The role of algae (Isochrysis galbana) enrichment on the bioaccumulation of benzo[a]pyrene and its effects on the blue mussel Mytilus edulis

Abstract

The role of algal concentration in the transfer of organic contaminants in a food chain has been studied using the ubiquitous model polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) as the contaminant, Isochrysis galbana as the phytoplankton food source, and the common mussel (Mytilus edulis) as the primary consumer. The effect of algal concentration on BaP uptake by M. edulis was determined by feeding M. edulis daily with I. galbana which had previously been kept in the presence of BaP for 24 h. Four combinations of concentrations of algae and BaP were used to give final exposure concentrations of 30,000 or 150,000 algal cells ml⁻¹ in combination with either 2 or 50 μg BaP l⁻¹. BaP concentrations were determined fluorometrically in rest tissues (excluding digestive glands) and digestive gland microsomal fractions of M. edulis after 1, 7 and 15 days exposure, and also in isolated algae. Potentially toxic effects of BaP on M. edulis were examined in terms of blood cell lysosomal membrane damage (neutral red dye retention assay) and induction of digestive gland microsomal mixed-function oxygenase (MFO) parameters [BaP hydroxylase (BPH) and NADPH-cytochrome c (P450) reductase activities]. BaP bioaccumulation in rest tissues (and to a lesser extent in digestive gland microsomes) of M. edulis increased with both increasing BaP and algal exposure concentrations, and over time, producing maximal bioconcentration factors in rest tissues after 15 days exposure to 150,000 algal cells ml⁻¹ and 50 μg BaP l⁻¹ of 250,000. The five-fold higher concentration of algae increased BaP bioaccumulation by a factor of approximately 2 for 50 μg BaP l⁻¹ at day 15. Blood cell neutral red dye retention time decreased linearly with increasing log₁₀ tissue BaP body burden, indicating an increased biological impact on M. edulis with increasing BaP exposure possibly due to a direct effect of BaP on blood cell lysosomal membrane integrity. An increase was seen in NADPH-cytochrome c reductase activity, and indicated in BPH activity, with 1 but not 7 or 15 days exposure to BaP, indicating a transient response of the digestive gland microsomal MFO system to BaP exposure.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) contaminate the aquatic environment and consequently those aquatic organisms consumed by man. Many PAHs are well-known mutagens and carcinogens. The US Environmental Protection Agency has recognised 16 PAHs as priority contaminants, and eight are included as possible carcinogens (Menzie et al., 1992). Benzo[a]pyrene (BaP) is a potentially toxic contaminant and a powerful carcinogen.

Mussels are suspension-feeders and filter food from the water column. All have a pumping and filtering mechanism capable of bioaccumulating lipophilic contaminants including PAHs. During recent decades, much research has focused on contaminant levels in mussels. The “Mussel Watch” monitoring programme is one approach to assessing environmental contamination (Farrington et al., 1983, Tavares et al., 1988, Livingstone, 1991, Livingstone, 1996, O'Connor, 1996, Livingstone and Goldfarb, 1998). In a comparative study of oyster, clam and mussel, Tenore et al. (1973) found mussels to have the highest feeding rate of the three bivalve mollusc species investigated (mussel Mytilus edulis L., American oyster Crassostrea virginica (Gmelin) and the hard clam Mercenaria mercenaria) The bioaccumulation mechanisms of BaP and other carcinogenic PAHs in marine organisms is of interest to aquaculture, and in the utilisation of these marine resources for human food, thus making it essential to understand the fate of such organic contaminants within these organisms (Moore et al., 1986).

Unicellular photosynthetic algae have recently been implicated in the metabolism and degradation of organic foreign compounds (xenobiotics) (Kumar et al., 1988, Peterson et al., 1994). Thus, phytoplankton, like other aquatic microorganisms (Quensen et al., 1998), may be important in reducing the exposure of higher organisms to contaminants via processes of adsorption, bioaccumulation and/or metabolism to non-toxic and/or water-soluble forms. Conversely, as a food-source, phytoplankton may facilitate uptake of water-insoluble contaminants into organisms, so increasing the possibility of toxicity. Quantitative knowledge of such processes is thus critical in determining the fate of contaminants in ecosystems. The effect of algal concentration on the accumulation of contaminants is especially important in eutrophic coastal waters where frequent algal blooms are observed. Relatively few studies have been conducted concerning the effects of algal concentration on the rate of accumulation of organic contaminants, such as oil (Widdows et al., 1982) and polychlorobiphenyls (PCBs) (Björk and Gilek, 1996, Björk and Gilek, 1997, Gilek et al., 1996) by mussels.

The main objective of this study was to examine whether the uptake of organic contaminants by a filter-feeding bivalve may be influenced by algal concentration, and therefore that algal concentration may play a role in contaminant transfer and bioaccumulation in food chains. The process was studied using the ubiquitous model PAH BaP as the contaminant, Isochrysis galbana as the phytoplankton food source, and the common mussel Mytilus edulis as the primary consumer. Toxicity of the BaP on Mytilus edulis were examined in terms of blood cell lysosomal membrane integrity and induction of the digestive gland microsomal mixed-function oxygenase (MFO) system. Damage to blood cell lysosomal membranes in Mytilus sp. (as measured by the neutral red dye retention assay) has been demonstrated for mixed-contaminants in the field (Lowe et al., 1995a) and the three-ring PAH fluoranthene in the laboratory (Lowe et al., 1995b), but nothing is known of the effects of the higher molecular weight five-ring BaP. Mytilus sp. are known to have an apparently inducible digestive gland MFO system with exposure to BaP and/or other related cytochrome P4501A (CYP1A)-type inducers (Livingstone, 1996, Livingstone and Goldfarb, 1998, Peters and Livingstone, 1999), including increases in microsomal NADPH-cytochrome c (P450) reductase (Livingstone, 1988, Michel et al., 1993) and BaP hydroxylase (BPH) (Michel et al., 1993, Livingstone et al., 1997, Solé et al., 1998) activities. Growing I. galbana was incubated with BaP for 24 h and the mixture then dosed daily into tanks containing Mytlius edulis to give different exposure concentrations of algae and BaP. Mytilus edulis were sampled at days 1, 7 and 15, and the tissue concentrations of BaP, blood cell lysosomal membrane stability and digestive gland NADPH-cytochrome c reductase and BPH activities determined. A separate study was also carried out on the effect of algal concentration on uptake and/or association of BaP by I. galbana. The results are discussed in relation to the effects of algal presence on processes of contaminant uptake, bioconcentration and toxicity.