Advances in Marine Biology (eBook)

3.2.3.1 Studies on growth inhibition with previously unexposed larvae

The first studies of bivalve larval growth in the presence of pollutants were conducted by Davis and Chanley (1955b), who investigated the effects of various antibiotics, and by Davis (1961), who demonstrated the toxicity of 32 pesticides on larvae of Crassostrea virginica and Mercenaria mercenaria. These experiments were concerned first with embryogenesis and the formation of D-larvae, and then with the subsequent development of D-larvae obtained in the absence of contaminants during a further 12 days of contaminant exposure. Growth and mortality were determined only on the twelfth day. Further laboratory studies were conducted at Milford by Hidu (1965) on the effects of synthetic surfactants on M. mercenaria,Calabrese and Davis (1966,1967) studied the effects of pH on C. virginica and M. mercenaria and of linear alkylate sulphonate (LAS), a surfactant, on C. virginica-,Calabrese (1972), Calabrese and Davis (1967), Calabrese and Nelson (1974) and Calabrese et al. (1977a) then investigated the effects of heavy metals and various other pollutants such as detergents and pesticides. Finally, Calabrese and Rhodes (1974) applied the same techniques in studies with embryos and larvae of the coot clam, Mulinia lateralis, demonstrating the advantages of using this species in bioassays. The results obtained at Milford laboratory were summarized by Calabrese et al. (1977b, 1982).

In studies of the effects of contaminants on larval growth using previously unexposed (healthy) larvae, the embryos were raised in an uncontaminated mass culture, collected on a sieve 48 hours after fertilization, counted and redistributed into the experimental beakers at concentrations of 5000 to 15 000 per litre. Incubation generally lasted about 10 days, except in the case of M. lateralis, which attains settlement competence after 6 to 8 days (cf. Calabrese, 1970b). The incubation water was changed every 2 days, always maintaining the appropriate contaminant concentration. To this end, the larvae were first collected on a sieve and sampled to determine mortality, possible abnormalities and shell size. Larval size was measured by ocular micrometer to a precision of ± 5 μm (cf. Calabrese, 1970b).

Measurements can also be made by photographing samples of larvae and later measuring the diameter of the shell under a binocular microscope, either on the photographs or on the negatives (e.g. His and Robert, 1981, 1982). This is usually done with a video imaging system (e.g. Widdows et al., 1989; McFadzen, 1992). Larval weight is rarely measured (Widdows et al., 1989; Beiras and His, 1994, 1995a) despite the fact that it is required to express lethal concentrations on a mass-specific basis, enabling comparison of data among larvae of different sizes (see Section 4.2.3).

In the ecotoxicological studies with Mytilus edulis and Pecten maximus by Beaumont et al. (1987), and those with Mercenaria mercenaria by Byrne and Calder (1977), larval feeding was based on the methods reviewed by Loosanoff and Davis (1963) (see Section 2.3.5). Renzoni (1975) studied the effects of the water soluble fraction of three petroleum products on growth in Mulinia lateralis during a period of 2 weeks, changing the water (and renewing the pollutant concentration) every 2 days and feeding the same algae as used by Calabrese (1970b). Laughlin et al. (1989) in studies of the effects of TBT on Mercenaria mercenaria fed the larvae exclusively with Isochrysis galbana (Tahiti strain). Finally, Gormly et al. (1996) studied the resistance of Mulinia lateralis to microbial pest control agents during 48 hours in microwells of 3 ml (15 to 20 larvae per well); the larvae were fed during these brief experiments on mortality and growth of the larvae.

Some authors obtain their larvae from commercial hatcheries and this may pose various problems. Such larvae are generally the progeny of a haphazard mix of parents, and they may also represent a mix of incubations of different ages which have ail been passed through the same sieves to obtain a homogeneous size; i.e., they may be the resuit of the culling procedure employed by commercial hatcheries to rid themselves of slow growers. Watling (1978, p. 126, and 1982) mentions that in the hatchery-reared larvae used “the range of individual sizes was restricted at the beginning of the experiments”, which may pose a problem in studies of larval growth.

3.2.3.2 Studies on growth inhibition with D-larvae already exposed to contaminants

Studies in which fertilization and embryogenesis were conducted in the presence of the contaminant being bioassayed are rare, even though they represent the best approximation of in situ conditions.

Granmö (1972) first distributed eggs of Mytilus edulis at a density of 10 per ml into vials with various concentrations of LAS (a surfactant) before proceeding to fertilize them. The water was subsequently changed daily during the first 8 days, the larvae being retained on a mesh of 60 μm at each change of water. Fertilization success was assessed after 20 to 24 hours, and larval mortality and size (“measured microscopically, the greatest length attained being recorded”, p. 356) were subsequently determined after 72 hours, 96 hours and 240 hours. Brereton et al. (1973) studied the effects of several concentrations of zinc sulphate on freshly fertilized eggs of Crassostrea gigas (15 000 per beaker of 1 litre) at a temperature of 20 °C to 22 °C and a salinity of 29 psu. Larval abnormalities were counted after 48 hours, and the D-larvae were subsequently passed through a 100 μm sieve to remove impurities, retained on a 45 μm sieve and incubated at a density of 5000 per litre at 26 °C. They were grown for 5 days in the presence of the contaminant and another 5 days in unpolluted seawater, and fed daily with Isochrysis galbana, Chaetoceros calcitrans, Monochrysis lutheri and Cyclotella nana at 100 cells per μl of larval culture volume. The water was changed every 48 hours, and at this time swimming behaviour was recorded, and mortality and growth were assessed. Shell size (“width across the valve”) was determined in 25 individuals of each of four replicates of each concentration. Laughlin et al. (1988) studied the toxicity of TBT to Mercenaria mercenaria; the eggs were exposed to the contaminant 4 hours after fertilization at densities of 150 per bowl of 50 ml, and the incubation lasted a total of 14 days. The solutions were renewed daily except during the first 48 hours, and the larvae were fed with Isochrysis galbana (Tahiti strain) at 40 000 cells per ml. Size was determined daily in sub-samples of 25 larvae per incubation.

The first studies of larval growth undertaken at Arcachon followed the methodology of Brereton et al. (1973) and Helm and Millican (1977). They were initially applied to investigate the effects of TBT on Crassostrea gigas and Mytilus galloprovincialis, showing the threat posed by this compound to coastal shellfish culture areas (His and Robert, 1980; Robert and His, 1981). These experiments were carried out with larvae that had been reared through embryogenesis in the presence of the toxicant, as well as with larvae reared to the D-larva stage in unpolluted water. The eggs were distributed into 2 litre beakers at a density of 60 per ml; oyster embryos were incubated for 24 hours at 20 °C and mussel embryos for 48 hours at 19 °C. After this period the contents of each beaker were passed through a 100 μm sieve to remove impurities, and the larvae were collected on a sieve with 40 μm mesh. After assessment of larval abnormalities the larvae were re-suspended in 250 ml graduated cylinders for recounting and incubation then continued at densities of eight individuals per ml for 7 days (only 5 days in the case of TBT, owing to the high toxicity of the concentrations tested). The water (0.2 μm filtered seawater) was changed daily and the larvae were fed daily as well. Mortality was assessed in each incubation, and larval size was determined with an ocular micrometer in sub-samples of 50 individuals.

Studies of growth were subsequently extended to 10 or 12 days, and the investigations with larvae formed in unpolluted water were aban- doned because of their lesser sensitivity. The feeding regimes of Helm and Millican (1977) were used at ail times. The water was changed only every second day, and mortality and growth were therefore equally assessed in intervais of 2 days; shell size was determined by photo-graphing sub-samples of larvae, rather than directly under the microscope. After studying the effects of TBT on larvae of Crassostrea gigas at concentrations around 1 μg per litre, further studies were conducted on the effects of copper sulphate, cadmium...