A First Comparison of Microplastics Abundance between Two Whale Shark Feeding Areas of the Gulf of California, Mexico.

Abundance and typology of microplastics (MPs) were measured in 12 water samples collected from two whale sharks feeding areas of the Gulf of California (Bahía de los Angeles (BLA) and Bahia de La Paz (LAP)). The concentration of MPs was 0.47 MPs/ m 3 in BLA and 0.63 MPs/ m 3 in LAP. Overall, lms were the most abundant type of MPs (41%; n = 74) followed by line (37%; n = 68), fragments (20%; n = 36) and pellet (2%; n = 3), with signicative differences between the types of microplastics. Theoretical ingestion rate per hour of MPs for the species was determined to be 288.58 and 386.82 in BLA and LAP, respectively. In faecal samples collected directly from animals of LAP (n = 3), 67% of the MPs were lms, 25% were fragments, and 8% were lines. Results from this study indicate that MPs are common in the feeding areas of this endangered species and that whale sharks are likely ingesting MPs from ltering the surface water.

) is the largest sh of the planet (Compagno 2001); it is a lter feeder with long life span and a circumglobal distribution (Stevens 2007; Rowat and Brooks 2012). In the Gulf of California, Mexico, feeding aggregations of whale shark are seasonally spotted in Bahía de los Angeles (Nelson and Eckert 2007) and Bahía de La Paz (Ketchum et al., 2013), where animals are seen feeding on dense patched of copepods, quetognats, and eufausids (Whitehead et al. 2019). Internationally, whale sharks are listed as "Endangered" by the International Union for the Conservation of Nature red list and in Mexico this species is protected under two national laws (DOF 2006(DOF , 2010. The presence of whale shark feeding aggregations in Bahia de los Angeles and Bahia de La Paz over the last two decades has risen to intense tourist activities and now represents an important economic source for the local community. In these areas, the increasing tourist pressure has forced local authorities to enforce management plans to guarantee the safety and conservation of the animals (SEMARNAT 2017). Despite these measures, recent studies have showed that whale sharks from Bahía de La Paz are exposed to toxic substances such as heavy metals (Pancaldi et al. 2019 a,b), persistent organic pollutants and plastic pollution (Fossi et al. 2017) as a result of continuous feeding activity on the sea surface near costal urban areas. The aim of this study is to provide the rst analysis of the abundance and type of MPs present in the surface waters in two whale sharks feeding areas of the Gulf of California (Bahía de los Angeles (BLA) and Bahia de La Paz (LAP)) during the period of maximum presence of the sharks in these areas. Analysis of MPs was also performed in faecal samples collected directly from animals in LAP.
Tows of water were performed in September 2016 in "La Mona" area, south of Bahía de los Angeles (BLA; 28.9519° N, 113.5624° W; Fig. 1) and from October to December 2016 in the "Mogote" area, south of Bahía de La Paz (LAP; 24.0832° N, 110.1839° W; Fig. 1). A manta net with a mesh of 333μm, 43.5 cm long and 14 cm wide was used to perform water tows. Tows were performed during the presence of foraging whale sharks, with wind speed between 2.5 and 7 knots. Whale shark presence was assessed visually and the estimation of total length (TL) of the individuals was established using an object (e.g., swimmer, boat) for comparison. Tows were trailed for 10 minutes at 2 knots, in a lineal path, parallel to the sharks' movement. Once the net was recovered, the material was placed in a glass baking dish and subsequently stored in an additional glass storage jar. To avoid any possible contamination from plastic, the cap of the jar was covered with an aluminium foil before closing, then frozen at -20°C for ahead of microscope analysis. The total volume of analysed water in each area was calculated by multiplying the length of the examined surface (distance between GPS start point and GPS end point of the trawling) and the area of the manta net mouth (which is calculated as the long side multiplied the short side) expressed in m 3 . To know the concentration of MPs per m 3 found in each area, the total number of MPs was divided by the total volume of water analysed.
Faecal samples were collected in November 2016 in LAP (Mogote area; Fig. 1). Feces, identi ed as compact brownish to reddish material, were collected from a swimmer during opportunistic eld observations after being defecated by the shark. The samples were collected in crystal jars and initially stored on ice packs, then transferred to long-term storage at -20°C before analyses. Samples were contained less than a minute after being defecated by the shark, nevertheless, environmental contamination from microplastics may have occurred when they were collected from the water.
In addition, the whale shark feeding aggregation season in Bahía de La Paz coincides with the period of strong north wind that could potentially accelerates the in ux of plastic debris from the urban area to the adjacent foraging area of the sharks.
Considering both study areas, lm was the MPs with the highest incidence followed by line, fragments, and pellet. Films were also the main MPs shape found in the whale shark feces collected in LAP, which con rms the high presence of these pollutants and the risks associated to them. Pellet was the MPs with a signi cantly lower incidence than the other types (P < 0.001). Pellets are primary MPs (Barnes et al. 2009) used mainly in cosmetic, pharmaceutical and textile industry (Gregory 1996;Fendall and Sewell 2009).
Considering that neither La Paz nor Bahia de los Angeles are industrial cities, it is probable why pellets showed such low incidence compared to the other MPs shapes. On the contrary, lm, fragment and line are secondary MPs (Barnes et al. 2009) derived from the breakup of daily use products (cloths, plastic bags, bottles, shing gears) and, in our study areas they are likely to ended up on local beaches and/or washed by currents into the whale shark feeding areas. Daily theoretical ingestion rate for whale sharks from this study was higher ( In conclusion, the presence of microplastics in whale shark feeding areas represents a threat to whale sharks which uptake these items by continuously lter feeding on the water surface. Local policies that regulate the use of plastic items should be enforced especially in areas adjacent to feeding grounds of protected megafauna. Further research should be carried out to evaluate the risks related to the ingestion of microplastics in this endangered species. Map of the study area: Bahía de los Angeles (BLA) and Bahía de La Paz (LAP), Mexico. Grey boxes indicate sampling areas. Map designed with Surfer Plot program. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Images of microplastics types: fragment (a), line (b), pellet (c) and lm (d) found in water samples from the whale shark feeding area in La Paz bay.

Figure 3
Percentage of fragment, line, pellet, and lm found in water samples collected in Bahía de los Angeles, Bahía de La Paz and in total.