Identifying the Presence of Microplastics in Maputo Bay Quantifying Microplastic Debris in a Field Setting

Identifying the presence of microplastics in Maputo Bay Quantifying microplastic debris in a field setting

Maja Karlsson

Degree project for Bachelor of Science in Biology

BIO603 Biology: Degree project 30 hec Spring-summer 2015

Department of Biological and Environmental Sciences University of Gothenburg

Examiner: Catharina Olsson Department of Biological and Environmental Sciences University of Gothenburg

Supervisor: Bethanie Carney Almroth Department of Biological and Environmental Sciences University of Gothenburg

External supervisor: Daniela de Abreu Department of Biological Sciences Eduardo Mondlane University

Front page image: Beach debris surrounding a ‘no littering’ sign in Catembe, Mozambique. Photographed by Sandra Toivio, used with permission.

Contents

Abstract – Swedish ...... 5 Abstract – English ...... 6 1. Introduction...... 7 2. Materials and methods ...... 9 2.1 Study area ...... 9 2.2 Biotic sampling ...... 9 2.3 Digestion of biotic tissues ...... 10 2.4 Beach surveys ...... 10 2.5 Sediment sampling ...... 11 2.6 Visual identification ...... 12 2.7 Statistical analysis ...... 12 3. Results ...... 12 3.1 Beach transects ...... 12 3.2 Sediment samples ...... 15 3.3 Biological samples ...... 16 4. Discussion ...... 17 4.1 Beach transects ...... 18 4.2 Sediment samples ...... 18 4.3 Biological samples ...... 19 4.4 Performing the study on Inhaca Island ...... 21 5. Conclusion ...... 21 Acknowledgements ...... 21 References ...... 22 Appendix ...... 25

Abstrakt – Svenska Under de senaste åren har medvetenheten hos forskare angående förekomsten av mikroplast i den marina miljön ökat nämnvärt och likaså intresset beträffande dess potentiellt skadliga effekter. “Mikroplast” är en samlingsterm för alla plastpartiklar som är mindre än 5 mm i storlek, oavsett ursprung, och kan således innefatta allt från tillverkade pellets, som används t.ex. i kosmetika eller som råmaterial vid tillverkning av plastartiklar, till fragment av större föremål som slitits ner. Denna studie undersöker förekomsten av mikroplastpartiklar i Maputo Bay i södra Moçambique. En jämförelse görs även mellan ett “förorenat” och två “oförorenade” områden, för att se om de skiljer sig åt. Varje område undersöktes ur tre aspekter: ”beach surveys” (direkt översatt ”strandinspektioner”), för att se mängden makroplast närvarande, sedimentprover, för att undersöka mikroplasten på stranden samt biologiska prover, för att se hur mycket mikroplast som intagits av marina djur. Resultaten visade en relativt jämn fördelning av mikroplast på alla tre platser och de enda signifikanta skillnaderna syntes hos makroplasten i strandinspektionerna, där det ”förorenade” området hade betydligt högre mängder (p<0,001). Mikroplast återfanns i totalt 32 % av de biologiska proverna och i 100 % av sedimentproverna. Resultaten måste dock tolkas i enlighet med förutsättningarna för studien, vilka begränsade utförandet något då de var relativt primitiva. Det råder dock inte mycket tvivel om att mikroplast i viss mån förekommer i Maputo Bay, till och med på den oexploaterade och ekologiskt värdefulla ön Inhaca.

Abstract – English In recent years, the awareness of microplastic pollution in the marine environment has spiked in the scientific community, and with it the interest and concern for its potential effects. ‘Microplastics’ is a collective term for any plastic particle less than 5 mm in size, whether being a fragment of a larger item or a pellet manufactured to be this size for use in factories or cosmetics. This study investigates the presence of microplastics in Maputo Bay in southern Mozambique, and compares the abundance in a ‘polluted’ area to that of a ‘pristine’ one. Three locations were chosen – one ‘polluted’ and two ‘pristine’, and three aspects were considered: beach surveys, for identifying macro-sized plastic debris, sediment samples, for quantifying micro-scale particles, and biological sampling by examining the GI-tracts of marine organisms to see how much microplastic they have ingested. Results showed a relatively even distribution of microplastics in all three sites. The only significant differences regarded macrodebris where the ‘polluted’ site was substantially higher in abundance than the others (p<0.001). Microplastics was found in 32 % of the biological samples and 100 % of the sediment samples in this study. The conditions under which it was performed put some constraints on the methodology and so some results have to be considered accordingly. There is however little doubt that microplastic pollution is to some degree present in Maputo Bay, even at the pristine and ecologically valuable island of Inhaca.

1. Introduction While it is well known that there are vast quantities of plastic garbage in the marine environment (Cozar et al., 2014; Eriksen et al., 2014; Gregory, 2009), much is still unclear about its ultimate fate in oceans and seas. Plastic debris can be found in almost every part of the ocean, from the deep sea to coastlines and surface water (Barnes et al., 2009), with a major accumulation point at the subtropical surface gyres (Cozar et al., 2014). It is also common to find marine animals entangled in discarded fishing gear, six- pack yokes or other plastic litter, as well as stranded marine animals that died from starvation after ingesting plastic items (Gregory, 2009). These types of debris, known as macroplastics, have received the most attention from the public due to the ethical and aesthetic problems they pose. As a result, many scientific studies have been performed to evaluate the harmful effects of macroplastics, particularly concerning marine birds, turtles and sea mammals (Gall & Thompson, 2015; van Franeker et al., 2011). However, marine debris is a difficult thing to monitor as it is patchy (Barnes et al., 2009), covers large areas, and is extremely time consuming to document. Therefore, knowledge about plastic quantities in the ocean is subject to much uncertainty. Though it has been reported that the levels of plastic debris in the open oceans seem to have stabilized, they have simultaneously increased on shorelines and in the deep sea (Barnes et al., 2009). Furthermore, as large items break down into smaller particles they become even harder to quantify and monitor since quite sophisticated equipment is needed for reliable identification (Hidalgo-Ruz et al., 2012). These small plastic fragments, pellets or fibres are known as microplastics and can be found in many parts of the ocean (Barnes et al., 2009; Browne et al., 2010; Cole et al., 2011; Collignon et al., 2012; Shaw & Day, 1994). Although the presence of microplastics has been recognised since the 1970’s (Carpenter & Smith, 1972), there has been no clear definition of the term until 2009, when the NOAA International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris suggested that it should be defined as “plastic particles smaller than 5 mm” (Arthur et al., 2009, page 10). There is, however, no lower size limit to the term and this has led to scientific reports differing in the size spectrum studied (Hidalgo-Ruz et al., 2012). Microplastics are sometimes divided into two categories: primary and secondary microplastics (Cole et al., 2011). Primary microplastics are particles manufactured to be microscopic, for use in cosmetics, air blasters and other products, and also includes pre- production raw plastic pellets. Secondary microplastics are fragments of larger plastic items that have been subjected to degradation (Cole et al., 2011). Marine degradation can occur through a number of mechanisms, both biological (Davidson, 2012) and non- biological, the most common likely being light-initiated oxidative degradation in coastal areas (Andrady, 2011). Degradation can also take place in the terrestrial environment, through everyday wear of synthetic products such as tires (Wik & Dave, 2009) or washing of clothes (Browne et al., 2011). Additionally, plastic debris of all sizes can end up in the oceans through poor waste management and river runoff from urban areas (Rech et al., 2014). Once the debris has entered the ocean, it can be transported long distances due to its positive buoyancy (Barnes et al., 2009). The transport of plastic debris can act as a vector for rafting organisms to travel far (Gall & Thompson, 2015; Goldstein et al., 2014; Gregory, 2009), potentially increasing the risk for invasive species in sensitive ecosystems. These are just a few of the many concerns the scientific community have regarding microplastics. Among the others are ingestion, bioaccumulation and toxicity of small particles in biota. Different size classes of debris have different qualities and can so

affect animals in different ways. Particles on a micro-scale can be easily ingested and act as pollutant vectors, and particles on a nano-scale are small enough to be transported through tissues and even inside cells (Maynard, 2006), potentially acting as a vector for toxins. Many types of plastic have shown potential for adsorbing PCBs (Endo et al., 2005), which can be transported into organisms as the particles are ingested. As well as adsorbed pollutants, some additives such as triclosan and PBDE-47 have been shown to transfer from ingested PVC into tissues of lugworms (Browne et al., 2013), resulting among other things in altered feeding patterns. Ingestion of plastic debris has been reported for various types of macrofauna, the most intensely studied being fulmar birds (van Franeker et al., 2011) but is has also been seen in pelagic fish (Boerger et al., 2010; Choy & Drazen, 2013; Davidson & Asch, 2011; Foekema et al., 2013). Studies of microplastic ingestion is a field that is still relatively new, yet reports have been made of microplastics found in animals ranging from cetaceans (Besseling et al., 2015; Lusher et al., 2015) to cultured Mytilus edulis meant for human consumption (Van Cauwenberghe & Janssen, 2014) to small animals such as zooplankton (Lima et al., 2014) and scleractinian corals (Hall et al., 2015). This report will focus on the presence of microplastics in biota and sediments from Maputo Bay in Mozambique. There is an overall lack of data from the east coast of Africa (Gall & Thompson, 2015), and though there is no reliable estimate of the biodiversity in Mozambique (Griffiths, 2005), it is known to be high. Maputo Bay is a shallow bay, the majority of it having a depth of less than 10 m (Canhanga & Dias, 2005). However, large cargo ships are able to enter the Maputo harbour and being the nation’s capital, Maputo is visited by many ships daily. The water in the Estuário do Espírito Santo, which is the name of the river mouth separating Maputo from Catembe (fig. 1), is very murky and oily with a lot of suspended sediment (pers. obs.). No published information regarding the water quality in Estuário do Espírito Santo was found, but the proximity to such a large city as Maputo, puts it at risk for heavy pollution by both macro- and microdebris. On the eastern end of Maputo Bay, approximately 35 km from Maputo and Catembe, lies Ilha da Inhaca (Inhaca Island) that together with Ponta Santa Maria acts as a barrier between the bay and the Indian Ocean (Armitage et al., 2006). It is considered a pristine and ecologically valuable area by scientists, and is home to a field biology station (Estaçao Biologia Maritima da Inhaca) belonging to the university (Universidade Eduardo Mondlane) in Maputo. This study aims to aid in mapping the presence of microplastics by investigating whether there are micro-plastics present in Maputo Bay. It also aims to compare the Fig. 1: The location of the three investigated sites relative abundance of both macro- and in Maputo bay: Catembe on the main land, and microplastics in a polluted area (Catembe), Marine Station and Saco on Inhaca Island. and a pristine area (Inhaca Island), to see Highlighted areas visible in fig. 2 (p. 11). Satellite whether the proximity to a highly image from Google Earth. populated area means higher levels of

pollution by microlitter in the ocean and subsequently a higher risk for marine animals. At Inhaca Island, two different sites will be investigated: one on the coastal beach outside the marine biology research station (hereafter referred to as ‘Marine Station’) and one located in the mangrove bay known as Saco da Inhaca (hereafter referred to as ‘Saco’). The different characteristics of the two pristine sites could have an impact on the amount of debris they contain and so a comparison between them would be of interest as well as the comparison with the polluted site. Additionally, investigating whether this type of study is at all possible to perform without a laboratory with advanced equipment would be very valuable for future projects aiming to map the presence of microplastics in remote areas or at low-budget field stations.

2. Materials and methods Many aspects of the methods had to be revised due to a lack of equipment at the field station where the study was conducted, and are therefore not optimal for maximum recovery but rather a simplified version adapted for use in field conditions. This is however important and will aid investigations in remote and primitive conditions in future studies. All equipment was washed in between samples with dishwashing detergent, tap water and rinsed with distilled water.

2.1. Study area This study was carried out between May and July 2015 on Ilha da Inhaca, or Inhaca Island, located at 26°S, 33°E in southern Mozambique (fig. 1). Inhaca is home to a variety of ecosystems, both terrestrial and marine, well described in Kalk (1995). Marine habitats range from seagrass beds and mangrove forests to coral reefs and tidal pools (pers. obs.). This island experiences seasonal tourism due to its pristine beaches and clear water. At the time of investigation, it was winter and so low season for tourists with only a few thousand local people inhabiting the island. Two sites were chosen on the island: one offshore of the Marine Biology station, open to the bay, and one in the mangrove bay of Saco da Inhaca, with only a narrow, relatively sheltered opening allowing water to flow. The remoteness and low level of human activity led these sites to be considered ‘pristine’ in the study. Samples were also collected from the coast of Catembe, a town located across the river from the nation capital Maputo. This site was considered ‘polluted’ due to the poor water quality and high human activity: fishing boats, ferries and cargo ships cruised the waters constantly and a constant stream of people used the beach to walk to and from the Maputo ferry. Furthermore, the proximity to Maputo puts this area at risk for runoff from the city of toxins as well as macro- and microdebris.

2.2 Biotic sampling No ethical permits were required for this study, still all animals were handled carefully in order to minimize stress while still alive. Marine animals caught in three locations were used for these analyses. Silver smelt (Sillago sihama), a commercially important species that usually inhabits estuarine and near-shore areas and is omnivorous (Taghavi Motlagh et al., 2012); Groovy mullet (Liza dumerilii), a detritivorous fish common in tropical and subtropical coastal waters (Blay, 1995); and the Mud crab or Mangrove crab Scylla serrata, a commercially important predator of macro-invertebrates (Hill, 1976) were used. Fish from ‘Marine Station’ and ‘Saco’ were bought, dead, from local fishermen in the respective areas. S. serrata from the mangrove swamp, used in a different study (S. Toivio, unpublished data), were

caught using metal cages and euthanized by being placed in a freezer overnight. From the pristine site ‘Marine Station’, 20 individuals of S. sihama were used, and from the polluted site ‘Catembe’, 20 individuals of L. dumerilii were used. All individuals were of similar size and kept in a freezer until use. They were measured and weighed while still frozen before the gastrointestinal (GI-) tract, from oesophagus to anus, was removed. The GI-tract was also frozen until further use. Plastic particles were identified by sifting through the GI-tract contents in a dissecting scope.

2.3 Digestion of biotic tissues Originally, the plan was to perform a chemical digestion of organic materials in order to get a clearer view of the non-organic particles present. During trials, the digestion process was derived from a previous methodology study (T. Karlsson, unpublished data) and adapted as needed under present circumstances. The frozen GI-tracts were weighed and cut into small pieces using scalpels and a pair of dissection scissors. They were placed in Erlenmeyer flasks with 10 ml homogenization buffer (400 mM Tris-HCL, pH 8, 0.5 % SDS), covered with aluminium foil to avoid contamination and incubated for 60 minutes in 60°C to denature proteins in preparation for the digestion process. Another 5 ml of the homogenization buffer was mixed with 500 μg/ml Proteinase K (3.0-15.0 unit/mg, T. album) and 5 mM CaCl2, and added to each sample. CaCl2 would activate the proteinase, which would then act to digest the proteins of biotic tissue in the sample. All samples were incubated for >2 hours in 50°C, shaken for 20 minutes in order to completely mix tissue samples and reagents and incubated for another 20 minutes. Finally, 30 ml H2O2 (30%) was added to break down chitin and other residual organic structures overnight. The next day, the samples were filtrated through a Whatman GF/C filter (1.2 μm, ⌀ 70 mm) using a Stermay HT-196 AC electric air pump and a MILLIPORE XX.10.047.04 filtrating system. Unfortunately, filtration was not possible due to the instant clogging of the filters and so this method had to be abandoned.

2.4 Beach surveys Appropriate beach transects were chosen in the same areas where fishes were caught (fig. 2). The surveys were carried out according to OSPAR guidelines for 100 meter transects (Wenneker & Oosterbaan, 2010). All the litter found was collected and categorized in one of 111 categories, from the edge of the water to the very back of the beach. Due to extreme tidal changes, surveys were timed to take place 1 hour after high tide, in order for the beach to be approximately the same width each time. During spring tide, the time between a survey and high tide had to be somewhat lengthened. In the ‘Saco’ mangrove swamp, only one beach area was available, so the remaining analyses were instead performed on the three substrates present: the mud, the sand and the riverbank, in order to get a complete view of the site. At the ‘Catembe’ site, some categories of litter had to be estimated due to their size and sheer abundance, and was done so by recording all items of these categories found in a 2 m wide section of the beach and multiplying. The categories in question were no. 15 (caps/lids), 32 (string and cord – diameter less than 1 cm), 117 (plastic/polystyrene pieces 0-2.5 cm) and 48 (other plastic/polystyrene pieces). Industrial plastic pellets were included in category 48, and in the first transect of the ‘Catembe’ site, these were even too abundant for the above- described estimation. In order to estimate their quantity, a 2x2 m square was measured and all pellets within it counted. This number was subsequently multiplied to cover the whole transect.

Fig. 2: Locations for transect 1-5 (T1-T5) at each investigated site. Satellite images from Google Earth.

2.5 Sediment sampling In association with the beach survey conducted, sediment samples were taken from each beach transect. Two 30 ml samples were collected from the top 2 cm layer at random points below (Sample 1) and above (Sample 2) the high tide mark, respectively, resulting in 4 samples per site. This was not possible in the ‘Saco’ swamp, since there was no distinguishable high tide mark. Instead, four samples were collected randomly at each transect. Each pair of samples was then mixed for homogenization purposes and 20 g (w.w.) were taken out for analysis. The amount of sediment chosen for analysis was derived from an earlier study (T. Karlsson, unpublished data). In order to separate the lighter plastic particles from the heavier sediment, a density separation approach was used. This has been previously shown to be an efficient way of retrieving microplastics from sediments (Claessens et al., 2013; Hidalgo-Ruz et al., 2012; Imhof et al., 2012), however, a simplified version had to be developed due to the lack of materials. A mixture of distilled water and NaCl with a density of ~1.2 g cm-3 was created, so that particles with a lower density than this would float while heavier particles would sink. Microplastics in general have a wide span of densities, but most are found between 0.8 g cm-3 and 1.4 g cm-3, while sand and sediment particles typically lie around 2.65 g cm-3 (Hidalgo-Ruz et al., 2012). 100 ml of the supersaturated mixture was added to a 500 ml graduated cylinder together with the 20 g of sediment, and one drop of vegetable oil. The oil served to attract the hydrophobic plastic particles rather than having them stick to the sides of the cylinder. With each pair of samples, a third cylinder was prepared (without sediment) as a blank sample to check for contamination. The cylinders were stirred well to ensure that all sediment would be suspended, leaving the plastic particles free to surface. The samples were then left overnight to allow for sedimentation. The next morning, >30 ml of supernatant was removed from each sample using a contraption made from a glass tube (⌀ 7 mm), a 20 ml syringe and various silicone tubes as a makeshift pipette and filtrated through a Whatman GF/C filter (1.2 μm, ⌀ 70 mm) using a Stermay HT-196 AC electric air pump and a MILLIPORE XX.10.047.04 filtrating

system. The glass tube, as well as the walls of the filtration system, was rinsed with distilled water and a small amount of dishwashing detergent to solubilize the oil. In samples with exceptionally high amounts of organic material, a small amount of H2O2 (3%) was added to avoid clogging as the sample was filtrated. The filter was then analysed through visual identification in a dissecting scope.

2.6 Visual identification Each filter from the sediment samples and the whole of the biotic samples were searched for plastic particles in a dissecting microscope. Particles were identified using criteria derived from a previous study (Nor & Obbard, 2014) and categorized according to their size, colour and shape. For the sediment samples, two size classes were used, as suggested in a microplastic methodology review (Hidalgo-Ruz et al., 2012). Group 1 consisted of particles <500 μm and group 2 of particles between 500 μm and 5 mm.

2.7 Statistical analysis Statistical analyses were performed using IBM SPSS Statistics v. 22.0.0.0. A Shapiro-Wilk normality test was used to check data for normality, and data from the variables used were found to not be normally distributed and did not display homogeneity of variance. To test differences between sites, non-parametric Kruskal-Wallis tests were used in combination with Mann-Whitney U tests, and to check for correlations a Spearman correlation test was used.

3. Results There was generally quite a lot of variation within the data and few results stood out clearly, apart from the results from the beach transects. This method is the only one with a standardized procedure possible to perform under relatively crude conditions and allows for very little possibility of bias.

3.1 Beach transects As the focus of this study is plastic debris, litter of other materials were not considered in the total number of categorized items (fig. 3). It is clear that a few categories are far more abundant than the rest, at ‘Marine Station’ being category 4 (drinks – bottles, containers and drums) and 32 (string and cord – diameter less than 1 cm). In ‘Saco’, the most abundant was category 19 (crisp/sweet packets and lolly sticks), and at ‘Catembe’, the site with predominantly more litter than the two others (fig. 4), some items were so abundant that counting them became impossible, and so some numbers are estimated. A Kruskal-Wallis test showed a significant difference in pollution degree between the three different sites (Kruskal Wallis H=25.073, df=2, p<0.001). A post-hoc Mann Whitney U test revealed that the differences between ‘Catembe’ and ‘Marine Station’ (U=8.534, df=1, p=0.003), ‘Catembe’ and ‘Saco’ (U=38.474, df=1, p<0.001), and ‘Marine Station’ and ‘Saco’ (U=18.638, df=1, p<0.001) were all statistically significant. In ‘Catembe’, the great majority of the items found in category 48 (other plastic/polystyrene pieces) were industrial pellets, but these are not yet recognized as a category in the OSPAR beach survey manual.

(a)

(b)

(c)

Fig. 3: Total number of plastic items found in sites (a) ‘Marine Station’ (b) ‘Catembe’ and (c) ‘Saco’, categorized according to OSPAR code (Wenneker & Oosterbaan, 2010), key found in appendix. Numbers marked with an asterisk (*) are estimations.

In ‘Catembe’, the great majority of the items found in category 48 (other plastic/polystyrene pieces) were industrial pellets, but these are not yet recognized as a category in the OSPAR beach survey manual. They were, however, extremely abundant at the first transect (T1) but abundance quickly lessened as surveys were performed further down the beach (fig. 5.a). In order to get a view of how the transects would compare without having the results skewed by the vast amount of pellets, the pellets were removed in fig. 5.b. (a) (b)

Fig. 4: (a) the arithmetic mean number of items found per transect at each site, with error bars showing SEM. (b) zoomed in view of the same graph.

(a) (b)

Fig. 5: (a) Comparison in litter abundance between the beach transects performed at the ‘Catembe ‘site. (b) Industrial pellets removed for better comparison between transects.

While there was a clear decrease in litter abundance in transects T4 and T5, these areas still contained approximately 700 and 1000 plastic items respectively per 100 m, which is a very high level of pollution. If the debris from ‘Catembe’ is compared with the other sites after removing the estimated high number of pellets, Mann-Whitney U tests showed this site to still be significantly more polluted than both ‘Marine Station’ (U=8.274, df=1, p=0.004) and ‘Saco’ (U=38.474, df=1, p<0.001) but with a smaller SEM value (fig. 6). Dividing the recorded debris by the area covered, the estimated number of plastic items per meter of beach (approximately 8 meters wide) is over 30, excluding pellets. Doing the same for the other two sites yields 0.75 items m-1 for ‘Marine Station’ and less than 0.05 items m-1 for ‘Saco’. Variation between transects was shown at the two other sites as well, but none varied as greatly as ‘Catembe’. (a) (b)

Fig. 6: (a) the arithmetic mean number of items found per transect at each site, excluding pellets, with error bars showing SEM. (b) zoomed in view of the same graph. At ‘Marine Station’, the fifth and southernmost transect had more than twice as much litter as the other transects (fig. 7.a), with levels still far from any transect in ‘Catembe’. The ‘Saco’ site was by far the cleanest, with litter present only at the first transect (fig. 7.b). This site was, however, not optimal for this type of survey due to the availability of only one beach area, with a road right next to it. At all sites, the majority of recorded litter was made from plastic or polystyrene. Disregarding the pellets, plastic comprised 95% of the overall recorded debris. Including the pellets, the number rises to 98% (table 1), which makes it by far the most common material among beached debris.

(a) (b)

Fig. 7: (a) Comparison in litter abundance between the beach transects performed at the ‘Marine Station ‘site. (b) Comparison in litter abundance between the beach transects performed at the ‘Saco ‘site.

Table 1: Proportion of plastic items among debris found. Beach 1: Marine Station; Beach 2: Catembe; Beach 3: Saco Plastic/polystyrene Beach Transect All debris (no. items) % plastic 3.2 Sediment samples (no. items) 1 1 40 58 69% While ‘Catembe’ was far more 1 2 35 43 81% polluted than the other sites regarding 1 3 27 40 68% 1 4 68 93 73% macrodebris, the 1 5 205 216 95% microdebris found in sediment 2 1 37575 38168 98% samples from the same beach 2 2 4680 4727 99% 2 3 4749 4792 99% transects were the most abundant in 2 4 693 726 95% samples from ‘Marine Station’ (fig. 8). 2 5 980 1022 96% 3 1 47 51 92% The site with the fewest particles was 3 2 0 0 N/A ‘Saco’. This site was not analysed as S1 3 3 1 1 100% (below high tide mark) and S2 (above 3 4 0 1 0% 3 5 0 0 N/A high tide mark) samples, since no such Total: 49100 49938 98% mark could be distinguished in the swamp. In both ‘Marine Station’ and ‘Catembe’, a slightly higher number of particles was found in S1 than in S2 (fig. 8). This was, however, not a significant difference (Kruskal Wallis H=0.519, df=1 p=0.471). Within the sites, the transects differed somewhat (fig. 9.a- c). Surprisingly, the mean number of particles per 20 g sample was sometimes lower than the number of Fig. 8: Total number of plastic particles found in the particles in the blank control filter (fig. sediment samples collected. The first group of bars 9.d). This indicates high levels of show the abundance at ‘Marine Station’; S1: below contamination during the process, and high tide mark; S2: above high tide mark. The second lowers the credibility of the results. Of group shows ‘Catembe’. At the ‘Saco’ site it was not all the particles found, the majority possible to clearly distinguish between the two zones, which is why these samples have not been divided into was smaller than 500 μm (fig. 10). At S1 and S2. The third group of bars shows the total ’Marine Station’, 100 of 132 particles number of particles found in all samples (200 g) from (76 %) were in size class 1. For ’Saco’ each site: ‘Marine Station’, ‘Catembe’ and ‘Saco’. and ’Catembe’, it was slightly over half,

(a) (b)

Fig. 9: (a) Mean number of particles found per 20 g of sediment (±SEM) and per control filter in each transect at the site ‘Marine Station’. (b) Mean number of particles found per 20 g of sediment (±SEM) and per control filter in each transect at the site ‘Catembe’. (c) Mean number of particles found per 20 g of sediment (±SEM) and per control filter in each transect at the site ‘Saco’, (d) The arithmetic mean number of particles found per 20 g sample at each site, compared with the mean number of particles found in the pairwise created control filters. 57 % and 56 % respectively. Comparing the mean of the samples from one site with the contaminants in the control filters paired with them, the only site having a higher number of particles in the sample filters is ‘Catembe’ (fig. 9.d). This is consistent with the results from the beach transects, where ‘Catembe’ was the most polluted. H owever, there was no significant difference in particle abundance between sites (Kruskal Wallis H=1.021, df=2, p=0.600).

3.3 Biological samples The majority of the sampled organisms did not contain any microdebris (fig. 11), and the ones containing the least were the fish originating from the ‘Catembe’ site. Of the specimen that had ingested particles, however, the fish from ‘Catembe’ had the highest average of 2.33 particles fish-1, compared to 1.75 particles fish-1 for ‘’Marine Station and 1.63 particles crab-1 for ‘Saco’. Overall, 32% (0.316666667) of the specimens contained microplastics. The number of particles found was highest in fish from the ‘Marine Station’ followed by the mud crabs from ‘Saco’ (fig. 12). Averaging the entire sample size, the fish from Fig. 10: Total size distribution of particles found in all ‘Marine Station’ contained 0.7 sediment samples, with a mark at 500 μm. particles per specimen, the fish from

‘Catembe’ 0.35 particles per specimen and the crabs from ‘Saco’ 0.65 particles per specimen. The difference between species was shown to be non-significant (Kruskal Wallis H=3.027, df=2, P=0.220). The average of all three sites was 0.57 particles ind-1. As shown in fig. 13, particle abundance in the GI-tract can be somewhat related to animal size. This was, however, found to be non-significant after performing a Spearman’s correlation test (ρ=0.000, p=1.000).

Fig. 11: Proportion of the specimen containing microplastic particles. S. sihama were caught at the site ‘Marine Station’, N=20; L. dumerilii were caught at ‘Catembe’ , N=20; S. serrata were caught at ‘Saco’, N=20

Fig. 12: Total number of microplastic Fig. 13: Number of particles found in the particles found in the sampled specimens. GI-tract of each individual, related to the S. sihama originated from ‘Marine length of that individual. Fork length was Station’, N=20; L. dumerilii originated measured on S. sihama and L. dumerilii, from ‘Catembe’, N=20; S. serrata and carapace width was measured on S. originated from ‘Saco’, N=20. serrata.

4. Discussion Microplastic pollution is an environmental threat receiving more and more attention in media and in the scientific community. While information concerning occurrence of microplastics in European, North American and Asian environments are increasing, there is a lack of data from different regions in Aftica (Gall & Thompson, 2015). This study set out to quantify the microplastics present in different aspects of the environment in Maputo Bay, Mozambique. A comparison was made regarding beached macrodebris, microdebris in beach sediment and stomach contents of marine animals in three sites: one close to a large city and two remote, pristine locations.

4.1 Beach transects The site originally classified as polluted, ‘Catembe’, did have an extremely high abundance of plastic debris on a macro scale (fig. 14), with more than 30 pieces of recorded debris per meter of beach, plastic pellets excluded. This is likely due to the high level of human activity, as the beach is located close to the ferry between Catembe and Maputo. Though there are regulations regarding littering, with visible signs on the beach (see front page image), there are few or no garbage bins available which makes it less likely for people to abide by these rules. A few restaurants and/or hotels do clean the beach in front of their establishments, but these parts of the beach and their immediate proximity were not used when conducting the beach surveys in order to avoid bias. The attitude towards littering in the studied parts of Mozambique seemed to be a general indifference, the only aspect considered being the aesthetic (pers. obs.). The amount of macrodebris found at ‘Marine Station’ was higher than expected, as this is considered a pristine area and is located relatively far from any village. All sites experience large tidal changes, with the mean spring tidal range being approximately 3.0 meters and the mean neap tidal range being 1.0 meters (Canhanga & Dias, 2005). It is therefore reasonable to argue that much of the debris on the beaches can originate from other parts of Mozambique or even other countries, being transported with currents and deposited on the beaches with the tide. The Agulhas current breaks off into Maputo Bay when going south along Mozambique and forms and eddy, creating a northward current along Inhaca’s west coast (Armitage et al., 2006). This makes deposition of transported debris a possibility. It also accounts for the difference between ‘Marine Station’ and ‘Saco’, since ‘Marine Station’ is a coastal area directly in contact with this current while ‘Saco’ is a bay with a relatively narrow mouth pointing towards the east (fig. 2, p. 11) and so more secluded from ocean currents. It could be speculated that ‘Catembe’ is more of a source for marine debris while ‘Marine Station’ is more of a sink, however more research would be needed in order to draw any conclusions about this. The fact that ‘Saco’ did not entirely fit the OSPAR criteria for a beach survey area should also be taken into account when interpreting the results, as only one beach area was available and this was located right next to a frequently used sand road. However, it still gives an indication of the pollution levels and since sediment samples from this site can still be used, the beach survey results were included in this report to allow for comparison.

4.4 Sediment samples In the results from the sediment samples, none of the sites differed significantly from each other. This is surprising when related to the results of the beach transects, ‘Catembe’ having far larger amounts of plastic debris than the other two sites. Since

Fig. 14: Beach debris at Catembe (left) and Inhaca (right)

plastic items on the beach are subjected to UV-radiation as well as mechanical stress, fragmentation is likely and much of the microplastics in the ocean in thought to have been yielded on beaches (Andrady, 2011). This indicates that ‘Catembe’ should in theory have higher levels of microplastics in the sediment than the other sites, unlike the results from this study. No significant difference was found between S1 and S2, but the S1 samples from ‘Marine Station’ did contain a slightiy higher level of microparticles than the S2 samples, which is another indication that the microplastics on Inhaca could potentially be originating from the ocean. In a previous study, Browne et al. (2011) found similar levels of microplastics (21-30 particles per sample) in Pemba, further north in Mozambique. However, the mentioned study used a different unit of measurement (a sample size of 250 ml) while this study had a sample size of 20 g (w.w.), restricting the possibility for comparison. The same is true for other previous studies (reviewed in Cole et al., 2011, Nor & Obbard, 2014). A standardized unit of measurement for quantifying microplastics in sediment samples is yet absent, but creating one would greatly help the potential for comparing different studies. When investigating the results more closely, the majority of microplastics found were smaller than 500 μm. This differs from the results by many previous studies where the majority was often between 1 and 5 mm (Hidalgo-Ruz et al., 2012). Particles of this size could potentially be airborne, which could be the reason for the high levels of contamination in the control samples. Should contamination by airborne particles be the case, however, it might still be an indication of the presence of microplastics at Inhaca, since all laboratory stages were performed on the island. The alternative of contamination originating from clothing or equipment used was minimised by using protective clothing (lab coat and gloves), having as many tools as possible being constructed from glass or other non-plastic materials and thorough cleaning and rinsing prior to use. Still, improvements could be made in the density separation step. First off, the salt used was regular cooking salt (NaCl) bought in a grocery store, meaning it had not been extensively purified or cleared of foreign particles. This is a possible source of contaminants, as well as the plastic containers used for storing distilled water, plastic graduated cylinders the density separation took place in and plastic gloves worn. However, even if only a portion of the microplastics found in the sample filters originate from the beach, it is reason enough to assume that microplastics are generally present in beach sediment. Moreover, high levels of airborne plastic particles would indicate a direct risk for uptake by humans by breathing, having unknown effects. Considering the fact that plastic bags are used for tinder to light fires on Inhaca and that garbage is disposed of in a large pile being lit on fire regularly (pers. obs.), there is a high risk of airborne particles being present on the island. These are, however, speculations that would require an additional study. The recovery could also be improved in some ways. Using NaI instead of NaCl gives the supersaturated water a higher density and so allows heavier particles to float, and elutriation lowers the risk of particles being trapped in the sediment (Claessens et al. 2013). Better ways of removing the supernatant could also be devised, again, with the access of additional equipment.

4.3 Biological samples The overall portion of animals containing microplastic debris (32 %) was higher or equal to previous studies investigating the plastic ingestion of fish (Boerger et al., 2010; Choy & Drazen, 2013; Davidson & Asch, 2011). Previous studies, however, had greater sample sizes and much higher levels of debris per studied fish. These studies were all

performed in the Central North Pacific, where the levels of plastic debris are high (Cozar et al., 2014), and investigated pelagic fish. One study (Foekema et al., 2013) was performed in the North Sea and found plastic in only 2.6 % of the investigated fishes. When interpreting the plastic contents of the individuals from different sites, not only the location but also the feeding patterns and the ecology of different species have to be taken into account. The highest number of particles was found in the specimens from the two sites on Inhaca Island, and both of these species are predators. S. sihama is an omnivore which is primarily planktonivorous (Taghavi Motlagh et al., 2012) but a lot of remains from ingested crustaceans, mainly shrimps, were found in their GI-tract together with polychaetes and nematodes. They did not contain a lot of sediment, which suggests that they feed in the water column, away from the sea floor. Quite a few of the GI-tracts of S. serrata were found to be empty or close to empty, which suggests that the individuals may have defecated their stomach contents due to stress after being captured, as measurements were taken for a different study prior to euthanization. S. serrata normally feeds on benthic macro-invertebrates, primarily molluscs (Hill, 1976). This is consistent with the stomach contents found, as they were mainly made up of shells or shell pieces from gastropods or bivalves. L. dumerilii differs from both these species by having a diet consisting mainly of detritus and microorganisms (Blay, 1995), which was evident by the stomach contents together with large amounts of sediment. It may be argued that more microplastics should be present in a sediment feeder than in a predator/omnivore due to the fact that particles often end up on the sea floor (Barnes et al., 2009), and the ‘Catembe’ fish that did contain microplastics had a slightly higher average per sample than the others. However, they still had a lower total of particles. The presence of sediment in the GI-tract could also make microplastics more difficult to find visually, even if the sample has gone through a digestion process, since it can also be largely inorganic. Furthermore, fouling by algae and microorganisms can lead planktonivorous animals to be attracted by floating particles and unknowingly ingest them, making these animals just as vulnerable to plastic debris as deposit feeders. In future studies, capturing the same species in different locations would allow for better comparison between sites, since the differences in diet and lifestyle would not be an issue. Using species within different trophic levels for the same site, and the same species for a compared site, would be the ultimate way of investigation. This was unfortunately not possible for this study, as the author could not control which species were caught by the fishermen. The levels of microplastics present in the GI-tracts of the sampled individuals were generally quite low. No significant differences were found between sites, although it is still possible that small particles may have been overlooked when examining the GI- tracts. Optimizing the method by performing the originally planned digestion protocol would lessen this risk, but it is a complicated procedure that requires specific equipment and chemicals. A simplification of this method would be extremely advantageous for future studies in a similar field setting. It is likely that the main reason that this attempt was unsuccessful could be the lack of a freeze dryer, which allows the biological material to soak up the digesting chemicals without diluting them with their own fluids. In addition, freeze drying the samples allows for pulverisation and so a larger surface:volume ratio for the chemicals to be absorbed. Possibly, using a filter with a larger mesh size could decrease the risk of clogging when a freeze dryer is unavailable, but this would also mean larger particles would have to be targeted as smaller ones would pass through the filter. If possible, sieving in steps using smaller and smaller mesh sizes could be advantageous as this would not only increase the recovery of

particles but it would also automatically divide the particles into size classes. In lack of these alternatives, however, which might be the case if the researcher is located in a remote area, the best option is the one performed in this study, namely visual inspection in a dissecting scope. Though a trend is visible suggesting that larger individuals contain higher amounts of debris (fig. 13, p. 17), the low sample size makes it hard to draw any conclusions. This is nonetheless interesting, and would be valuable to investigate in a different study.

4.4 Performing the study on Inhaca Island As there are many possible sources for contamination and bias even in a laboratory setting, performing this type of study in the field yields a great number of factors that need to be taken into account when interpreting the data. First off, since the methods used were less than optimal, it is difficult to with any certainty quantify the microplastic present. The accessibility of different types of equipment could increase recovery in both biotic and sediment samples. Secondly, avoiding contamination in the samples is crucial for getting reliable results, and this, too, is problematic in a field setting. It is, however, possible to draw conclusions even from numbers that are not absolute. Microplastic particles were found to be present in all (100 %) of the sediment samples collected, 40 % of the biological samples from the ‘pristine’ site, and 15 % of the biological samples from the ‘polluted’ site. Even if the number of particles in each sample cannot be guaranteed to be exact, the purpose of the study can still be reached when performed under field conditions, as the presence itself of microplastics in Maputo Bay can be mapped.

5. Conclusion Despite the heavy pollution of plastic macrodebris in Catembe, no significant difference in microplastic abundance was found between the three sites. Neither the sediment samples collected nor the GI-tracts of animals caught in the different locations differed significantly. Although the presence of microplastic debris in Maputo Bay is difficult to quantify in absolute numbers due to contamination and less than optimal materials, it is possible to consider the aspect of presence vs. non-presence, in which case microplastics were found in 100 % of sediment samples and 32 % of biological samples from all sites. This allows for the conclusion that pollution of plastic microdebris is a fact in Maputo Bay, with yet unknown impacts on the ecosystems.

Acknowledgements I would like to thank Sida and Joachim Sturve for the financial support, Bethanie Carney Almroth for supervising, Abdul Adá for his invaluable help in the field, Daniela De Abreu for giving advice, Adriano Macia for helping with logistics and equipment, and Kristoffer Stedt and Sandra Toivio for assistance with beach surveys and lots of moral support.

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Appendix Key to beach debris (fig. 3, p. 12). (Wenneker & Oosterbaan, 2010) Code Plastic & polystyrene 1 4/6-pack yokes 2 bags (e.g. shopping) 3 small plasticbags, e.g. freezer bags 112 plastic bag ends 4 drinks (bottles, containers and drums) 5 cleaner (bottles, containers and drums) 6 food containers incl. fast food containers 7 cosmetics (bottles & containers e.g. sun lotion, shampoo, shower gel, deodorant) 8 engine oil containers and drums < 50 cm 9 engine oil containers and drums > 50 cm 10 jerry cans (square plastic containers with handle) 11 injection gun containers 12 other bottles, containers and drums 13 crates 14 car parts 15 caps/lids 16 cigarette lighters 17 pens 18 combs/hair brushes 19 crisp/sweet packets and lolly sticks 20 toys & party poppers 21 cups 22 cutlery/trays/straws 23 fertiliser/animal feed bags 24 mesh vegetable bags 25 gloves (typical washing up gloves) 113 gloves (industrial/professional gloves) 114 lobster and fish tags 26 crab/lobster pots 27 octupus pots 28 oyster nets or mussel bags including plastic stoppers 29 oyster trays (round from oyster cultures) 30 plastic sheeting from mussel culture (tahitians) 31 rope (diameter more than 1 cm) 32 string and cord (diameter less than 1 cm) 115 nets and pieces of nets < 50 cm 116 nets and pieces of net > 50 cm 33 tangles nets/rope/cord and string 34 fish boxes 35 fishing line (angling) 36 light sticks (tubes with fluid) 37 floats/buoys

38 buckets 39 strapping bands 40 industrial packaging, plastic sheeting 41 fibre glass 42 hard hats 43 shotgun cartridges 44 shoes/sandals 45 foam sponge 117 plastic/polystyrene pieces 0-2.5 cm 46 plastic/polystyrene pieces 2.5-50 cm 47 plastic/polystyrene pieces > 50 cm 48 other plastic/polystyrene pieces