ICES CM 2013/A:05
Microplastics: effects on oyster physiology at adult and larval stages
Rossana Sussarellu, Philippe Soudant, Christophe Lambert, Caroline Fabioux, Charlotte Corporeau,
Charlotte Laot, Nelly Le Goïc, Virgile Quillien, Jean-Yves Daniel, Pierre Boudry, Marc Long, Christian
Mingant, Bruno Petton, Thomas Maes, Dick Vethaak, Johan Robbens & Arnaud Huvet.
(1) Ifremer, Centre de Bretagne, LEMAR UMR 6539 UBO-CNRS-IRD-IFREMER, Plouzané France ; (2) LEMAR
UMR 6539 UBO-CNRS-IRD-IFREMER, Institut Européen de la Mer, Plouzané, France ; (3) CEFAS, Centre for
Environment, Fisheries & Aquaculture Science, Lowestoft Laboratory, Lowestoft, Suffolk, UK; (4) Deltares, Delft,
Netherlands; (5) ILVO, Oostende, Belgium. [email protected], Phone: +33 (0)2 98 22 46 93
Summary
Plastics are persistent synthetic materials, which can accumulate in the marine environment, although the
consequences of macroplastic debris for wildlife are well documented, the impacts of microplastics (MP)
on marine life are still largely unknown. In this study a chronic exposure of adult Pacific oysters
(Crassostrea gigas) to MP, during two months, was performed under controlled conditions using a mix of
yellow-green fluorescent polystyrene MP (2 and 6 µm). The distribution and accumulation of the MP in
oyster hemolymph and tissues was monitored using histology and flow cytometry. Appropriate
experimental conditions were designed to induce gametogenesis and production of mature gametes in
order to test the effects of MP on oyster reproduction, gamete quality and subsequent larval development.
Physiological perturbations in adults were assessed by measuring ecophysiological behavior, growth,
defense mechanisms and reproductive allocation. These measurements would help defining toxic
endpoints and to guide future studies on the effects of MP on Pacific oyster physiology.
Introduction
Plastics are persistent materials, which tend to accumulate in the marine environment and can affect
marine life as they remain there for years. Plastic materials degrade to smaller macro- and microplastic
particles, or can enter the environment as small fragments (e.g. scrubbers). MP can cause particular
problems when ingested by different organisms. MP also contain additives such as UV-stabilizers,
colorings, flame retardants, plasticizers, and can accumulate persistent pollutants which are indeed
susceptible for uptake and accumulation by living organisms (Teuten et al., 2007). Filter feeders organisms
are likely to be impacted by MP pollution as they filter large volumes of water and can ingest little
particles while feeding. Occurrence and effects of MP filtration are already been studied in several filter
feeding species as in mussels (Browne et al., 2008; von Moos et al., 2012), in sea cucumbers (Graham and
Thompson, 2009), lungworms (Besseling et al., 2012). These studies mainly evidenced reduction of the
feeding activity, inflammatory response and translocation of MP in the circulatory system. Longer term
effects on growth, survival or reproduction are still unknown. As part of the MICRO EU-Interreg project
(MicroPlastics – Is it a threat for the 2 seas Area?), biological effects of MP were assessed through an
integrative approach on the Pacific oyster C. gigas at adult and larval stages. A two months exposure of
adult oysters to MP, was performed under controlled conditions while inducing gametogenesis in order
to evaluate effects on physiology, quality of gamete production and subsequent larval development.
Materials and Methods
A chronic exposure of adult oysters (18 months) to MP during two months (March-May 2013) was
performed under controlled conditions using a mix of yellow-green fluorescent polystyrene MP (2 and 6
µm) at a concentration of 2000 MP/mL (1800 of 2 µm, 200 of 6 µm). Triplicates of 50 L tanks (40 oysters per
tank) were employed for MP-exposed and control oysters. Seawater was preliminary filtered (1 µm) and
UV-treated, average temperature was 17°C. Flow-through seawater systems (30% seawater renewal/h)
provided continually MP and algae (1:1 Isochrysis galbana clone Tahitian (T-Iso) and Chaetocerosgracilis at a
ratio of dry mass of algae to oyster of 8%). Filter activity was assessed by measuring twice a day algae
concentration by a Coulter Counter system. MP concentration in the seawater was measured by flow
cytometry once a day. Histology was performed in order to detect MP in various tissues of oysters
(digestive tracts, gills, palps) and determine the maturity stage of gonads. MP presence in hemolymph
was assesed by flow cytometry. Sperm and oocytes were collected by stripping of the gonads with
seawater and counted (Huvet et al., 2002). Fecundation was performed using 9 females per condition,
each of them in triplicate, and a pool of control sperm with a ratio of 300 spermatozoids per oocyte. The
D-larval yield (at 48 h) was used to estimate the fertilization success (number of D-larvae total number of
embryos−1). Larval rearing was performed in flow-through seawater system (50% seawater renewal/h,
Rico-Villa et al., 2008, temperature 25°C) providing the same algae (concentrations according to Rico-Villa
et al., 2009) and MP concentration than in adult experiment. Six 5 L tanks were run with larvae issued
from exposed and non-exposed parents at a density of 30 larvae/ml. Three tanks for each parental
conditioning were exposed to MP. Larvae were sampled each 2-3 days in order to measure their length by
image analysis (WinImager 2.0 and Imaq Vision Builder 6.0 software for images capture and treatment,
respectively). Each experiment ended when ≥ 50% of the whole population as eyed larvae.
Results and Discussion
Initial results show a significant increase in food intake in adult oysters exposed to MP (+4%, p=0,009). The
MP concentrations in seawater suggest that oyster filtered 20% of 2 µm MP, 85% of 6 µm MP. MP were
found in in the digestive tract, feces and hemolymph (data to be confirmed). The gametes produced under
MP exposure led to significantly reduced fertilization rates compared with controls (Control: 49.8±16.7%,
MP: 29.6±14.9%, p<0.0000). Larval rearing was slower for larvae issued from MP-exposed parents.
Preliminary results suggest that MP can affect reproduction capacities of C. gigas, further analysis are
needed in order to understand the perturbation mechanisms.
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