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depth-dependent toxicity and bioaccumulation of cadmium in marine benthic protist communities
Cadmium toxicity Bioaccumulation Protist communities Density Number of species Biomass Depth levels
Gregorio Fernandez-Leborans1' Regina Gabilondo1 Soledad Ruiz-Alvarez2
1 Department of Zoology, Faculty of Biology, Pnta 9, Complutense University,
Madrid - 28040, Spain;
e-mail: firstname.lastname@example.org * corresponding author
2 Department of Cellular Biology and Ecology,
Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela - 15740, A Coruna, Spain
Received 16 May 2007, revised 17 July 2007, accepted 7 September 2007.
The toxicity and bioaccumulation of cadmium in a marine benthic protist community were examined at different depths within the sediment. For this purpose, sediment-water microcosms with 1000 ^gCd dm~3 of the pollutant were used in two assays. The addition of cadmium caused a significant reduction in protist density, number of species and biomass. There was also a decrease in these three parameters with depth. During the treatment the density of protist groups was strongly depth-dependent. The dominant groups of protists at the different
depths during the assay were also considered. The most dominant protist group in terms of density were the heterotrophic flagellates, both in the control and in the treatment with cadmium. In the treatments with cadmium, these were followed by ciliates and by dinoflagellates in both assays. In the control, all protist groups were present during the assay, whereas in the treatments with cadmium, autotrophic flagellates, diatoms and sarcodines were found in reduced proportion or not at all. Cadmium bioaccumulation increased towards the end of the assay. At any time during the assay, the proportion of cadmium bioaccumulated was an increasing function of depth.
Some natural components of marine sediments could be regarded as pollutants at concentrations high enough to cause adverse biological effects. These are: nutrients, including phosphorus, and nitrogen compounds such as ammonia; metals, including iron, manganese, lead, cadmium, zinc and mercury; and metalloids such as arsenic and selenium. These contaminants do not ordinarily biomagnify in marine food webs, but at extremely high concentrations they are able to travel up the food chain. All have been linked to health problems in humans. Cadmium is a relatively rare element that acts as a minor nutrient for plants at low concentrations (Price & Morel 1990, Lee et al. 1995, Lane & Morel 2000), but is toxic to aquatic life at only slightly higher concentrations. Typical maximum cadmium concentrations in bays and estuaries range from 0.3 to 862 ppm (Forstner & Wittmann 1983), although the typical Cd concentration range for relatively uncontaminated marine sediments lies between 0.1 to 0.6 ppm (Warren 1981). Cadmium can enter the environment from various anthropogenic sources: by-products of zinc refining, coal combustion, mine wastes, electroplating processes, iron and steel production, as well as the pigment, metal, plastics, battery, fertiliser and pesticide industries (Hutton 1983, Clark, 1992, EPA 2001, Rubinelli et al. 2002). The impact of cadmium on aquatic organisms depends on its occurrence among different chemical forms (Callahan et al. 1979), which can have different toxicities and bioconcentration factors. In saltwaters with salinities from about 10 to 35gkg_1, cadmium chloride complexes predominate. Because of chloride complexation, the adsorption of cadmium to particles decreases with increasing salinity (Li et al. 1984). In both fresh and saltwaters, particulate matter and dissolved organic material may bind a substantial portion of the cadmium, and under these conditions cadmium may not be bioavailable as a result of this binding (Callahan et al. 1979, Kramer et al. 1997). In fact, up to about 80%, but usually less, of the cadmium in coastal seawater is complexed with dissolved or colloidal organic matter (Muller 1996, 1998).
Protozoa have been used to analyse cadmium toxicity in the marine environment because of their physiological and ecological characteristics: they are eukaryotic; their biology is well-known; the physiology and biochemistry of several species has been studied extensively; they are easily maintained in the laboratory and in large numbers; life-cycles and generation times are short; they are able to accumulate high levels of metals in their organelles; they are conveniently handled in the laboratory (Nilsson 1979, Fisher et al. 1983, 1995, Dive & Persoone 1984, Fernandez-Leborans & Novillo 1995). Several authors have described the effects of certain heavy metals, cadmium among them (Fernandez-Leborans & Novillo 1994), using species such as Tetrahymena pyriformis and Acanthamoeba castellani (Krawczynska et al. 1989), or Uronema marinum (Coppelloti 1994). In addition to the biochemical and physiological effects of cadmium on protozoans, its toxicity has also been determined at the community level (Fernandez-Leborans & Olalla-Herrero 1999). In sandy bottom coastal areas, heavy metals are subject to enrichment processes, and the concentration of cadmium in several areas decreases from the surface to deeper levels (Erlenkeuser et al. 1974). A number of studies have focused on the mobilisation of heavy metals from marine sediments. Microorganisms, algae, annelids, molluscs, echinoderms and other organisms have been implicated in cadmium removal (Forstner & Wittmann 1983, EPA 2001, Perez-Rama et al. 2002). However, there are no studies on the behaviour of protist communities at different depths of the sediment after the addition of a toxic metal such as cadmium. For this reason, the effects of Cd on these communities with respect to their density, biomass and species diversity were analysed in the present study. One key finding was that protist communities responded differently, depending on the depth within the sediment at which they were found.
2. Material and methods
Sample collection. In October 2002 and February 2004 samples were obtained from Da Seras beach (42°41'N, 9°02'W) (Coruna, Galicia, Spain), a beach open to the Atlantic Ocean, close to the southern entrance to the Muros estuary. Near-shore sediment samples were taken with a drag at a depth of 1 m. Samples were carried to the laboratory in 5 dm3 polyurethane containers with the least possible disturbance of the sediment, adding seawater over the upper surface of the sediment. The sediment profile was preserved as far as possible during sample collection and transport.
Microcosms. In the laboratory, the samples were stored in 20 dm3 microcosms (80% of sandy sediment and 20% of seawater). Enrichment was
with sterilised wheat grains (4 grains per dm3). After an acclimatisation period of 7-9 days, the benthic community of protists exhibited a high density.
Bioassay. After the acclimatisation period, cadmium was added as cadmium acetate from a stock solution of 1 g dm-3.Two sets of experiments were carried out in the presence and absence of cadmium:
(1) control without cadmium;
(2) nominal concentration of 1000 fgCd dm-3 at time 0.
Three replicate samples were taken from the control as well as from the cadmium treatment.
Every 24 h the following variables were measured:
- density, number of species and biomass of protists;
- physicochemical parameters: temperature (°C), pH, oxidation-reduction potential (mV), salinity (PSU), conductivity (mS cm-1), and mean grain size.
These variables were measured at the following depths: 0, 4, 8 and 12 cm from the surface of the sediment.
A high concentration of cadmium was added in order to ensure rapid diffusion to the deeper layers of the sediment.
Extraction of subsamples. The subsamples (5 cm3) from the different depths of the sediment were obtained using a sucking pipette with a closing device at the end introduced within the sediment. Subsamples were obtained so as to minimise sediment disturbance.
Protist count. This was carried out with a Lackey Drop Microtransect (APHA 1989), using 100 fil subsamples. Dilutions were used for high densities of microorganisms. A minimum of three subsamples per depth were examined.
Abundance. This was expressed as the number of cells per cm3.Amini-mum of three subsamples per depth in each microcosm were examined.
Classification of protists. This was done by light microscopy (X100, X1000) in bright field and in phase contrast. Additionally, an Image Analysis System (KS300 Zeiss) was used to support identification. Various fixation and staining techniques were used:
a) Silver impregnation techniques for the observation of ciliate morphology: protargol and silver carbonate techniques (Truffau 1967, Fernandez-Galiano 1976).
b) Lugol, acridine orange, cresyl blue and Noland's technique for flagellate identification.
c) Methyl green (Gabe 1968).
Biomass. This was obtained from the biovolume. Cell dimensions were measured using a micrometer eye-piece and the biovolume was obtained from the most appropriate formula for its geometrical shape. Volumes were converted into biomass using only one conversion factor: 0.15 pgC fim-3. A number of conversion factors have been proposed for different protist groups, but this factor represents a well-documented standard value (Bal-
dock et al. 1983, Baldock & Sleigh 1988, Montagnes et al. 1988, Butler
& Rogerson 1996). The biomass is expressed in mg m-3 of dry weight.
Functional groups. The protists were classified into the following groups to represent the diversity of marine protist communities: heterotrophic flagellates (HFLA), autotrophic flagellates (AFLA), dinoflagellates (DIN), diatoms (DIAT), ciliates (CIL) and sarcodines (SARC).
Physicochemical parameter analysis. The pH and oxidation-reduction potential were measured using a pH meter (Crison 507) with a calomel electrode. The temperature, conductivity and salinity were measured with a conductivity meter (WTW LF196). All chemical products used were of a high degree of purity (proanalysis). A vibrating screen was used for the granulometric analysis of the sand, which was passed through a series of sieves (dry sieving), according to the Wentworth scale (Giere 1993). The mean grain size was calculated from the phi value (Krumbein 1939). A minimum of three subsamples per depth in each microcosm were examined.
Cadmium bioaccumulation. Pore water samples free ofsediment (achieved with the help of a stereoscopic microscope and a pipette) were filtered (1.2 pore diameter) to harvest the protists. Filters were used that had been treated with nitric acid in a Millestone digestor. The cadmium bioaccumulated on the protists was measured using atomic absorption spectrophotometry (Perkin Elmer 4100 ZL with automatic sampler-injector AS70) coupled to a graphite furnace corrected for the Zeeman effect at 229 nm, using palladium as matrix modifier.
Statistical analysis of data. This was performed using the Statgraphics, SPSS and Multicua (Cuadras et al. 1991) programs.
Physicochemical variables. In general, there were few significant differences between the control and microcosms with cadmium in either year (Tables 1 and 2). In October 2002, there were significant differences with
Table 1. Physico-chemical parameters during the 2002 assay
Mean ± standard deviation (above, treatments with cadmium; below, control)
20.5 ± 02
-31.2 ± 05
39.8 ± 05
54.1 ± 05
7.5 ± 05
16.7 ± 02
-27.4 ± 05
40.1 ± 05
54.2 ± 05
7.5 ± 01
15.6 ± 05
2.6 ± 02
33.2 ± 05
45.9 ± 01
6.5 ± 02
16.8 ± 04
81.6 ± 05
40.8 ± 05
55.0 ± 02
5.6 ± 01
16.0 ± 05
5.8 ± 05
32.6 ± 01
45.2 ± 01
7.0 ± 05
16.8 ± 00
36.9 ± 05
40.0 ± 05
54.0 ± 05
6.5 ± 06
15.5 ± 00
14.9 ± 01
32.6 ± 05
45.3 ± 01
6.8 ± 03
15.0 ± 04
32.0 ± 05
39.8 ± 05
54.0 ± 05
6.5 ± 01
16.9 ± 04
55.5 ± 00
33.0 ± 05
45.5 ± 05
5.7 ± 05
16.4 ± 04
43.5 ± 00
38.9 ± 01
52.7 ± 05
6.3 ± 05
16.4 ± 05
-27.4 ± 05
35.2 ± 05
44.8 ± 00
7.6 ± 03
16.2 ± 02
-21.2 ± 01
26.8 ± 05
43.2 ± 01
7.4 ± 02
16.0 ± 05
03.1 ± 02
36.7 ± 05
50.0 ± 05
6.5 ± 03
16.4 ± 00