See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/278814507 Assessing cockle shells (Anadara granosa) for reconstruction subdaily environmental parameters: Implication for paleoclimate studies Article in Historical Biology · June 2015 DOI: 10.1080/08912963.2015.1052806 CITATIONS READS 3 574 2 authors: MReza Mirzaei Tan shau hwai Iranian Fisheries Science Research Institue Universiti Sains Malaysia 20 PUBLICATIONS 48 CITATIONS 136 PUBLICATIONS 1,277 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Sea hare and its life cycle View project Substitution of herbal drugs instead of chemical agents in aquaculture View project All content following this page was uploaded by MReza Mirzaei on 21 June 2015. The user has requested enhancement of the downloaded file. SEE PROFILE This article was downloaded by: [5.112.239.86] On: 19 June 2015, At: 14:41 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Historical Biology: An International Journal of Paleobiology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ghbi20 Assessing cockle shells (Anadara granosa) for reconstruction subdaily environmental parameters: Implication for paleoclimate studies a a M.-Reza Mirzaei & Aileen Tan Shau Hwai a Marine Sciences Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Published online: 19 Jun 2015. 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Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions Historical Biology, 2015 http://dx.doi.org/10.1080/08912963.2015.1052806 Assessing cockle shells (Anadara granosa) for reconstruction subdaily environmental parameters: Implication for paleoclimate studies M.-Reza Mirzaei* and Aileen Tan Shau Hwai Marine Sciences Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia (Received 20 April 2015; accepted 15 May 2015) This study aims to investigate the potential of cockle shells as an environmental recorder, examining the environmental factors controlling the shell growth of the intertidal Anadara granosa from west coast of Malaysia. Subdaily environmental factors were recorded from December 2011 to November 2012. A total of 600 individuals were collected on a monthly basis and the shells sectioned from umbo to ventral margin, polished, etched and photographed under a light microscope to observe microgrowth bands and increments. Comparison of correlation matrix between mean increment width and each environmental factor indicated that shell growth had the highest positive correlation with seawater temperature (þ 0.72) and weak positive correlation with salinity (þ0.53). Multiple regression analysis was used to assess independent associations between shell mean increment width and environmental parameters. Study model showed that 60.8% of the variation in shell growth could be explained by temperature, salinity, rainfall and tidal change. Individually, temperature and salinity made the greatest unique contribution to explain shell growth, respectively ( p , 0.01). Laboratory results showed shell growth was in a linear trend to optimum temperature and salinity. These findings provide a basis for the interpretation of the temporal changes in shell microgrowth patterns in terms of environmental conditions of cockle shells. Keywords: growth pattern; shell microgrowth increments; intertidal; temperature; salinity; shell cross section Historical Biology 1. Introduction The reconstruction of environmental factors and life history characteristics in invertebrates that produce accretionary skeletons is a valuable exploration in biological studies. Bivalves are widely distributed in different geographical latitudes and their shells are able to record environmental factors with high resolution. Recently, many paleoecological studies have investigated the effects of environmental factors on shell growth with high accuracy using the shell growth bands of molluscs (Brock & Bourget 1989; Richardson 1989; Goodwin et al. 2001; Schöne et al. 2006). The cockle, Anadara granosa is a widespread species with the highest distribution throughout South East Asia, especially in the strait Malacca between the west coast of Malaysia and Indonesia (Mirzaei et al. 2014a). Anadara granosa is a suitable species to study the relationship between shell growth and environmental parameters based on microgrowth bands and increment widths (Richardson 1987). Growth rate depending on these types of periodic bands is generally associated with regional and local environmental conditions. Therefore, mollusc shells can be a remarkable archive of past climatic changes (Gibson et al. 2001; Schöne et al. 2003). Bivalve growth can be affected by several environmental parameters such as seawater temperature, salinity and food availability (Yaroslavtseva & Sergeeva 2006; *Corresponding author. Email: mirzaei.mr@gmail.com q 2015 Taylor & Francis Matozzo & Marin 2011). Sato (1999) that showed shell microgrowth bands were affected by periodic changes in food availability, while recent researchers have found that food availability is dependant on tidal change (Simpson & Sharples 2012) and emersion period (Cerrato et al. 1991). Furthermore, geographical and temporal variations are one of the main factors which effect shell growth at different latitudes due to different temperatures (Wanink & Zwarts 1993; Mirzaei et al. 2014b). Variations in minerals and the structure of growth bands are thus potential recorders of different environmental factors, such as salinity, light and dark periods, water current, rainfall, dissolved oxygen, pH and suspended sediments (Marchitto Jr et al. 2000; Epplé et al. 2006). Most studies on molluscs used external shell layer have focused on morphological examination (Selin 2000), pigments (Kozminsky & Lezin 2007) and chemical structure (Gillikin et al. 2005; Mubiana & Blust 2007). Only a few studies have used the shell cross sections as proxies for environmental factors (Richardson et al. 1980). The major problem with this method was the effects of environmental factors such as seawater temperature, tidal change and salinity on the shell growth at the same time. As a result, there was an overlap between all environmental factors affecting shell growth. However, examinations in the current study were conducted on microgrowth bands and increments of the cockle shells which were produced during tidal changes. This 2 M.-R. Mirzaei and A.T. Shau Hwai Historical Biology Figure 1. Location of study site, Penang Island, west coast of Malaysia. investigation was based on marked and recovered samples. In order to determine the effects of environmental parameters on the shell growth of Anadara granosa, shell increment widths were compared with environmental parameters in Penang Island, Malaysia. The multivariate technique was used to interpret the data and determine the relationship between shell growth and different independent variables. A standard multiple regression was used to show how much the variance in the dependent variable (increment width) could be explained by independent variables (environmental parameters). This method also demonstrated how much unique variance in the dependent variable could be explained by each of the independent variables. Therefore, the main objectives of this study were (1) to investigate the relationship between shell Figure 2. Cutting direction from Umbo to ventral margin along the maximum growth line. microgrowth patterns and daily environmental variables in Anadara granosa cockles and (2) to determine which environmental variable was the most prominent among the factors affecting the shell growth of Anadara granosa from Penang Island, Malaysia. 2. Material and methods 2.1 Site preparation and sample collection A total of 600 similar age (< 4 months) and size (< 10 mm) Anadara granosa were stained with shell dye (Alizarin Red) at a concentration of 30 ppm before being transferred to the study site. The staining technique was designed to date-mark one or more bands in the shell structure so that subsequent growth could be related to the staining point. Cockles were placed in a plastic mesh cage (1.5 m £ 1.5 m £ 2 m) located at an intertidal site (58300 05.5000 N 1008110 35.3200 E), where it could easily be accessed by boat from Balik Pulau in Penang Island, Malaysia (Figure 1). A total of 40 individuals of Anadara granosa were collected monthly (40 individuals £ 12 months) between December 2011 and November 2012. In the laboratory, soft tissues were gently removed from inside the shell. A single valve of each specimen was rinsed and numbered for shell cross section preparation. Furthermore, a total of 25 samples of Anadara granosa were collected fortnightly between mid-December 2011 and the end of January 2012 (4 sample collections £ 25 individuals £ 1 site). Historical Biology Historical Biology 2.2 Shell cross section preparation Radial cross sections were prepared from one valve of any specimen based on standard methods by Neves and Moyer (1988). The valve of each specimen was marked by a pencil from the umbo to the ventral margin along the maximum growth band at a point from the shell posterior (Figure 2). Marked valves were embedded in an epoxy resin (a combination of epoxy resin to epoxy hardener with the proportion of 2:1) for 24 h to protect the valves against cutting and grinding procedures. Valve resin mouldings were then fixed on the low speed saw with a diamond-impregnated blade (Buehler Ltd, Lake Bluff, IL, USA). A low speed made it possible to cut fragile materials that would otherwise fracture. The valves were cut through the pencil mark from the umbo to shell ventral margin. Shell sections were then grounded by sequential grit sandpapers (240, 400, 600, 800 and 1200 Buehler carborundum grits) to remove epoxy resin from the cut surfaces of the valves. The finer grits (800 and 1200) were used to reduce scratches from the coarser grit papers. T-cut valve surfaces were polished with Aluminum oxide powder on a semi automatic polishing machine (FORCIMAT – FORCIPOL 300-1V). The specimen was then removed, rinsed in tap water and dried. Etching was conducted so as to leave the aragonite granules on the surface of the valve section that distort the section image. As a result, the polished surfaces of the valves were etched by immersing the specimens in a dilute solution of hydrochloric acid (0.1% HCl) for 1 min. 2.3 Growth patterns periodicity The experiment was designed to examine whether tidal change, emersion and daily rhythms had any effect on the shell banding formation in Anadara granosa. The total number of growth bands was counted in each shell between the staining point and the shell margin. A sample t test was used to test whether the banding was daily, tidal or produced by emersion. The mean difference between pairs of expected and observed values was used to determine whether the values differed significantly from zero. 2.4 Increment width measurement The etched surface of each section was observed with light microscope-stereo (Olympus SZ61 – Olympus Optical Co. Ltd, Tokyo, Japan) at 100 £ magnification and photographed (Xcam Alpha-The imaging source GmbH). Based on Alizarin red staining on the shell, in each sample collections the microgrowth increments were successively measured from the shell margin toward staining point using microscopic image analysis software (Analysis Image Processing Version 5.1 – @Olympus soft imaging solutions 1989 –2008). 3 2.5 Environmental data Daily seawater temperature was measured using HOBO Pendant Temp/Light logger fixed to the cage in the study area. The logger was calibrated to record hourly seawater temperatures during the experiment. Seawater salinity was measured daily with a hand-held refractometer (RHSN10ATC BUILT) at the study site. Rainfall measurements were recorded to the nearest 0.1 millimetre with the daily measurements taken by Penang Island Regional Meteorological Office. Monthly tide tables of the sampling site were obtained from Tide Tables Malaysia (2011–2012) published by the National Hydrographica Centre, Royal Malaysian Navy. 2.6 Laboratory experiment A total of 160 individuals of Anadara granosa were stained using Alizarin red combined in seawater for 24 h (Riascos et al. 2007). Factorial design (A 4 £ 3) was conducted to compare the effects of four different temperatures (25, 27, 30 and 328C) and three salinity treatments (26, 28 and 31 ppt) on the shell increment growth. Treatment levels fall within the natural range of temperature and salinity in the distributional range of the cockles in study site. The system consists of 50-l tanks including optional water controller to produce simple tidal fluctuations. Water was emptied every 11 h and cockles were exposed for 1 h and then replacing with water of appropriate salinity. Throughout the 30-day experimental period, the cockles were fed daily with marine microalgae. At the end of experiment, cockles were collected from each tank, labelled and shell sections were prepared from each group. The shell increment widths were measured from the staining point to the last increment in shell margin. 2.7 Statistical analysis The SPSS (Statistical programme for social scientist) software (IBM SPSS statistics version 20) was used to analyse data from the shell increment and environmental factors. The effects of environmental parameters on the shell growth were investigated in detail using regression analysis on the mean increment widths as a dependent variable and environmental factors such as seawater temperature, salinity, rainfall and tidal change as independent variables. This method was based on correlation, but allowed some advanced statistical analysis to find the interrelationship between independent variables. The two factorial ANOVA was used to analyse significant differences between treatment levels in laboratory experiment. 3. Results 3.1 Microgrowth pattern periodicity The mean total number of microgrowth bands deposited in the shell layer was almost the same as the total number of 4 M.-R. Mirzaei and A.T. Shau Hwai Figure 3. Shell microgrowth pattern formed from 6 to 31 December 2011 for the individual (shell length: 13 mm, age: 5 month) of Anadara granosa collected from Pinang Island, Malaysia (modified from Mirzaei et al. 2014b). Historical Biology tidal emersions (Figure 3). In the entire semi-monthly periods from December 2011 to January 2012, there was no significant different at p . 0.01 between the observed microgrowth lines and number of tidal emersion for shells in the intertidal area (Table 1). 3.2 Shell increment width measurement The mean shell increment widths showed frequent high fluctuations between 32.20 ^ 2.21 and 65.08 ^ 3.01 mm from December 2011 to March 2012. The mean shell increment widths remained relatively stable with very little change from 53.23 ^ 2.05 to 59.16 ^ 3.57 mm from April 2012 to early July 2012. It decreased slightly from 59.16 ^ 3.57 mm (July 2012) and remained at the lowest level, which was from 25.31 ^ 2.86 to 55.09 ^ 2.18 mm until late September 2012. Mean shell increment widths became thicker when they achieved a width of around 58.76 ^ 3.21 to 61.06 ^ 2.13 mm over the last two months of the study duration (Figure 4). 3.3 Environmental factors Daily variations in the seawater temperature ranged from a maximum of 338C to a minimum of 228C during the study period. Figure 5 shows the frequent high fluctuations of seawater temperature during December 2011 and January 2012. Seawater temperature was almost constant, with slight variations from 28 to 338C during late January 2012 to late June 2012. It slowly declined from early July 2012 (28.58C) and remained at its lowest point, which varied from 22 to 278C. Subsequently, seawater temperature increased slightly to 308C in late September 2012. During the last two months, the temperature remained relatively stable from 28 to 338C. Seawater salinity was almost constant in the range from salinity of 29 to 31 during the one-year study period. Three frequent high fluctuations of seawater salinity were observed in December 2011, from April 2012 to late May 2012 and from late September 2012 to late November 2012. Seawater salinity dropped sharply and reached to the lowest point (11 ppt) on 12 December 2011. The second Table 1. Observed and expected numbers of growth bands in the shells of Anadara granosa kept in intertidal area from December 2011 to the end of January 2012. Excepted no. of bands assuming the following periodicity Sampling date No. of shells examined 15 Dec 2011 30 Dec 2011 14 Jan 2012 31Jan 2012 25 25 25 25 Observed mean total no. of bands (^SD) Daily Tidal Emersion 8.2 ^ 1.81 37.53 ^ 2.18 51.90 ^ 3.04 76.33 ^ 3.06 15 31 45 62 29 60 87 119 9 39 54 78 p Value Daily Tidal þ p , 0.01 p , 0.01þ p , 0.01þ p , 0.01þ Emersion þ p , 0.01 p , 0.01þ p , 0.01þ p , 0.01þ Note: *No significant difference from an emersion periodicity p . 0.01; þsignificant difference from tidal and daily periodicity p , 0.01. 0.0824* 0.0163* 0.0230* 0.0267* Historical Biology Historical Biology Figure 4. Mean shell increment widths of Anadara granosa from Penang Island, Malaysia (December 2011– November 2012). 5 6 M.-R. Mirzaei and A.T. Shau Hwai Historical Biology Figure 5. Annual pattern of daily seawater temperature from December 2011 to November 2012 in Penang Island, Malaysia. lowest seawater salinity was 14 ppt on 6 March 2012 and 27 October 2012 (Figure 6). A mixed semidiurnal pattern of the tidal cycle was observed in Penang Island, Malaysia. Tidal fluctuations of between þ 0.1 and þ 3.0 m were recorded during the spring tides, and þ 1.1 to þ 1.9 m during the neap tides. Due to the cage location at the study site (þ 1.0 m), the samples were exposed during all low tides (Figure 7). A total of 147 rainy days were observed throughout the study period. The highest average rainfalls were 328 and 336 mm in September and October 2012, respectively. However, the lowest average rainfall was in January and February 2012 at 65 and 69 mm, respectively (Figure 8). 3.4 Correlation analysis The correlation between the environmental factors (IVs) and mean shell increment width (DV) are shown in Table 2. The correlation coefficient was used to determine the correlation between the two variables. Based on Pallant (2004) and Tabassi et al. (2012), when correlation values were greater than 0.7, the scales were considered to Figure 6. have a high degree of reliability. The correlation matrix showed that there was a strong positive correlation between mean shell increment width and seawater temperature (þ 0.72) and a positive correlation between mean increment width and salinity (þ 0.53). Furthermore, there was a very weak negative correlation between mean increment width and rainfall (– 0.469), while there was no correlation between mean shell growth increments and tidal change (þ 0.071). 3.5 Multiple regression analysis Multiple regression analysis was used to predict a continuous dependent variable (increment width) on the basis of several independent variables (environmental factors). The study model was designed to find up how much of the variance in the shell growth was explained by the model which includes the variables of temperature, salinity, rainfall and tidal change. Table 3 shows the strength of the relationship between the combination of environmental factors and mean shell increment width. Multiple correlation coefficient between Annual pattern of daily seawater salinity from December 2011 to November 2012 in Penang Island, Malaysia. Historical Biology Historical Biology Figure 7. Annual pattern of daily tidal change between December 2011 and November 2012 in Penang Island, Malaysia. all environmental factors and mean increment width (R-value) was 0.780 which was highly acceptable in the current study. The R 2 showed 60.8% of the variance in shell increment width (DV) could be explained by environmental variables (IVs). This was an overall measure of the strength of association and did not reflect the extent to which any particular environmental parameter was associated with the increment width. In order to evaluate the significance of the model summary, it is essential to check the ANOVA table from the output of multiple regression analysis. The ANOVA table indicated that the research model was significant (F (4,703) ¼ 273.03, P , 0.001) (Table 4). Subsequently, coefficient table values were used to establish how well each environmental factor (IVs) in the research model contributed to the prediction of the increment width (dependent variable) (Table 5). It worth mentioning that beta values represented the unique contribution of each environmental factor, when the overlapping effects of all other variables were statistically removed. As standardised coefficients converted all environmental parameters to the same scale by SPSS equations, the beta value was used to compare the different Figure 8. 7 environmental variables. Temperature was the maximum beta coefficient (0.617) and, as a result, made the greatest unique contribution to shell growth. The lower beta value (0.346) for salinity showed that it was the second factor affecting shell growth (increment width). The results of the Sig. column showed that temperature and salinity were statistically significant in terms of their contribution to shell growth ( p , 0.01), while tidal change and rainfall did not contribute significantly to shell growth ( p . 0.01). 3.6 Laboratory experiment Two-way ANOVA test showed that temperature ( p , 0.01) and salinity ( p , 0.01) significantly influence on the increment width of Anadara granosa. After 30 days of exposure to experimental conditions, the mean increment widths of individuals in 31 ppt and 308C were significantly higher than mean increment width at the other temperature and salinity combinations, while, individuals in salinity of 26 ppt at 258C showed a mean increment width significantly below other groups (Figure 9). Therefore, trends of these interactive effects were an increase of shell increment with increasing temperatures (optimum at 308C), and at high Average monthly rainfall between December 2011 and November 2012 in Penang Island, Malaysia. 8 M.-R. Mirzaei and A.T. Shau Hwai Table 2. Correlation matrix between mean shell increment width of Anadara granosa and each environment factor from December 2011 to November 2012. Parameters Increment width Tidal change Salinity Rainfall Temperature 1 0.071 1 0.530 0.071 1 2 0.46 0.024 2 0.828 1 0.72 0.09 0.35 –0.36 1 . 0.030 . 0.000 0.030 . 0.000 0.259 0.000 . Pearson correlation Increment width Tidal change Salinity Rain fall Temperature Sig. (1-tailed) Increment width Tidal change Salinity Rain fall Temperature Historical Biology (31 ppt) salinity. A scatter diagram was used to determine the relationship between mean shell increment width and two environmental parameters. The mean increment width measurement showed that maximum shell growth was at 308C. However, mean increment width slowly decreased at seawater temperatures higher than 308C (Figure 10), while maximum shell growth was at salinity of 31 ppt (Figure 11). 4. Discussion Cockle shell growth is a function of multiple environmental factors. It is affected by several parameters such as food supply, temperature, population density, tidal change, pH and water depth. In terms of the effect of environmental factors on shell growth, there may be some synergistic interaction between environmental parameters such as temperature, rainfall and salinity. Along with environment factors, endogenous aspects also influence shell growth. While it is difficult to estimate the actual influence of a single environmental factor on the shell growth of cockles, multiple regression statistical analysis can show reliable relationships between the shell growth (dependent variable) and environmental factors (independent variables). In order to assess the environmental impact on shell microgrowth increment, a year-long microgrowth increments pattern was compared with environmental records. In order to examine the annual pattern, the following conditions needs to be considered. First, the periodicity rhythm of shell increment should not change during the Table 3. Model summarya for environmental factors (IVs) and mean increment width (DV) in the research model. Model 1 a b R R2 0.780b 0.608 Adjusted R 2 Std. error of the estimate 0.606 4.013 Dependent variable: increment width. Predictors: (constant), temperature, tidal change, salinity and rainfall. 0.000 0.006 0.000 0.000 . study period. Second, the shells must show continuous growth throughout the study without cessation of growth in different environmental conditions. In this study, the findings indicated the precise timing of growth line and growth increment formation in intertidal zone. It was found that the microgrowth lines in shell sections of Anadara granosa in intertidal zone consist of uniform microgrowth lines. It seems possible that these results are due to a mixed semidiurnal pattern of the tidal cycle in Penang Island. Therefore, cockles produced two microgrowth bands when they were emersed twice a day (24 h). This finding is in agreement with Richardson’s (1989) and Mahé et al.’s (2010) findings which showed that shell microgrowth patterns in intertidal zone mainly reflected tidal periodicity. Analysis of environmental parameters showed that the daily seawater temperature was fairly constant at around 30 – 338C during the study period. However, the seawater temperature had slightly decreased to a low range of 22 –278C from July 2012 to late September 2012. Seawater salinity was almost constant in the range of 29– 31 ppt, except for three frequent high fluctuations in the range of 11– 31 ppt during December 2011, April 2012 to late May 2012 and late September 2012 to late November 2012. Rainfall investigation showed that the highest average rainfall was 328 and 336 mm in September 2012 and October 2012, respectively, while the lowest average rainfall was in January 2012 and February 2012 with a total of 65 and 69 mm, respectively. Tidal change examination showed a mixed semidiurnal pattern of the tidal cycle in Penang Island, Malaysia. Shell examination showed that increment width remained relatively stable from 53.23 ^ 2.05 to 65.08 ^ 3.01 mm during the study period. Increment widths decreased slightly in July 2012 and remained at the lowest level which varied from 25.31 ^ 2.86 to 55.09 ^ 2.18 mm until late September 2012. Regarding the multiple regression analysis, it was found that temperature was the main factor affecting shell Historical Biology Table 4. Model 1 a Historical Biology b Regression Residual Total 9 ANOVAa test to assess research model. Sum of squares df Mean square F Sig. 17594.64 11325.44 28920.09 4 703 707 4398.66 16.11 273.036 0.00b Dependent variable: increment width. Predictors: (constant), temperature, tidal change, salinity, rain fall. growth (Beta value: 0.61). In addition, correlation matrix analysis showed the shell increment width was strongly and positively correlated to seawater temperature (0.72 coefficients out of 1). The results of this study showed that the maximum increment width (65.08 ^ 3.01 mm) was at the optimum temperature (308C), while continuous, narrow increment widths from 25.31 ^ 2.86 to 55.09 ^ 2.18 mm were recorded during the lowest seawater temperatures (22 – 278C). Therefore, lower shell growth corresponded to lower water temperature. Nevertheless, the distance between growth bands (increment widths) did not increase at temperatures above 308C and in fact began to decline. The results of this study were consistent with those of Gosling (2008) who found that shell growth increased in a linear manner until optimum temperature was achieved, after which the growth rate decreased rapidly. Hiebenthal et al. (2011) reported that high seawater temperature increased phytoplankton concentration, thus increasing the increment width in mollusc shells. Moreover, Page and Hubbard (1987) found a relationship between seawater temperature and the increment width of molluscs due to the effects of food availability on shell increment width. A scatter plot between temperature and increment width showed that temperature was an important growth regulator between 25 and 308C. However, shell growth rate decreased sharply at temperatures above 308C. A possible reason for the shell growth reduction above the optimum temperature was a decrease in feeding activity, leading to a decline in food availability, thus leading to a breakdown in the mechanism of metabolic activity. Isono et al. (1998) confirmed that high seawater temperatures (. 308C) affected respiratory activities such as heartbeat and ciliary movement of marine organisms. As a result, when the seawater temperature was below 308C, the shell growth (increment width) of Anadara granosa was controlled by seawater temperature because feeding and other metabolic activities increased. Moreover, there was a similarity between optimum temperatures expressed in this study and those described by Broom (1985) who had stated that the optimum seawater temperature for oxygen consumption of Anadara granosa ranged from 28 to 308C; however, when the seawater temperature was above 308C, shell growth (increment width) was affected by other factors. Due to the interaction between temperature and salinity, it is essential to consider salinity as an affective parameter during non-optimal temperatures in the study site. Based on Multiple regression analysis and the correlation between environmental factors and increment widths, salinity was the second factor affecting cockle shell growth. Salinity fluctuations at Balik Pulau showed a constant trend except during the heavy rainfalls of the intermonsoon period when it was lower than other times of the year, leading to shell valves staying closed for long periods due to the exposure to low salinity. Consequently, reduced the time available for feeding activity and changed the normal metabolic rate, which later become the main reason for lower growth rates. As a result, thinner increments were deposited during low periods of low salinity, as well as heavy rainfall. The present findings seem to be consistent with other studies which had been reported that when salinity dropped from 31 to 10 ppt, there was severe physiological damage to the molluscs (Kim et al. 2001). Moreover, Hamer et al. (2008) found that cockles respond to low salinity by closing their valves tightly when salinity is low. In addition, these findings concur with Riisgård et al. Table 5. Coefficienta table for the variables contributes in research model one. Unstandardised coefficients Model Tidal change Salinity Rain fall Temperature a B Std. Error Standardised coefficients Beta t Sig. 20.14 0.94 0.03 2.06 0.25 0.11 0.03 0.08 2 0.01 0.34 0.04 0.61 2 0.56 8.07 1.03 24.09 0.57 0.00 0.30 0.00 Dependent variable: increment width. 10 M.-R. Mirzaei and A.T. Shau Hwai Historical Biology Figure 9. Shell increment rate of all individuals in the laboratory experiment at temperature and salinity combinations. (2012) who studied the seasonal changes in increment width in cockle shells. They had indicated that a decline in seawater salinity created a sudden reduction in the growth of cockle shells. These results can be compared to those of Pathansali (1966) who stated that seawater salinity changes in Penang Island were minimal during the dry season. However, seawater salinity during the rainy season had a maximum oscillation. In addition, Pathansali (1966) made a study of the salinity requirements and preferences of Anadara granosa in a laboratory where the feeding rates of Anadara granosa under different salinity levels were studied. The feeding rate was reduced by 85% at a salinity of 17.6 ppt and was nil at 12 ppt. The findings indicated that the shell valves were closed at very low seawater dilutions. It was reported that Anadara granosa was able to function relatively at salinity above 23 ppt although young specimens seemed to be able to continue their normal feeding activity at lower salinity levels compared to older specimens. This study concerning Anadara granosa shell growth provides new insights for the understanding of growth patterns in Penang Islands, west coast of Malaysia. The sclerochronological approach appears as a powerful tool to characterise high-frequency variations in the shell growth in relation to environmental parameters. This approach allowed us to prospective analysis of culture location and environmental assessment of farm related to tidal levels. The results of this investigation show that analysis of increment width in the shell cross section of Anadara granosa leads to an accurate estimation of the environmental effects during their life history. One of the most significant findings to emerge from this study is that cockle shell growth was mainly controlled by temperature and less by salinity, while rainfall and tidal changes were insignificant. The second major finding is that the growth rate of Anadara granosa was in a linear trend to optimum temperature, while salinity contributes to shell growth as a moderator at temperatures above the optimum. In addition, Anadara granosa responded to variations in seawater salinity by closing its shell valves and changing its feeding activity and normal metabolism. This study shows that shell microgrowth increments were well defined and deposited with a tidal periodicity in the intertidal zone, providing the calendar base for high-resolution ecological studies and environmental reconstruction from shell cockles Anadara granosa. Figure 10. Scatter plot of the shell increment rate of all individuals in the laboratory experiment versus different water temperatures. Figure 11. Scatter plot of the shell increment rate of all individuals in the laboratory experiment versus different seawater salinity. Historical Biology Acknowledgements The authors would like to acknowledge the Marine Science Laboratory, School of Biological Sciences, Universiti Sains Malaysia, for their support and contribution to this study. Disclosure statement No potential conflict of interest was reported by the author(s). Historical Biology References Brock V, Bourget E. 1989. 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