Feeding ratio and frequency affects cadmium bioaccumulation in black sea bream Acanthopagrus schlegeli

Feeding ratio and frequency have substantial effects on food digestion and assimilation in fish, yet few attempts have been made to determine their influences on the bioaccumulation of trace metals. In this study, juvenile Acanthopagrus schlegeli were exposed to either waterborne or dietary cadmium (Cd) at different feeding ratios (0, 2, or 4% body weight d−1 [BW d−1] under waterborne Cd exposure and 1, 2.5, or 4% BW d−1 under dietary Cd exposure) or feeding frequencies (1, 2 or 8 times d−1) for 5 wk. Under waterborne Cd exposure, the fish fed 0% BW d−1 or 8 times d−1 showed significantly lower growth rates and those fed 0% BW d−1 or 1 time d−1 exhibited higher Cd body burdens and Cd uptake rates compared to the other groups. The gut showed a significantly higher waterborne Cd uptake rate than the gills when fish were fasted or fed only 1 time d−1. These results suggest that starvation and low feeding frequency facilitate waterborne Cd uptake. Under dietary Cd exposure, the fish fed 4% BW d−1 or 2 times d−1 grew faster. Cd body burden and Cd assimilation were higher in fish fed 2.5% BW d−1 or 2 times d−1. Dietary Cd retention was positively correlated with feed efficiency, suggesting the utilization of Cd probably coincides with the essential nutrients in fish. Overall, this study demonstrates that different feeding strategies significantly influence waterborne and dietary Cd bioaccumulation in marine fish. Therefore, feeding conditions have to be considered carefully for managing trace metal contamination in marine fish farming.


INTRODUCTION
Globally, 128 million tons of fish provided about 3 billion people with almost 20% of their average per capita intake of high-quality animal protein in 2010 (FAO 2012).However, the wide spread of trace metal pollution in agricultural and urban soils (Wei & Yang 2010, Luo et al. 2012) and estuarine and coastal regions (Pan & Wang 2012) has resulted in an increasing risk of trace metal contamination in aquaculture environments (e.g.sediments and seawater; Pan & Wang 2012), as well as in fish feed ingredients (Mai et al. 2006, Dang & Wang 2009, Wei & Yang 2010), and thus the high risk of excessive trace metal con-tents in aquatic products.For ex ample, several investigations have found high Cd contents in aquatic foods and the high risk of Cd exposure to humans by aquatic food consumption in coastal areas (Yu et al. 2006, Wu et al. 2013).Previous studies have largely focused on how physico-chemical environmental factors affect Cd bio accumulation in aquatic organisms.Yet few at tempts have been made to determine the effects of realistic management practices on Cd bioaccumulation in fish.Feeding ratio and frequency, for instance, are critical for production costs and water quality in intensive fish farming.Earlier studies have indicated that feeding ratio and frequency have significant effects on the nitrogen and energy budget (Sun et al. 2003), growth rate and body composition of black sea bream (Acanthopagrus schlegeli; Lou et al. 2006Lou et al. , 2007)), whereas it is unclear how these parameters influence trace metal bioaccumulation in farmed fish in trace metal-contaminated environments.
Aquatic animals accumulate trace metals from both dietary and dissolved phases.Diet is the predominant route of exposure for most aquatic organisms (Rainbow 2007, Wang et al. 2012).Several studies have demonstrated that dietary trace metal assimilation is closely related to the ingestion rate (IR) and gut passage time (GPT) of diets (Zhang & Wang 2006a, Croteau et al. 2007).Feeding ratio and frequency have a substantial influence on the IR and GPT of diets in fish (Lee et al. 2000, Riche et al. 2004), and thus probably affect the assimilation process of dietary trace metals.Although a growing body of study has revealed that changes in food quality have a significant effect on dietary metal assi milation in farmed marine fish (e.g.food type [Wang & Wong 2003, Zhang & Wang 2006a] and dietary protein sources [Mai et al. 2006, Wang et al. 2012]), little is known about the influence of food quantity management (e.g.feeding ratio and frequency adjustments) on dietary metal bioaccumulation in farmed fish.
In addition, feeding patterns may also impact waterborne metal uptake in fish (Hashemi et al. 2008, Wood et al. 2010).Since most marine fish drink seawater continuously to maintain osmotic homeostasis (Grosell 2006) and intestines are the major sites for the uptake of dissolved metals (Zhang & Wang 2007a), feeding and digestion can alter the waterdrinking rate (Wood et al. 2010) and physico-chemical environment of fish intestines (e.g.pH and acid−base levels [Taylor et al. 2007, Bucking et al. 2009] and major ion concentrations [Bucking et al. 2011]).For instance, Wood et al. (2010) found that gulf toadfish Opsanus beta under daily satiation feeding accumulated much less waterborne Ag in whole body, liver and white muscle samples than those without feeding.To the best of our knowledge, however, there is a surprising paucity of available literature addressing the impacts of realistic feeding management actions on waterborne heavy metal uptake in marine fish.
Black sea bream Acanthopagrus schlegeli is a popular and valuable commercial fish in East Asia (e.g.China, Japan, Korea and some other countries of Southeast Asia) due, for example, to its high growth rate, good meat quality, excellent adaptability to environments and resistance to diseases (Gonzalez et al. 2008, Zhang et al. 2012).Earlier studies indicated that feeding ratio and frequency have significant effects on the nitrogen and energy budget (Sun et al. 2003) and the growth rate and body biochemical composition of black sea bream in uncontaminated environments (Lou et al. 2006(Lou et al. , 2007)).With respect to Cd bioaccumulation in black sea bream, our previous studies demonstrated that the gastrointestinal tract is the main site for dissolved Cd uptake (Zhang & Wang 2007a) and dissolved Cd bioaccumulation is apparently affected by metal pre-exposure (Zhang & Wang 2005, 2006b), water salinities (Zhang & Wang 2007b) and body size (Zhang & Wang 2007c).Nevertheless, it remains unclear how feeding ratio and frequency influence growth and Cd bioaccumulation in black sea bream.In the present study, we therefore conducted a 5 wk feeding experiment to examine the effects of feeding ratio and frequency on (1) growth performance and feed utilization, (2) Cd body burden and (3) waterborne Cd uptake and dietary Cd assimilation efficiency in juvenile black sea bream.

Cd exposure and feeding ratio and frequency
In the present study, there were 4 series of experiments to test the effects of feeding ratio and feeding frequency on waterborne and dietary exposure separately (Table 2).There were 2 control groups of 25 fish fed 2 times d −1 at 3% body weight d −1 (BW d −1 ) without Cd exposure.The control groups were mainly used to monitor the aquaculture system and to provide background values for Cd concentrations in fish under the present experimental conditions (Table 2).
Series 1: waterborne Cd exposure -effect of feeding ratio In waterborne Cd exposure experiments, the waterborne Cd concentration was 100 µg l −1 (the measured value was 97.2 ± 2.5 µg l −1 as CdCl 2 , 99.5%; Guangzhou Chemical Reagent Factory).Three levels of feed-ing ratios (0, 2 and 4% BW d −1 ) were chosen to test the effects of feeding ratios on fish growth and waterborne Cd bioaccumulation (the feeding frequency was at a normal level, i.e. 2 times d −1 ; Table 2).

Fish feeding and sampling procedures
The fish in each tank were weighed in bulk at the middle of the feeding experiment to re-adjust feeding ratios.All fish were fed set feed weights at set times using automatic fish feeders (Pet Wang Products).Uneaten food was collected 1 h after feeding, then dried and re-weighed to calculate the weight of feed ingested by fish.In the waterborne Cd exposure, the water with Cd (100 µg l −1 ) added was first artificially renewed with normal seawater before fish were fed; subsequently, fish were provided with a natural diet (Table 1) in seawater without additional Cd.After fish feeding, a CdCl 2 solution was added to the seawater to keep the dissolved Cd concentration at 100 µg l −1 .A water renewal rate of once per hour was maintained in dietary Cd exposure groups by running seawater.The feeding experiment lasted 5 wk.
Upon completion of the experiment, all fish were weighed and 3 fish from each tank (6 fish per treatment) were transferred to other aquaria for determination of waterborne Cd uptake rate and dietary Cd assimilation efficiency (AE).The remaining fish were deprived of feed for another day and then sacrificed by an overdose of MS-222 (200 µg l −1 ) for whole body composition analysis and Cd concentration determination in selected tissues (gills, gut, liver, dorsal muscle, carcass [the fish without gills and viscera] and whole body [whole fish]).

Cd uptake rate and assimilation efficiency measurement
To determine waterborne Cd uptake rates, 3 fish from each waterborne Cd exposure tank (6 fish per treatment) were placed in 10 l aquaria containing 8 l of natural aerated seawater filtered using Whatman glass filters (pore size: 0.22 µm).The water was spiked with 20 µg l −1 113 Cd (International Atomic Energy Agency Office at USA, New York, in CdO form, and dissolved in 1 mol l −1 HNO 3 to 16.7 ± 0.6 µg Cd l −1 ).Since our previous results suggested that juvenile black sea bream take up dissolved Cd linearly with exposure time without saturation over 4 h (see details in Zhang & Wang 2006b, 2007b), the fish in the present study were sampled at 4 h for 113 Cd content measurement in fish tissue.To determine the AE of dietary Cd, 4 fish from each dietary Cd exposure tank (8 fish per treatment) were kept individually in 2.5 l aquaria (containing 2 l natural aerated seawater) for 3 d to acclimate to experimental conditions.During those 3 d, the fish were fed at the same feeding ratio/frequency as those in previous experiments.Then, the fish were fed a diet spiked with 10 µg g −1 113 Cd (the measured value was 9.8 ± 0.6 µg g −1 ) plus 240 µg g −1 natural Cd (to produce the same concentration as in the long-term dietary exposure) in the next 24 h using the corresponding feeding ratio or frequency.After feeding with spiked diets, the fish were fed diets containing natural Cd for 60 h and then sampled for 113 Cd content measurements in fish tissues.

Proximate composition and Cd content analysis
All diet and fish tissue samples were dried at 60°C for 24 h.The proximate composition analysis of fish and diets were conducted as described by the AOAC (1995).Specifically, dry matter content was determined by oven-drying at 105°C to constant weight.Crude ash content was determined by incineration in a muffle furnace (550°C for 12 h).Crude protein content (N × 6.25) was determined using an Automatic Kjeldahl System (2300 Kjeltec Analyzer Unit, FOSS Tecator).Crude lipid content was determined by ether extraction in a Soxtec System HT6 (Tecator).
Fish tissue and diet samples were ground and then digested using concentrated HNO 3 (69%, ultrapure, Fisher Scientific) for 48 h at 80°C.The total Cd concentrations in water and digested samples were quantified by inductively coupled plasma-mass spectroscopy (ICP-MS, 7700X, Agilent Technologies).The net concentration of 113 Cd (Δ 113 Cd) in the samples with respect to Cd uptake rate and AE were determined using the methods described by Croteau et al. (2007).The equations are given in the Appendix.

Data calculation and statistical analysis
The parameters regarding growth performance and feed utilization were calculated as follows: SGR = 100 × (ln mBW final -ln mBW initial )/N (1 where SGR is specific growth rate (% d -1 ), mBW initial and mBW final are intial and final mean body weight (in g), respectively and N is the number of days; where FI d is daily feed intake (% BW d -1 ), FI total is total feed intake (% BW); where FE is feed efficiency (%), DW gain is dry weight gain of fish.
The parameters regarding the kinetics of Cd bioaccumulation were calculated as follows: where J w is waterborne Cd uptake rate (ng g -1 h -1 ); where AE diet is the dietary Cd assimilation efficiency (%), DW tissue is the dry weight of fish tissue, Cd spike is the 113 Cd concentration in spiked diets; where RE is the dietary Cd retention efficiency (%), Cd initial and Cd final are the initial and final Cd concentration in fish, respectively, and Cd diet is the Cd concentration in the diet.
The data were analyzed by 1-way analysis of variance (ANOVA) followed by Tukey's HSD (honestly significant difference) post hoc test for multiple comparisons between different treatments (Table 2).The only exception is that the differences between 2 and 4% BW d −1 fish in waterborne Cd exposure groups were determined by t-test since there were only 2 groups.Normality and homogeneity of data were determined using a 1-sample Kolmogorov-Smirnov test and Levene's test.The data were log(x + 1) transformed if normality and homogeneity were not assumed.The difference was regarded as significant when p < 0.05.All statistical analyses were performed with the SPSS 18.0 software package.

Waterborne Cd exposure -effects of feeding ratio
The juvenile black sea bream in the control groups showed normal growth performance (SGR: 1.55 ± 0.08% d -1 ; mean ± SD), suggesting that the present feeding experiment was conducted in a well-regulated culture condition.In the control groups, the Cd concentration was low (0.17 ± 0.23 µg g −1 ; mean ± SD).
When the fish were exposed to waterborne Cd exposure, the group without feeding (0% BW d −1 ) showed the lowest and even negative SGR (Table 2).The fish fed 4% BW d −1 grew fastest.The FE of the fish fed 2% BW d −1 was significantly higher than that of the fish fed 4% BW d −1 .The group without feeding displayed the lowest crude lipid content (CL) but the highest ash content in whole body samples (Table 2).139 Fig. 1.Cadmium concentration (µg Cd g -1 in dry matter) in the gills, gut, liver, muscle, carcass and whole body of black sea bream Acanthopagrus schlegeli exposed to waterborne Cd (97.2 µg l -1 ) for 5 wk at 3 different levels of feeding ratio (FR) or frequency (FF).(A) FR treatment: fish were fed 0, 2 and 4% body weight d -1 at 2 times d -1 .(B) FF treatment: fish were fed 1, 2 and 8 times d -1 at a feeding ratio of 3% body weight d -1 .Mean ± SD, n = 6 (3 fish per replicate tank); means of the same tissue with different superscripts are significantly different among treatments (p < 0.05) The fish without feeding had the highest Cd concentrations in their gut (22.8 µg g −1 ), and also ac cumulated significantly higher Cd concentration in the muscle, carcass and whole body (Fig. 1A).The up take rate of waterborne Cd was also highest in non-fed fish (Fig. 2A).The waterborne Cd uptake rate in gut was about 6-fold higher than that in the gills when the fish was not feeding, whereas those 2 organs showed a similar Cd uptake rate in fish fed 2 or 4% BW d −1 (Fig. 2A).Moreover, the fish without feeding showed a higher percentage of Cd in their gut (31.5%), but lower values in their gills (4.9%) and liver (10.6%) than other groups (Fig. 3A).

Waterborne Cd exposure -effects of feeding frequency
Under waterborne Cd exposure, the SGR and FE were highest when fish were fed 2 times d −1 (Table 2), but there was no significant difference in fish body composition between the groups with different feeding frequencies.
The Cd concentration was significantly higher in the gills, gut, liver and whole body when fish were fed 1 time d −1 compared with those fed 2 or 8 times d −1 (Fig. 1B).The fish fed 1 time d −1 took up waterborne Cd in the gut (7.6 ng g −1 h −1 ) approximately 3-fold faster than those fed 2 (2.4 ng g −1 h −1 ) or 8 times d −1 (2.6 ng g −1 h −1 ) (Fig. 2B).Moreover, the Cd uptake rates in the gut of fish fed 1 time d −1 were clearly higher than those in the gills (4.1 ng g −1 h −1 ), but the guts of fish fed 2 and 8 times d −1 showed an even slightly lower Cd uptake rate than did the gills (Fig. 2B).Additionally, the proportions of Cd in gut decreased steadily when the feeding frequency increased (Fig. 3C).

Dietary Cd exposure -effects of feeding ratio
In dietary Cd exposure groups, the SGR of fish steadily increased with the increasing feeding ratios.
The group with 1% BW d −1 exhibited a significantly higher FE and ash content than the others (Table 2).
The fish fed 4% BW d −1 showed the lowest dietary Cd AE (3.8%), while AE values were similar between the other 2 groups (6.1 and 6.6% in fish fed 1 and 2.5% BW d −1 , respectively).After 5 wk of dietary Cd exposure, the fish fed 1% BW d −1 accumulated the least Cd in all body parts, and the fish fed 2 and 4% BW d −1 accumulated comparable Cd (Fig. 4A).Moreover, the gut and carcass accounted for most of the Cd body burden (> 80%), but the proportions of Cd partitioned into different tissues were similar between different feeding ratios (Fig. 3B).

Dietary Cd exposure -effects of feeding frequency
When the fish were exposed to dietary Cd at different feeding frequencies, the SGR, FE and CL were highest in the fish fed 2 times d −1 (Table 2).
The AE in fish fed 2 times d −1 (6.0%) was about 2fold that of the other 2 groups (3.2 and 3.5% in fish fed 1 and 8 times d −1 , respectively).At the end of 5 wk of dietary Cd exposure, the fish fed 1 time d −1 accumulated significantly lower Cd contents in all body parts except the gills, and those fed 2 times d −1 exhibited highest Cd in the gut (194.3 µg g −1 ), carcass (7.5 µg g −1 ) and whole body (11.5 µg g −1 ; Fig. 4B), whereas feeding frequency did not sig nificantly impact the Cd partitioning among tissues (Fig. 3D).
Moreover, when the fish were exposed to dietary Cd at different feeding ratios or feeding frequencies, dietary CRE (%) was significantly lower at higher feeding ratios (Fig. 5A), and was highest in the fish fed twice daily, compared to 1 and 8 times d −1 (Fig. 5B).CRE was positively related to FE (%), across all ratios and frequencies tested (Fig. 5C).Fig. 4. Cadmium concentration (µg Cd g -1 in dry matter) in the gills, gut, liver, muscle, carcass and whole body of black sea bream Acanthopagrus schlegeli exposed to dietary Cd (254.4 µg g -1 ) for 5 wk at 3 different levels of feeding ratio (FR) or frequency (FF).(A) FR treatment: fish were fed 1, 2.5 and 4% body weight d -1 at a FF of 2 times d -1 .(B) FF treatment: fish were fed 1, 2 and 8 times d -1 at a FR of 3% body weight d -1 .Mean ± SD, n = 6 (3 fish per replicate tank); means of the same tissue with different superscripts are significantly different among treatments (p < 0.05).Note: quadratic scale of y-axis

Starvation and waterborne Cd bioaccumulation
In the present study, the fish starved showed significantly higher waterborne Cd accumulation and uptake rates compared with those fed diets, notably in the gut.Consistently, Wood et al. (2010) observed the same phenomenon in marine gulf toadfish (Opsanus beta) when it was exposed to waterborne Ag for 22 d.Their results indicated that the net whole body accumulation of Ag was reduced by > 50% in fish fed daily to satiation compared with those fasted.In freshwater common carp (Cyprinus carpio), similar results were obtained (Hashemi et al. 2007(Hashemi et al. , 2008)).Here, the most obvious interpretation for the ob -served protection by feeding against metal accumulation is that the ingestion, digestion and assimilation of food prevents the uptake of waterborne metals in the gastro intestinal tract by decreasing the drinking of water (Wood et al. 2010) and/or changing the physico−chemical environment of the intestines (e.g.pH and acid−base disturbances [Taylor et al. 2007, Bucking et al. 2009], gut fluid volume [Wood et al. 2010], bulk ion exchange and ion concentration [Bucking et al. 2011]).Alternatively, it is noteworthy that the fish in all these studies (including the present study) were fasted for >10 d, which usually resulted in an insufficient intake of essential nutrients, thus resulting in po tential disturbances in normal phy siological processes/functions, such as energy metabolism and partitioning (Sun et al. 2003), osmoregulation (e.g.Na + regulation), metal detoxication (e.g.metallothionein synthesis; Hashemi et al. 2007Hashemi et al. , 2008) ) and homeostatic regulation of trace metals (e.g.biliary discharge of Ag; Wood et al. 2010).All these physiological alterations are likely to be the causes of increasing metal bioaccumulation in the fish when they were fasted.
Furthermore, this study is the first one to report that feeding frequency could significantly affect waterborne Cd bioaccumulation in fish.The fish fed once per day showed the highest Cd body burden and Cd uptake rates in the gut.This finding can probably be attributed to the occurrence of empty guts between the feeding intervals when no food is ingested after gastric evacuation, with gastric evacuation times being strongly correlated with feeding intervals (Lee et al. 2000, Riche et al. 2004).In tilapia (Oreochromis niloticus), for instance, the stomachs of fish fed 5 times d −1 still contained some of the food at 24 h, whereas stomachs were empty and flaccid in fish fed 3 times d −1 (Riche et al. 2004).Thus, in the present study, the gastrointestinal tract of the fish fed once a day was probably empty for a much longer time compared to those fed 2 or 8 times d -1 .If fish guts are empty, with no further food ingestion during feeding intervals, the fish may be

Feeding ratio and frequency affecting dietary Cd bioaccumulation
Dietary Cd assimilation efficiency (AE) ranged from 3.2 to 6.6% when fish were fed with artificial diets in this study, which agreed well with the values in the study by Zhang & Wang (2005) (4.2 to 6.2%).Both feeding ratio and feeding frequency had significant effects on fish Cd body burden and AE.The ingestion rate (IR, equivalent to FR in the present study) and gut passage time (GPT) are suggested to be the 2 most important factors determining the AEs of metals in many aquatic organisms (e.g.Zhang & Wang 2006a, Croteau et al. 2007).In most cases, the AEs of metals increased with prolonged GPT, and AEs were higher at a lower IR and vice versa (Zhang & Wang 2006a, Croteau et al. 2007).In our study, the higher AEs at a lower FR probably resulted from the greater efficiency of the limited number of transporters on the intestine epithelium and/or enhanced Cd digestion due to the numerous digestive enzyme contacts and reactions (Zhang & Wang 2006a).Similarly, black sea bream fed 2 times d −1 showed higher Cd AEs than those fed 1 or 8 times d −1 when FR was fixed at 3% BW d −1 .A somewhat more complex diges tive process was noted with re gard to changes in IR and GPT.The low Cd AEs in fish fed 1 time d −1 might be the main factor at tributing to the high IR for each meal (i.e. when the diets of 3% BW d -1 were divided to 2 or 8 times d -1 , the IR is lower for each meal compared with the diets fed once a day).However, when the 3% BW diet was divided to allow feeding 8 times d −1 , i.e. a feeding interval of only 3 h, fish may have experienced gastric overload (Riche et al. 2004).Thus, the GPT of each meal was very short, possibly leading to inadequate digestion of food and thus low AEs of Cd (Zhang & Wang 2006a).
In our study, when exposed to waterborne Cd, the fish undergoing starvation showed Cd values of 0.14 µg g −1 in muscle wet weight (WW), which exceeded the permissible level of 0.1 µg g −1 according to China's national standard (China National Standards Management Department 2001) and that of the European Union (2001).When the black sea bream were exposed to dietary Cd, only those fed once a day showed the lowest Cd content of 0.083 µg g −1 WW; the Cd contents in the other groups were higher than the permissible level of 0.1 µg g −1 (i.e.0.23 to 0.58 µg g −1 WW).

Suitable feeding ratio and frequency for fish growth performance
When exposed to waterborne or dietary Cd for 5 wk, the growth and feed utilization of the black sea bream was often lower than that detected in similar studies on black sea bream (Sun et al. 2003, Lou et al. 2006, 2007, Zhang et al. 2012), suggesting the potentially toxic effects of waterborne and dietary Cd to fish.The fish showed the best growth performance when fed at 4% BW d −1 twice a day, which was consistent with those previous studies.For instance, Lou et al. (2007) found that the SGR of fish were highest at 3% BW d −1 and then decreased slightly when the feeding ratio reached 4% BW d −1 .Additionally, we found the same effect of feeding frequency on SGR and FE as in Lou et al. (2007), i.e. the optimal feeding frequency was 2 times d −1 and high feeding frequency (6 times or 8 times d −1 ) led to reductions in growth rate and feed utilization, regardless of Cd exposure.

Implication for feeding management in marine fish farming
Estuarine and coastal areas are the main regions of marine aquaculture and, concurrently, are the ultimate receptacles of the bulk of anthropogenic pollutants; specifically, they are often subjected to Cd pollution (e.g.Cheung et al. 2008, Pan & Wang 2012).Furthermore, the Cd pollution of the environment may result in increased Cd bioaccumulation in some kinds of feed ingredients (e.g.Mai et al. 2006, Dang & Wang 2009).Thus, marine fish farming is likely to be subjected to Cd pollution, occasionally via a dissolved or dietary route.
The feeding frequency and feeding ratio in realistic management protocols often vary greatly with seasons, feeding strategies and the marketable demands of targeted fish.For instance, feed restriction− refeeding cycles are widely used in aquaculture to induce compensatory growth in numerous fish species as a way of increasing profits and reducing waste load to the environment (Ali et al. 2003, Sevgili et al. 2012).The present study suggested that starvation or a decrease in feeding frequency may substantially increase the risk of excessive metal accumulation in farmed fish, if feed or water are contaminated by trace metals.Therefore, in Cd-contaminated seawater environments (fish feeds are free of cadmium contamination), feeding frequency is recommended to be 2 times d −1 and feeding ratio to be 3 to 4% BW d −1 for juvenile black sea bream, from a product safety and cost-efficiency perspective.
Furthermore, this study found that feeding ratio and frequency were vital factors influencing dietary Cd bioaccumulation.The digestion and assimilation of non-essential metals probably coincides with the assimilation of essential nutrients.It is well recognized that feed efficiency should be kept as high as possible to promote cost-efficiency in commercial fish farming.Therefore, it appears difficult to decrease dietary metal bioaccumulation via the management of feeding ratio and frequency without imposing a negative effect on feed efficiency and fish growth performance.Moreover, it is widely acknowledged that diet is the predominant route of metal exposure for most aquatic organisms (Rainbow 2007, Wang et al. 2012).Thus, the best way to prevent dietary metal bioaccumulation is to decrease metal contents in fish diets.
In summary, our findings clearly demonstrated that feeding ratio and feeding frequency were important factors affecting Cd bioaccumulation in juvenile black sea bream.Fasting or feeding less frequently may expose fish to higher risks of waterborne Cd contamination, due to an increase in waterborne Cd uptake through the gastrointestinal tract.Changing feeding ratios and frequencies can also affect dietary Cd accumulation in fish.However, it may not be a practical way to reduce dietary Cd contamination, since it affects Cd assimilation and nutrient utilization in parallel.As the predominant source of Cd bioaccumulation in fish is their diet, it is important to control the Cd contents in feed.

Fig. 2 .
Fig.2.Uptake rate (ng g -1 h -1 ) of waterborne Cd in various tissues of black sea bream Acanthopagrus schlegeli at 3 different feeding ratios or frequencies.Experimental treatments and statistical significance as in Fig.1