population fitness. Moreover, MP can be transferred from lower to higher food web levels €la € et al., 2014). In the marine biota, MP have been shown to (Seta cause physical and chemical adverse effects, to interfere with the toxicity of other environmental contaminants, and to transfer chemicals to organisms (Antunes et al., 2013; Oliveira et al., 2013; Rochman et al., 2014; Wright et al., 2013). Three hypotheses were tested here: (i) early 0þ age group Pomatoschistus microps juveniles can ingest MP; (ii) the presence of MP simultaneously to real prey may decrease the fish predatory performance and efficiency; and (iii) the environmental conditions during the onthogenic development may influence the capability of juveniles to discriminate real prey from MP. P. microps was selected as model species mainly because it is an important intermediary ~o et al., 2006; Mehner, predator in several European estuaries (Leita 1992; Monteiro et al., 2007), including some located in urban and industrial regions where MP concentrations are expected to be high.
360
et al. / Environmental Pollution 196 (2015) 359e362 L.C. de Sa
2. Material and methods
2.4. Statistical analysis
2.1. Microplastics
Data were tested for variance homogeneity and normality of distribution and data transformations were made when necessary. Percentages were arcsine transformed, using the Bartlett's correction (Zar, 1996). The percentage of overall preys ingested by fish from distinct estuaries was compared using a two-way Analysis of Variance (ANOVA) with interaction (main factors: treatments and fish provenience - estuary). The Student's t test was used to compare M-est and L-est fish inside treatments, and their length and weight. A one-way ANOVA was carried out with data from each estuary separately, followed by the Tukey's test to compare fish performance towards different prey types. SPSS software, version 20, was used. The significant level was 0.05.
Polyethylene microspheres (PMP) were from Cospheric - Innovations in Microtechnology (USA). All PMP were spherical (>90%), opaque and had 420e500 mm diameter; white PMP (WPMP): 1.2 g/cc density; black PMP (B-PMP): paramagnetic, 1.15 g/cc density; red PMP (R-PMP): 0.98 g/cc density (manufacturer indications). 2.2. Fish collection and acclimatization Juveniles were collected (spring 2013) in the estuaries of Minho (M-est) and Lima (L-est) Rivers (NW Iberian coast) that have several ~es et al., 2012). differences in environmental conditions (Guimara Fish were transported to the laboratory and acclimatized for 2 weeks (Oliveira et al., 2013). Briefly, conditions were: 14 h light/10 h dark photoperiod, artificial salt water 18 g/l salinity (ASW), 21 ± 1 C, continuous water aeration and filtration, 98 l glass aquaria (200 fish each). 2.3. Bioassay After the acclimation, 126 fish (63 from each estuary) with about 1.5e2 cm long were selected. Bioassay conditions were similar to those above indicated, with some changes: food supply was stopped 24 h before the bioassay starting; randomly selected fish were put individually in glass beakers containing 500 ml of ASW where they were maintained for 96 h (static conditions, with aeration, no food). Water parameters were measured at 0 h and at each 24 h. After 96 h, a predatory behaviour assay was done. Seven prey types (treatments, with 9 fish from each estuary per treatment) were: 30 24 h old Artemia franciscana nauplii (Art) alone (controls); 30 W-PMP, R-PMP or B-PMP alone; 15 Art in combination with 15 W-PMP, B-PMP or R-PMP. Briefly, each fish was transferred to a prey-exposure chamber containing 300 ml of ASW. After 5 min, 30 preys were introduced into the test chamber (A), and the total number of preys ingested by the fish in a 3 min time interval was recorded. The fish was removed and the number of preys remaining in the prey-exposure chamber was counted by visual observation (B). The number of ingested prey was calculated (A - B). Fish predatory performance was determined as the percentage of the overall ingested preys relatively to the total number of preys offered (30) (IP/A*100). Fish predatory efficiency was calculated as the percentage of Art ingested relatively to the total number of preys ingested. The relative percentage of PMP ingested was calculated relatively to the total number of preys ingested. The hypotheses were tested by investigating if: fish ingested PMP (first hypothesis); the predatory performance and efficiency were lower under simultaneous exposure to Art and PMP relatively to control groups (second hypothesis); comparing the predatory performance and efficiency of M-est and L-est fish (third hypothesis). White, red and black PMP were used because colour may be important in the perception of particles as prey by fish: W-PMP because the main natural prey of early P. microps juveniles (zooplankton) are whitish; R-PMP because some of their potential benthic preys may have reddish colours; B-PMP because they have lower colour similarity to juveniles natural preys. In preliminary assays, 30 Art was found to be ad libitum food for a 3 min predation time; the ingestion of 420e500 mm PMP by 24 h old Art was never observed.
3. Results and discussion 3.1. General conditions No fish mortality occurred. Water pH and temperature variations were lower than 1 pH unity and 1 C, respectively; water dissolved oxygen was higher than 8 mg/l. Mean and standard deviation of total length and weight were: 2.017 ± 0.194 cm and 0.087 ± 0.025 g, respectively, for M-est fish; 1.938 ± 0.214 cm and 0.088 ± 0.028 g, respectively, for L-est fish. No significant differences in length (N ¼ 63; t ¼ 0.939; d.f. ¼ 124, p > 0.05) or weight (N ¼ 63; t ¼ 0.039; d.f. ¼ 124, p > 0.05) were found. 3.2. MP ingestion and confusion with real prey Fish ingested all PMP types, either under individual or combined exposures (Figs. 1e2), thus corroborating our first hypothesis. These results are in good agreement with data from previous studies reporting the ingestion of small plastic debris by other fish
Fig. 1. Percentage of the total amount of prey (Artemia nauplii þ microplastics) ingested by juveniles of the common goby (Pomatoschistus microps) from the estuaries of Minho (M-est) and Lima Rivers (L-est) when exposed to a total number of 30 preys in the water for 3 min. The values indicate the mean of 9 fish per treatment with the corresponding standard error bars. Art e Artemia nauplii (control group); W e exposure to white polyethylene micro-plastic spheres (PMP) alone; B e exposure to black PMP alone; R e exposure to red PMP alone; Art þ W e combined exposure to Artemia nauplii and white PMP; Art þ B e combined exposure to Artemia nauplii and black PMP; Art þ R e combined exposure to Artemia nauplii and red PMP; different letters (A and B e Minho estuary; a and b e Lima estuary) indicate statistical significant differences relatively to the respective control group as indicated by the Tukey multicomparisons test (p 0.05). The predatory performance of M-est and L-est fish exposed to each treatment was compared by the Student's t-test and significant differences (p < 0.05) between fish from distinct estuaries were only found under Art þ BPMP and Art þ R-PMP exposures.
et al. / Environmental Pollution 196 (2015) 359e362 L.C. de Sa
361
Table 1 Results of the two-way ANOVA on the overall percentage of preys ingested by fish from the estuaries of the Minho and Lima Rivers under exposure to different treatments (Artemia nauplii only, white microplastics alone, black microplastics alone, red microplastics alone, Artemia nauplii þ white microplastics, Artemia nauplii þ black microplastics; Artemia nauplii þ red micro-plastics). Comparison
F
P
Type of preys offered (treatments) Origin of the fish (estuaries) Interaction
F(6,112) ¼ 19.908 F(1,112) ¼ 6.963 F(6,112) ¼ 1.183
p < 0.05 p < 0.05 p < 0.05
performance and efficiency of early P. microps juveniles, depending on the MP type and fish condition.
Fig. 2. Relative percentages of Artemia nauplii and microplastics ingested by juveniles of the common goby (Pomatoschistus microps) from the estuaries of Minho and Lima Rivers in combined exposures. The values indicate the mean of 9 fish per treatment with the corresponding standard error bar. Art þ W e combined exposure to Artemia nauplii and white PMP; Art þ B e combined exposure to Artemia nauplii and black PMP; Art þ R e combined exposure to Artemia nauplii and red PMP.
species (Boerger et al., 2010; Dantas et al., 2012; Lusher et al., 2013; Possato et al., 2011). Significant differences among treatments, between fish from distinct estuaries and a significant interaction between the two factors were found (Table 1). Relatively to the respective control feed with Art only, fish showed a significant reduction (M-est: F(6,56) ¼ 6.563, p < 0.05; L-est F(6,56) ¼ 16.492, p < 0.05) of the predatory performance when exposed to PMP alone (Fig. 1). Moreover, in all the combined treatments, M-est fish ingested considerably more Art than PMP (Fig. 2). Thus, fish showed prey selectivity, preferring Art over PMP. They were able to select Art among PMP when both were present in the water. Thus, they can discriminate food from MP, despite making some mistakes. Early P. microps juveniles are visual predators feeding mainly on zooplankton (Mehner, 1992). Art were alive and moved while PMP did not. Thus, Art motion may have helped fish to discriminate it from PMP. In the wild, MP are likely to be in movement too as the result of water currents and other factors, potentially increasing the difficulty of selecting food among MP. In combined exposures, L-est fish ingested more W-PMP than BPMP or R-PMP (Fig. 2) suggesting that they confuse W-PMP with food more than the other PMP. W-PMP are more similar to the whitish colour of early P. microps natural preys than the other PMP. Thus, white MP may have a high likelihood of being ingested by early P. microps due to their colour and visibility, as suggest for other plastic types and fish species (Carpenter et al., 1972; Boerger et al., 2010).
3.3. The presence of MP simultaneously to prey reduces fish predatory performance and efficiency In combined exposures, M-est fish were effective in selecting Art from MP during the 3 min measurement period, ingesting only a small proportion (z10%) of PMP (Fig. 2). Thus, they seem to confuse PMP with preys only in a small extent, and the presence of PMP did not reduce significantly their predatory performance (Fig. 1). On the contrary, under combined exposures, L-est fish predatory performance was significantly reduced in the presence of B-PMP or RPMP, and their predatory efficiency was reduced in the presence of all PMP types (Figs. 1e2). Overall, the above findings indicate that the presence of MP in water may decrease the predatory
3.4. The environmental conditions during early developmental phases may influence the capability of juveniles to discriminate real prey from MP Under combined treatments, L-est fish ingested less Art and more MP than M-est fish, particularly W-PMP (Figs. 1e2). This can be due to a lower capability of L-est fish to discriminate PMP from the real prey or because they were less able in capturing Art during its attempt to escape from the predator than M-est fish. Because no significant differences in the predatory performance between fish from distinct estuaries were found under exposure to Art alone (Fig. 1), the second possibility can be excluded. Therefore, L-est fish showed a reduced capability in discriminating PMP from the real prey relatively to M-est animals. Fish from the two estuaries were collected in the same week, they have no significant differences in weight and size, and acclimation and experimental conditions were the same. Thus, the differences in the predatory performance and efficiency found were likely due to the influence of distinct environmental conditions during previous developmental phases. Mest and L-est have several differences, including in their levels of ~es et al., 2012). Previous studies with older pollution (Guimara P. microps wild juveniles indicated a decreased health status of L-est fish relatively to those from the M-est, likely caused by higher levels of background pollution in the L-est, among other possible factors ~es et al., 2012). Despite the difsuch as prey availability (Guimara ferences in feeding ecology between early and latter P. microps ~o et al., 2006), these studies support juveniles (Mehner, 1992; Leita our hypothesis that onthogenic developmental conditions may influence the capability of fish to discriminate real prey from MP. Acknowledgements We thank Prof. Natividade Vieira for providing parental Artemia, and Dr. Miguel Oliveira for discussions. This study was funded by the Portuguese Foundation for Science and Technology (FCT) with national and European Regional Development Fund (ERDF) funds through the Operational Competitiveness Programme under the projects “SIGNAL” (PTDC/AAC-AMB/110331/2009; FCOMP-01-0124FEDER-013876) and PEst-C/MAR/LA0015/2013. References Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62, 1596e1605. Antunes, J.C., Frias, J.G.L., Micaelo, A.C., Sobral, P., 2013. Resin pellets from beaches of the Portuguese coast and adsorbed persistent organic pollutants. Estuar. Coast. Shelf Sci. 130, 62e69. Besseling, E., Wegner, A., Foekema, E.M., van den Heuvel-Greve, M.J., Koelmans, A., 2013. Effects of microplastic on fitness and PCB bioaccumulation by the lugworm Arenicola marina (L.). Environ. Sci. Technol. 47, 593e600. Boerger, C.M., Lattin, G.L., Moore, S.L., Moore, C.J., 2010. Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre. Mar. Pollut. Bull. 60, 2275e2278.
362
et al. / Environmental Pollution 196 (2015) 359e362 L.C. de Sa
Carpenter, E.J., Smith, K.L., 1972. Plastics on the Sargasso sea surface. Science 175, 1240e1241. Cole, M., Lindeque, P., Halsband, C., Galloway, T.S., 2011. Microplastics as contaminants in the marine environment: a review. Mar. Pollut. Bull. 62, 2588e2597. Cole, M., Lindeque, P., Fileman, E., Halsband, C., Goodhead, R., Moger, J., Galloway, T.S., 2013. Microplastic ingestion by zooplankton. Environ. Sci. Technol. 47, 6646e6655. Dantas, D.V., Barletta, M., Ferreira da Costa, M., 2012. The seasonal and spatial patterns of ingestion of polyfilament nylon fragments by estuarine drums (sciaenidae). Environ. Sci. Pollut. Res. 19, 600e606. ~es, L., Medina, M.H., Guilhermino, L., 2012. Health status of Pomatoschistus Guimara microps populations in relation to pollution and natural stressors: implications for ecological risk assessment. Biomarkers 17, 62e77. Leit~ ao, R., Martinho, F., Neto, J.M., Cabral, H., Marques, J.C., Pardal, M.A., 2006. Feeding ecology, population structure and distribution of Pomatoschistus microps (Krøyer, 1838) and Pomatoschistus minutus (Pallas, 1770) in a temperate estuary, Portugal. Estuar. Coast. Shelf Sci. 66, 231e239. Lusher, A.L., McHugh, M., Thompson, R.C., 2013. Occurrence of microplastics in the gastrointestinal tract of pelagic and demersal fish from the English Channel. Mar. Pollut. Bull. 67, 94e99. Mehner, T., 1992. Diet spectra of Pomatoschistus microps (Krøyer) and Pomatoschistus minutus (Pallas) (teleostei, gobiidae) during first weeks after hatching. Zool. Anz. 229, 13e20.
Monteiro, M., Quintaneiro, C., Nogueira, A.J.A., Morgado, F., Soares, A.M.V.M., Guilhermino, L., 2007. Impact of chemical exposure on the fish Pomatoschistus micropsis Krøyer (1838) in estuaries of the Portuguese Northwest coast. Chemosphere 66, 514e522. Oliveira, M., Ribeiro, A., Hylland, K., Guilhermino, L., 2013. Single and combined effects of microplastics and pyrene on juveniles (0þ group) of the common goby Pomatoschistus microps (teleostei: gobiidae). Ecol. Indic. 34, 641e647. Possatto, F.E., Barletta, M., Costa, M.F., Ivar do Sul, J.A., Dantas, D.V., 2011. Plastic debris ingestion by marine catfish: an unexpected fisheries impact. Mar. Pollut. Bull. 62, 1098e1102. Rochman, C.M., Hentschel, B.T., The, S.J., 2014. Long-Term sorption of metals is similar among plastic types: implications for plastic debris in aquatic environments. PLoS ONE 9, e85433. http://dx.doi.org/10.1371/ journal.pone.0085433y1. €l€ Seta a, O., Fleming-Lehtinen, V., Lehtiniemi, M., 2014. Ingestion and transfer of microplastics in the planktonic food web. Environ. Pollut. 185, 77e83. Wright, S.L., Thompson, R.C., Galloway, T.S., 2013. The physical impacts of microplastics on marine organisms: a review. Environ. Pollut. 178, 483e492. Zar, J.H., 1996. Biostatistical Analysis. Prentice Hall, United Kingdom. Zarfl, C., Matthies, M., 2010. Are marine plastic particles transport vectors for organic pollutants to the Arctic? Mar. Pollut. Bull. 60, 1810e1814.