Temporal and spatial variation in heavy metal concentrations in the bivalve Donax deltoides from the Ninety Mile Beach, Victoria, Australia

Volume 3 0 / N u m b e r 6 / J u n e 1995 Marine Pollution Bulletin, Vol. 30, No. 6, pp. 419-424, 1995

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Temporal and Spatial Variation in Heavy Metal Concentrations in the Bivalve Donax deltoides from the Ninety Mile Beach, Victoria, Australia DAVID HAYNES*, JOHN LEEDER~ and PHILIP RAYMENT:~ *Gippsland Water, P O. Box 348, Traralgon 3844, Australia t Amdel Laboratories, 508 City Road, South Melbourne 3205, Australia ~Monash University, Gippsland, Switchback Road, Churchill 3842, Australia The accumulation of metals from the water column by bivalve molluscs has been shown to be relatively rapid and to reflect ambient exposure levels closely (Phillips, 1990). As a consequence, the use of bivalves as biomonitors of heavy metal pollution has been well established (Phillips, 1980). The mussel Mytilus edulis has been used to measure metal concentrations at sites with hard substrates along the southern Australian coast (Harris et al., 1979; Smith et al., 1981; Ritz et al., 1982; Talbot & Chegwidden, 1982) and several tropical subtidal bivalves from hard substrates have been investigated as potential indicators of heavy metal pollution along the north-eastern Australian coastline

(Klumpp & Burdon-Jones, 1982). The use of bivalves from soft substrates as biomonitors in Australian waters has received less attention. Subtidal species including the mud oyster Ostrea angasi (Talbot & Chegwidden, 1982), the pearl oyster Pinctada carchariarium (McConchie & Lawrance, 1991) and the razor clam Pinna bicolor (Ward et al., 1986; McConchie & Lawrance, 1991) have been assessed as potential indicator species, but no work has been published to date on the biomonitoring potential of bivalves inhabiting Australian sandy intertidal shores. As approximately half of the Australian coastline consists of sandy beaches (Fairweather, 1990), the potential use of such bivalves as indicators of pollutant concentrations along this type of coastline is large. Temporal and spatial variations are described here for heavy metal concentrations in the sandy intertidal bivalve Donax deltoides collected from the Ninety Mile Beach, Victoria, Australia. This Donacid bivalve species is found along the south-eastern coast of Australia (Macpherson & Gabriel, 1962) and although the autecology of the species is not well known, the genus has been extensively studied (Ansell, 1983). Donax deltoides were collected from a 2 km stretch of the lower intertidal zone from each of two sites (Seaspray and Paradise Beaches) along the Ninety Mile Beach in November 1991, April and November 1992, and April 1993 (Fig. 1). These sites are free of any obvious anthropogenic impacts. Approximately 45 bivalves with a shell length of between 3 and 4 cm were collected from each beach on each sampling occasion. The collected bivalves were stored frozen in acidwashed plastic containers until their analysis. After thawing, the shellfish collected from each beach were

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Fig. 1 Bivalve sampling locations, Ninety Mile Beach.

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Marine Pollution Bulletin

divided at random into three replicates per beach for each sampling occasion. Shell lengths were measured to the nearest millimetre and the flesh and any liquid present in the mantle cavity of each animal shucked from the shell and composited into a plastic Whirl-pac using an acid-washed plastic knife. Total wet tissue weight and total shell volume were recorded for each replicate processed. The three composited tissue replicates from each beach for each survey were freeze-dried and the total dry tissue weight of each replicate was recorded. Tissue dry weights and shell cavity volumes were used to calculate an average body condition index (Widdows & Johnson, 1988) for each replicate sample: Average body condition index (BCI) = 1000 (average tissue dry weight)/ (average shell cavity volume). This index was used to quantitatively rank bivalve condition over time and space. The freeze-dried tissue was ground to a fine powder using a pestle and mortar and 2 g of homogenized tissue weighed into a 250 ml Erlenmeyer flask. Samples were digested with 15 ml of aqua regia (1 part nitric:3 parts hydrochloric acid) and three drops of concentrated sulphuric acid at room temperature for 2 h. Digested samples were heated slowly to 90°C; the sample volume reduced to 5 ml; and then cooled, before adding 2 ml of hydrogen peroxide. After reheating and cooling, the final solution was filtered through an acid-washed cellulose fibre filter and diluted to a volume of 10 ml. Inductively Coupled Plasma Atomic Emission Spectrometric Analysis (ICP-AES) was used to analyse for arsenic, cadmium, copper, molybdenum, nickel, lead, selenium and zinc. Mercury was analysed using a LDC Long Pathlength UV analyser. Tissue nickel and selenium concentrations were not assessed in samples collected in November 1991. All analyses were carried out in duplicate, and the average of the two results are reported here. All glassware used in metal analysis was pre-cleaned with Pyroneg detergent, thoroughly rinsed and soaked in 10% nitric acid for at least 1 week, before rinsing four times with deionized water prior to use. A Certified Standard Reference Material (National Bureau of Standards SRM 1566a, certified oyster tissue) and reagent blanks were analysed concurrently with bivalve tissues to validate the methods used. The

ICP-AES results compared favourably with results obtained by electrothermal atomic absorption spectrometry. All recoveries of metals from the standard reference material were within the 95% confidence interval quoted for the reference material (Table 1). Statistical analyses were carried out using the SYSTAT V5.03 statistical package (Wilkinson, 1990). Data were checked for normality and logm transformed where necessary, prior to analysis. Pearson correlation coefficients and the Bonferroni adjusted probability associated with each correlation coefficient were calculated for bivalve tissue metal concentrations and a two-way analysis of variance (ANOVA) was used to check for differences in tissue metal concentration over time and between sampling sites, using the body condition index as a covariate. Significant effects were located using a Tukey HSD multiple comparison test with an experimentwise type 1 error probability of 0.05. The variations in body condition index and tissue metal concentration over time and between sites in D. deltoides collected from the Ninety Mile Beach are illustrated in Figs 2-4. A summary of the results of twoway ANOVAs comparing mean body condition indices and tissue metal concentrations across sampling sites and times is presented in Table 2. The average condition index was significantly higher in samples collected in April compared with November for the Seaspray Beach site. In contrast, the average condition index of bivalves collected from the Paradise Beach site in April 1992 was not significantly higher than in 180 r~

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Fig. 2 Donax deltoides condition indices (n = 3: error bars - 1 SEM).

TABLE ! Average recoveries (gg g-J dry wt) of metals in the standard reference material.

Metal Arsenic Cadmium Copper Mercury Molybdenum Nickel Selenium Zinc

NBS standard reference material 1566a (mean +_SD)

November 1991

April 1992

November 1992

April 1993

14 _+ 1.2 4.15 _+0.38 66.3 -+ 4.3 0.064 _+0.0067 NC 2,25 _+0.44 2,21 +_0.24 830 +_ 57

9.44 2.67 47.1 NA < 0.5 2.00 2.19 795

12.00 3.0 50.0 NA < 0.5 1.9 2.71 620

9.55 2.69 47.6 0.(/724 < 0.3 1.44 2.64 629

9.44 2.67 47.1 (l.0724 < (1.3 1.72 2,19 620

NC, Not certified; NA, not assessed.

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Volume 30/Number 6 / J u n e 1995 TABLE 2 ANOVA F values and significance levels for analyses of bivalve body condition indices and tissue metal concentrations.

Variate BCI

As Cdf Cu$ Hg$

Mo$

Site 15.32** 0.01 5.86* 28.83*** 0.77 87.96***

Ni$

0.07

Se$

3.72 0.08

Zn$

Interaction (site X time)

Time 14.95"** 16.33*** 104.78"** 156.08*** 2.34 109.50"** 22.81"** 1.47 12.63***

6.19** 1.49 59.95*** 11.49*** 0.61 68.67*** 1.57 9.46** 1.13

Covariate (BCI) (/.53 0.16 0.01 0.13 21.59'** 3.12 0.64 0.63

•0.1 < p < 0.05. •"0.001

SData logu~ transformed prior to analysis.

samples collected at this site in the two November surveys (Fig. 2). The average tissue cadmium concentration was higher in samples collected at the Paradise Beach site in April (Fig. 3a). However, multiple results below the detection limit of 0.1 ~g g-~ dry wt make it difficult to draw firm conclusions about temporal and spatial variation in tissue concentrations

of this metal. The average tissue concentrations of molybdenum, nickel and zinc were significantly higher in individuals collected in November (Figs 3b-d). Differences in tissue molybdenum concentrations also existed between sampling sites on one sampling occasion (November 1991). Average mercury concentrations were also higher in November samples than those from April, but not significantly so (Table 2, Fig. 4a). Arsenic, copper and selenium concentrations did not display consistent temporal periodicity, although significant differences in arsenic and copper concentrations existed between sampling times and sites (Figs 4b-d). Inter-element, body condition and tissue weight correlations are presented in Table 3. Tissue weight was negatively correlated with all metal concentrations, although only significantly so with those of arsenic following a Bonferroni adjustment. Following a Bonferroni adjustment, the condition index only correlated significantly with zinc concentrations. After a Bonferroni adjustment, inter-element correlations of significance included zinc with mercury, nickel and selenium; selenium with copper and mercury; and nickel with copper (Table 3). Few data are available on background concentrations of heavy metals in the bivalve genus Donax. Data that are available are often problematic, in that the metal concentrations reported are from pooled samples of C, r

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deltoides cadmium, molybdenum, nickel and zinc concentrations. All concentrations expressed as ~tg g-~ dry wt. (November 1991 and November 1992 Cd results set at half detection limit, n = 3: error bars --1 SEM.)

Fig'.3 Donax

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Fig. 4 Donax deltoides mdrcury, arsenic, copper and selenium concentrations (n = 3; error bars - 1 SEM).

TABLE 3 Person correlation coefficient matrix, including Bonferroni adjusted probabilities for tissue metal concentrations.

Tissue dry wt BCI As Cd Cu

Hg Mo

Ni Se Zn

Tissue dry wt

BCI

As

1,000 0,748** --0,669* -0,054 --0,051 --0.050 -0.091 --0.381 -0.298 --0.495

1.000 --0.311 0.224 0.310 --0.204 --0.333 --0.553 --0.465 --0.678*

1.000 0.086 0.415 0.003 0.133 0.100 0.214 0.292

Cd

1.000 0.165 --0.309 -0.182 --0.283 --0.207 --0.397

Cu

1.000 -0,406 --0,307 --0.776** -0.714"* --0.478

Hg

Mo

Ni

Se

Zn

1.000 0.599 0.332 0.470* 0.618"*

1.000 0.434 0.666 0.736

1.000 0.895 0.727**

1.000 0.775**

1.000

*0.01 < p < 0.05. **0.001 < p <0.01.

shellfish with a wide range of shell sizes, and hence ages and exposure periods (e.g. Hornung & Oren, 19801981; Mauri & Orlando, 1983; Watling & Watling, 1983). Shellfish used in the analyses were also collected over an extended and/or varied sampling period (e.g. Hornung & Oren, 1980-1981; Watling & Watling, 1983). However, limited comparisons may still be made between the average metal concentrations in D. deltoides collected from the Ninety Mile Beach and those of other species of Donax sampled elsewhere (Table 4). The present study indicates that tissue metal 422

concentrations in D. deltoides collected from the Ninety Mile Beach are comparable to, or lower than, metal concentrations present in bivalves of this genus taken elsewhere. No data are available for comparison of arsenic, molybdenum or" selenium concentrations present in this genus. Selenium was found in elevated concentrations in D. deltoides compared to levels found in other Australian molluscs (Maher et al., 1992) and to tissue concentrations in bivalves collected elsewhere (Johns et al., 1988). By contrast, molybdenum and arsenic were present in concentrations similar to those

Volume 30/Number 6/June 1995 TABLE 4

Comparison of average metal concentrations in Donax deltoides from the Ninety Mile Beach, Victoria, with Donax spp. from elsewhere. All concentrations are shown as Ixg g-~ dry wt except where specified.

Date Length (ram) n Arsenic Cadmium Copper Mercury Molybdenum Nickel Selenium Zinc Reference

Donax trunculus

Donax trunculus

Donax serra

Donax trunculus

Donax deltoides

Donax deltoides

Mediterranean (industrialized)

Mediterranean (unpolluted)

South Africa (unpolluted)

Atlantic (unpolluted)

Bass Strait (unpolluted)

Bass Strait (unpolluted)

Nov. 1979Sept. 1978 30-40

Feb. and June

Aug. 1978Aug. 1979

Feb. 1987

Nov. 1991 and Nov. 1992 30-40 261 17.6 < 0.18 6.4 0.08

April 1992 and April 1993 30-40 223 15.8 < 0.27 7.2 0.05

3.22 1.39 6.58 127 Present study

1.4 0.92 7.03 76 Present study

0.34 15.9 0.33 (wet wt)

Commercial size

Small 0.22 5.1 0.45

10

0.6 11.8

2.04 65 Hornung & Oren (1980-1981)

63 Mauri & Orlando (1983)

69 105 Watling & Watling Romeo & Gnassia(1983) Barelli (1988)

recorded for other mollusc species living in unpolluted waters (Moore & Ramamoorthy, 1984; GESAMP, 1988). Similarities between sampling sites in the tissue concentrations of arsenic, mercury, molybdenum, nickel and zinc on any given sampling occasion suggest that D. deltoides has some potential as a biomonitor of these metals. However, inter-site variability in selenium and copper concentrations may limit the use of this species to monitor ambient concentrations of these metals. The spring seasonal maxima in mercury, molybdenum, nickel and zinc concentrations in D. deltoides is consistent with the explanation that seasonal variation in metal concentrations of bivalves follows a sinusoidal curve, with enhanced levels being present in winter. In general, dry tissue mass in D o n a x spp. peaks in the summer months (Ansell et al., 1980) and corresponds with the maturation of gonads and an increased tissue lipid content. As a consequence, metal concentrations may be lowered during the summer, as the growth period dilutes trace metals by increasing biomass (Fabris et al., 1978; Borchardt et al., 1988). Arsenic, copper and selenium concentrations in D. deltoides did not display any consistent temporal periodicity over the survey period. This is in contrast to the seasonal variation found to occur in the mussel Mytilus edulis in concentrations of copper (Fabris et al., 1978; Amiard et al., 1986) and arsenic (La Touche & Mix, 1982). Bivalves collected from the Seaspray Beach site tended to display higher body condition indices, which exhibited temporal periodicity. Elevated biomass in the white sand mussel Donax serra collected from sites along the eastern coast of South Africa has been associated with the discharge of organic matter from adjacent rivers (McLachlan, 1977). The Seaspray Beach sampling site is adjacent to the mouth of Merrymans Creek, which drains cleared land used for livestock grazing in southern Gippsland. The creek discharge ranges in nutrient status from mesotrophic to

eutrophic (Victorian Environment Protection Authority, unpublished data). It is postulated that elevated condition indices in D. deltoides collected from the Seaspray Beach site reflect locally elevated phytoplankton densities in the water column, as a consequence of the nutrient-rich river discharge.

The staff of Marine Science & Ecology assisted in collection of bivalve samples for analysis. Kerrie Lee and Peta Woolan (Gippsland Water) assisted in bivalve collection and tissue preparation. Tissue samples were freeze-dried by Mary Cole (Monash University). Steve Shinners (Gippsland Water) is thanked for critically reviewing the draft manuscript. Trevor Blake (Victorian EPA) kindly made unpublished nutrient data available.

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