Towards a safe standard for heavy metals in reclaimed water used for fish aquaculture

Aquaculture 284 (2008) 115–126

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Towards a safe standard for heavy metals in reclaimed water used for fish aquaculture M. Feldlite a,⁎, M. Juanicó b, I. Karplus c, A. Milstein d a

Israel Water Works Association, Ramat-Hashron Water Plant, Morasha, Petah-Tikwa, 49001 Israel Juanicó — Environmental Consultants Ltd., 2 Aliah St., Afula 18392, Israel Aquaculture Research Unit, Agriculture Research Organization, P.O.B. 6, Beit-Dagan, 50520 Israel d Fish & Aquaculture Research Station, Dor. M.P. Hof HaCarmel, 30820, Israel b c

a r t i c l e

i n f o

Article history: Received 20 January 2008 Received in revised form 20 July 2008 Accepted 21 July 2008 Keywords: Arsenic Aquaculture Cadmium Fish Lead Mercury Wastewater

a b s t r a c t The objective of the present study is to provide a basis for the development of a standard on the concentration of heavy metals in reclaimed water used for edible fish aquaculture, focusing on the four heavy metals addressed by the Israel standard for food quality (arsenic, cadmium, lead and mercury). A series of experiments were carried out on three commercial fish species of differing feeding habits and complementary measurements were made in fish growing in a reservoir with secondary treated effluent. In a field experiment the fish were raised on natural food and on pelleted feed, in reclaimed wastewater and in freshwater. In laboratory experiments, the fish were raised in aquaria while exposing them to heavy metals supplied in the pelleted feed or dissolved in the water. In the field experiment with fish growing in tertiary treated reclaimed water during five months, no detectable levels of the four tested heavy metals were found in fish flesh (no analyses in internal organs performed). In fish reared during two years in secondary treated reclaimed water, no detectable levels of heavy metals were found in fish flesh, but the concentrations of Cd and Pb in liver and bones of some fish were above the food standard. Laboratory experiments on rearing fish while exposing them to water and pelleted feed with increasing levels of heavy metals, revealed detectable levels of these heavy metals in fish flesh, bone and mainly in liver. The standards on reclaimed water for unrestricted irrigation are a good basis for the development of a water quality standard for growing fish in reclaimed water, because fish can be grown in the reservoirs used to storage the reclaimed water and/or this is the reclaimed water that can be used to fill fish ponds. Except for Hg, the proposed Israel requirements for heavy metals in reclaimed water used for unrestricted irrigation (As =0.1 mg/l, Cd =0.01 mg/l, Pb=0.1 mg/l, Hg=0.002 mg/l) are safe regarding the consumption of flesh from fish reared in reclaimed water, even if the growing season is long (up to two years). These requirements are not equally safe regarding the consumption of liver (and probably other viscera). © 2008 Elsevier B.V. All rights reserved.

1. Introduction The objective of the present study is to provide a basis for the development of a standard on the concentration of heavy metals in reclaimed water used for fish aquaculture. Israel suffers from chronic water shortage and reclaimed water is the most immediately available non-conventional water source. Today, nearly 50% of the water used for irrigation in the country is reclaimed water, while freshwater resources are more and more dedicated to satisfy the increasing domestic demand. On the other hand, the amount of reclaimed water produced in the country increases with population growth, and its quality improves in conformity with new, stricter quality standards (Juanicó, 2008). Most Israeli aquaculture is based on earthen ponds and reservoirs for the polyculture of common carp (Cyprinus carpio), tilapia hybrid ⁎ Corresponding author. Tel.: +972 50 3750674; fax: +972 4 6247905. E-mail addresses: [email protected] (M. Feldlite), [email protected] (M. Juanicó), [email protected] (I. Karplus), [email protected] (A. Milstein). 0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.07.036

(Oreochromis niloticus×O. aureus), gray mullet (Mugil cephalus), filter feeder hybrid Chinese carp (a hybrid of silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis). Reclaimed water has been used for freshwater fish culture for hundreds of years both in the Far East (e.g.: Edwards, 1987; CIFA, 1997; Jana, 1998; Tripathi and Sharma, 2001; Saha and Jana, 2003), Europe (e.g.: Kovacs and Olah, 1984; Strauss and Blumenthal, 1988; Guterstam et al., 1998), Latin America (Moscoso and Florez, 1991; Nava, 2001) and also experimentally in the USA (CostaPierce, 1998). Still, fish rearing in reclaimed water is not practiced in most countries around the world, and existing guidelines do not address heavy metals and other toxic compounds (e.g., Blumenthal et al., 2000). In Israel, the single record for fish culture in reclaimed water is very old (Sarig,1956) and was discontinued; reclaimed water aquaculture is not a present practice nor has Israel any guidelines for it. However, as the sources of freshwater presently dedicated to aquaculture continue to decline, reclaimed water stands as the single source of water potentially available to maintain aquaculture in the country. Thus, the development of guidelines for this practice is urgently required.

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It is already known that use of reclaimed water in fish culture is possible and can also improve yields (Feldlite, 1987). Numerous studies have dealt with the potential microbial contamination of fish reared in reclaimed water and have concluded that the concentration of pathogens in fish is a function of its concentration in water (e.g., Buras et al., 1987). Fattal et al. (1989) proposed to maintain the concentration of Faecal coliforms in the water below 10,000 per 100 ml as water quality guideline for growing fish in wastewater, but a WHO Scientific Group (1989) finally proposed a guideline of 1000 Faecal coliforms/100 ml; this guideline was later confirmed by Mara et al. (1993) and Blumenthal et al. (2000). The potential accumulation of toxic compounds in cultured freshwater fishes has been addressed in a much more limited number of studies, and some of the experiments such as those reported by Liang et al. (1999) were performed for a too short growing season of only two months. The toxic compounds that could potentially be found in reclaimed wastewater can be grouped into organic compounds and heavy metals. The present study deals with the four heavy metals included in the Israel standard for food quality: arsenic, cadmium, lead and mercury. It is difficult to determine whether bioaccumulation of heavy metals differs among fish species due to their differing feeding habits. On the other hand, it is already known that heavy metals in aquatic systems are trapped in the sediment (Juanicó et al., 1995) and might bioaccumulate through organisms that feed on it (Tarifeno et al., 1982; Liang et al., 1999). It is expected that the accumulation of heavy metals in fish fed with pelleted feeds would differ from that in fish that feed on the natural food web. Thus, in this study a series of experiments were carried out in three fish species of differing feeding habits. To see whether there is accumulation of heavy metal in fish exposed to reclaimed wastewater, in a field experiment the fish were raised during five months on natural food and on pelleted feed, in reclaimed wastewater and in freshwater (as control). This was complemented

with measurements performed on fish that had been living in reclaimed wastewater for two years. In order to determine the maximum concentrations of heavy metals that should be adopted in reclaimed water for edible fish culture, fish were raised in laboratory experiments exposing them to a wide range of concentrations of heavy metals supplied in the feed or dissolved in the water. The main characteristics of the performed experiments are presented in Table 1. 2. Materials and methods 2.1. “Field experiment” (fish reared for five month in tertiary reclaimed water) The field experiment was carried out in an operational reservoir that receives reclaimed water from the municipal sewage treatment plant of Ramat HaSharon — Central Israel. The reservoir has a volume of 120,000 m3 and a mean depth of 6 m; the hydraulic residence time of the reclaimed water within the reservoir is 14 days. The sewage treatment process is activated sludge including nitrification/denitrification and releases secondary effluents with biological oxygen demand (BOD5) b20 mg/l and total suspended solids (TSS) b30 mg/l. The effluents are then filtered in deep granular media and chlorinated. The typical quality of the final reclaimed water entering the reservoir where the experiment was carried out is BOD5 b 5 mg/l, TSS b 5 mg/l, ammonium b 3 mg/l, nitrite b 1 mg/l and nitrate b 2 mg/l. A fish pond at Kibbutz Gan Shmuel fish farm (Central–Northern Israel) which receives freshwater, was used as a control. In each location six cages (1.5 m3, 5 mm pore) were stocked on 8th June 2003 with the filter feeder hybrid Chinese carp, the omnivorous tilapia and the bottom feeder common carp, at the densities and stocking weights indicated in Table 1. Half of the cages received 30% protein commercial floating feed pellets at a daily rate of 10% of the fish biomass; the other half did not receive pelleted feed and the fish depended on natural food. The

Table 1 Characteristics of the field studies and experiments performed Field experiment Objective

Where was carried out

Duration Treatments

Field measurement

Lab experiment feed

Lab experiment water

Relationship between heavy metal concentration in feed and its accumulation in fish. 100 liter plastic containers at Ornamental fish laboratory of Gan Shmuel fish farm

Relationship between heavy metal concentration in water and its accumulation in fish. 100 liter plastic containers at Ornamental fish laboratory of Gan Shmuel fish farm

4 months Heavy metals added to feed: Cd: 8, 9, 10, 18 Pb: 0.5, 1, 2, 10 times the maximum concentration allowed in the Israeli food standard for human consumption.

5.5 months Heavy metals added to water: As, Cd, Hg, Pb 1, 2, 10 times the maximum concentration allowed in the Israeli standard for unrestricted irrigation, containers with bottom sediment, and 10 times that standard, containers without bottom sediment.

7–9 fish/cont.(28 g) 7–9 fish/cont. (10 g) 7–9 fish/cont. (18 g) Feed pellets at a daily rate of 10% of the fish biomass at stocking, kept constant until harvest.

7–9 fish/cont.(27 g) 7–9 fish/cont.(19 g) 7–9 fish/cont.(16 g) Feed pellets at a daily rate of 10% of the fish biomass at stocking, kept constant until harvest.

As, Cd, Hg, Pb

Cd, Pb

As, Cd, Hg, Pb

Flesh Liver Bone

Flesh Liver Bone

Sediment Flesh Liver Bone

Check heavy metal accumulation in Complementary measurements of heavy fish reared in reclaimed wastewater. metals in fish reared for a long period in reclaimed wastewater. Reclaimed wastewater reservoir Burgata Reclaimed wastewater reservoir (after secondary treatment) Mishmar HaSharon (after tertiary treatment) and freshwater fishpond in Gan Shmuel fish farm (control) 5 months 2 years Cage culture in: Sampling of fish that were stocked Reclaimed water + feed 2 years before. Reclaimed water − feed Freshw. fishpond + feed Freshw. fishpond − feed

Fish⁎ C. carp H. Chin. carp Tilapia Feed supplied

30 fish/cage (12–22 g) 20 fish/cage (7–17 g) 30 fish/cage (25–30 g) Half of the cages received floating feed pellets at a daily rate of 10% of the fish biomass, adjusted monthly. Heavy metals As, Cd, Hg, Pb studied Heavy metals Water sampled in Sediment Fish flesh

⁎Number and stocking weight (g). ⁎⁎Number and mean weight of sampled fish.

⁎⁎ 5 (1.7 kg) 1 (3 kg) 2 (400 g) Not supplied

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fish were reared for five months (June–October). Once a month the fish were weighed, and the feeding rate was adjusted according to their weight. Water quality was monitored weekly – during the morning in the fish pond and at noon in the reclaimed water reservoir – for dissolved oxygen, ammonium, nitrite, pH, temperature and transparency (Secchi disc). 2.2. “Field measurements” (fish reared for 2 years in secondary reclaimed water) Eight fish were sampled for the analysis of heavy metals in flesh, liver and bones, from the 4.5 million cubic meter “Burgata” reservoir that receives secondary treated effluents from the Natania Sewage Treatment Plant (Table 1). These fish were stocked two years before in the reservoir for biological control of water quality and grew on natural food. The effluents entering the reservoir have BOD ~ 9 mg/l, TSS~ 11 mg/l, Total N ~ 17 mg/l and Total P ~ 4.5 mg/l. The heavy metal concentrations in the effluents entering the reservoir and in those within the reservoir are below the detection level of the analytical procedure: As b 0.015 mg/l, Cdb 0.003 mg/l, Pbb 0.01 mg/l and Hgb 0.001 mg/l.

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food quality were diluted in the water of the containers as cadmium acetate dihydrate, lead acetate, arsenic acid and methylmercury chloride. Three levels of heavy metals were applied in three replicates to the containers with sediments, and the highest level was also applied to the aquaria without sediments (“no-sed”). Each three days the water was changed to eliminate dirt and metabolites, and heavy metals were again added to the clean water. The concentration of the four heavy metals allowed in the Israeli requirements for unrestricted irrigation with reclaimed water (Inbar Commission, 2005) was used as a reference concentration (1WStd = Water Standard): As = 0.1 mg/l, Cd = 0.01 mg/l, Pb = 0.1 mg/l and Hg = 0.002 mg/l. The other treatments had double (2WStd) and 10 fold (10WStd and 10WStd-no-sed) that concentration. Initially, a 100 fold concentration level was also tried, but all fish died during the first 2 days and that treatment was dropped. The standard for unrestricted irrigation with reclaimed water was selected because in many cases fish are reared in reservoirs during the storage of runoff water for irrigation (Milstein et al., 1992) and reservoirs for the storage of reclaimed water for irrigation are a good potential place to rear fish in the future. 2.4. Sampling for heavy metal determinations

2.3. “Feed” and “water” laboratory experiments (fish reared in aquaria for 4–6 months) The laboratory experiments were carried out at the ornamental fish laboratory of Gan Shmuel fish farm, the “Feed” experiment (heavy metals added to commercial pelleted feed) during 4 months from 22nd Mar to 18th Jul 2004, and the ‘Water’ experiment (heavy metals added to water) during 5.5 months from 19th Aug 2004 to 25th Jan 2005. In both experiments twelve plastic containers of 100 l were stocked with hybrid Chinese carp, tilapia and common carp, at the densities and stocking weights indicated in Table 1. To avoid disturbing the fish, fish were not weighed during the experiment and a constant amount of commercial feed pellets equivalent to 10% of the fish biomass at stocking were supplied until harvest. It was observed that fish actually ate the feed. To eliminate dirt and metabolites, water was exchanged and dirt siphoned out periodically. Water temperature was kept constant at 23–24 °C. In the ‘Feed’ experiment, the accumulation of heavy metals in fish ingested via the feed was checked. Since the bioavailable forms of heavy metals are salts and organic compounds, but not the elemental forms (ARMCANZ, 2000) cadmium was added to the pellets as cadmium acetate dihydrate and lead as lead acetate. A posteriori we discovered that the commercial feed used in this experiment had a cadmium concentration 8 times higher than the standard for human food while the lead concentration in the feed was below detection level. The fish were fed with four different levels of heavy metals in the feed. For cadmium, the Food Standard is 0.05 mg kg− 1 dry weight and the levels tested were 8, 9, 10 and 18 times the food standard (treatments 8FStd, 9FStd, 10FStd and 18FStd respectively). For lead, the Food Standard is 0.2 mg kg− 1 dry weight and the levels tested were 0.5, 1, 2 and 10 times the food standard (treatments 0.5FStd, 1FStd, 2FStd and 10FStd respectively). To keep the environment clean, water was slowly and continuously replaced. In the ‘Water’ experiment, the accumulation of waterborn heavy metals in fish was checked. Sediments are a ‘trap’ for heavy metals that then accumulate in the bottom of water bodies (Juanicó et al., 1995; Storelli and Marcotrigiano, 2001; Wenchuan et al., 2001), and phytoplankton may also absorb waterborn heavy metals. To reproduce these conditions, nine of the containers had a small layer of sediments on the bottom and artificial illumination during 12 h a day with 3 light-bulbs of 500 W each with wavelength similar to that of natural light. The remaining three containers had no sediment on the bottom and no added illumination. Fish were fed commercial pelleted feed that had heavy metal concentrations below detection level. In this experiment the four heavy metals addressed by the Israel standard for

In the field experiment, samples of fish flesh (dorsal muscle without skin) were taken four times during the experiment. At the beginning, one fish of each species was sampled for initial values in the reclaimed water reservoir and fish pond. In the following sampling dates, one fish of each species was sampled from each cage. Water and sediment samples for heavy metal determinations were taken one month before fish stocking, and further sediment samples were taken at the end of the culture period. In the laboratory experiments, samples of fish bone (vertebral column) liver and flesh were taken at the beginning and end of the experiments. At the beginning, three fish of each species were collected, each tissue of all fish of the same species were combined in one sample, and these composite samples were considered to be representative of the corresponding initial values in all treatments. At harvest, up to three fish of each species were sampled from each container, and each tissue of all those fish were combined in one sample. In the ‘Water’ experiment samples of sediments were collected from each aquaria at the end of the experiment.

Table 2 Field experiment SGR (% day− 1)a

Survival (%)a

Carp

Hybrid Chinese carp

Tilapia

Carp

Hybrid Chinese carp

Tilapia

⁎⁎ 0.85 Sign %SS ⁎ 16 ⁎⁎ 60 ⁎ 24

⁎⁎⁎ 0.95 Sign %SS ⁎⁎ 12 ⁎⁎⁎ 85 ns 3

⁎⁎⁎ 0.91 Sign %SS ⁎⁎⁎ 94 ns 3 ns 3

⁎⁎⁎ 0.90 Sign %SS ns 5 ⁎⁎⁎ 74 ⁎⁎ 21

⁎⁎⁎ 0.91 Sign %SS ⁎9 ⁎⁎⁎ 76 ⁎⁎ 15

ns 0.40 Sign ns ns ns

Mean multicomparisons by location (n = 6) Reclaimed water 1.09 a_ 1.59 a_ Fish pond 0.99 _b 1.51 _b

1.71 a_ 1.47 _b

30 a 36 a

28 _b 36 a_

79 a 91 a

Mean multicomparisons by feed (n = 6) Feed 1.12 a_ 1.66 a_ No feed 0.92 _b 1.44 _b

1.57 a 1.61 a

50 a_ 15 _b

45 a_ 19 _b

88 a 82 a

ANOVA models Significance r2 Variance source Location Feed Location⁎feed

ANOVA and Scheffe mean multicomparisons of specific growth rate (SGR) and survival of each species. Significance levels: *b 0.05, **b 0.01, ***b 0.001, ns=not significant. %SS=percentage of total sums of squares accounted by each variance source. Mean multicomparisons: same letters within a column indicate no significant differences at the 0.05 level. a Statistical tests performed on transformed data. Values of means given untransformed.

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Table 3 Field experiment

Table 5 Field experiment

Element

Concentrations found in both the reclaimed water and the freshwater of the field experiment (mg/l)

Maximum concentrations allowed in drinking water standard — Israel (mg/l)

Maximum concentrations in proposed standard for unrestricted irrigation with reclaimed watera (mg/l)

Arsenic Cadmium Lead Mercury

b 0.015 b 0.003 b 0.01 b 0.001

0.05 0.005 0.01 0.001

0.1 0.01 0.1 0.002

Heavy metals found in the reclaimed water and the freshwater of the fishpond, compared with maximum allowed concentrations in drinking water and reclaimed water for unrestricted irrigation in Israel. a Proposed reclaimed water quality for unrestricted irrigation in Israel (The Inbar Commission, 2005).

2.5. Heavy metal determinations and data analyses In all experiments the fish, water and sediment samples were deep frozen pending analysis. Analyses of arsenic, cadmium and lead were performed with ICP-OES (Inductively Coupled Plasma — Optical Emission Spectroscopy) and those of mercury with Flame Atomic Absorption, at Bactochem laboratory. The detection limits of the measurements on liquid samples were 0.015, 0.003, 0.01 and 0.001 mg/kg dry weight respectively for As, Cd, Pb and Hg. The detection limits of the measurements on solid samples (fish tissues and sediments) were 0.2, 0.003, 0.1 and 0.05 mg/kg dry weight respectively for As, Cd, Pb and Hg. In the ‘Feed’ experiment only cadmium and lead were studied. Fish parameters and heavy metal concentrations were subjected to ANOVA. Tests on survival and specific growth rate (percentages) were performed on normalized data, obtained with the arcsine of the square root transformation (arcsin(sqrt(variable/100))). Regressions of heavy metals in fish on heavy metals in the environment (food and water) were run on the combined data of both laboratory experiments. The data of the ‘Water’ experiment provided the lower range environmental metal concentrations (Cd: 0.01–0.1, Pb: 0.1–1, Hg: 0.002– 0.02 mg/l = mg/kg) and the data of the ‘Feed’ experiment the higher concentrations (Cd: 0.4–0.9, Pb: 0.1–2 mg/kg; no Hg data available). 3. Results

Element

Arsenic Cadmium Lead Mercury

Field experiment: concentrations found in fish flesh (a)

Maximum allowed concentration in food standard in Israel

Concentrations commonly found in fish flesh (b)

Concentrations found in flesh of fish from pristine freshwater (d)

(mg/kg dry weight)

(mg/kg dry weight)

(mg/kg dry weight)

(mg/kg dry weight)

b 0.2 b 0.003 b 0.1 b 0.05

1 0.05 0.2 0.5

0.4–2 b 0.1 (c) 0.3–0.6 0.1–0.4

0.005–0.2 0.007–0.07 0.02–0.54 0.04–0.6

Heavy metals found in the flesh of fish reared in reclaimed wastewater, compared with the maximum concentrations allowed in food in Israel. (a) Concentrations in fish flesh were always below the detection limit. (b) Based on the Sidwell et al. (1978) review of 224 publications. (c) Values N 0 were rounded to 0.1 in the tables by Sidwell et al. (1978). (d) From Gutleb et al. (2002).

50 cm in October; the dissolved oxygen concentration at noon was usually oversaturated and never dropped below 5 mg/l; pH varied between 7.3 and 8.6. The nitrification/denitrification process in the sewage treatment plant that supplied the reclaimed water failed from mid-June to mid-August. As a result, ammonium concentrations reached 15–30 mg NH4/l within the reservoir (containing 1.7–3.3 mg NH3/l of toxic un-ionized ammonia at the recorded pH and temperature), and high nitrite concentrations (up to 5 mg NO2/l) occurred throughout the study period, which may account for the recorded low fish survival rates. In the fish pond a large phytoplankton population developed, dominated by the blue-green Mycrocistis sp; the algae decreased the Secchi disc visibility to 20–25 cm and increased the pH that varied from 7.9 to 9.5. The dissolved oxygen concentration was always high: 6 to 12 mg/l. Inorganic nitrogen compounds were low: the highest recorded concentrations were 0.5 mg NH4/l and 0.1 mg NO2/l. Almost no fish mortality occurred in this pond. 3.1.2. Fish growth and survival Table 2 shows the results of ANOVA on specific growth rate (SGR) and survival of each species. There were significantly higher SGRs of all species in the reclaimed water reservoir than in the fish pond. The survival rates of carp and hybrid Chinese carp were low in all treatments and that of tilapia was reasonable.

3.1. Field experiment (reclaimed water reservoir versus freshwater fishpond) 3.1.1. Water quality In both locations the minimum/maximum temperatures were 26/ 33 °C during the summer and decreased to 20/27 °C by late October. In the reclaimed water reservoir Secchi disc visibility decreased from 40 cm in early June to 25 cm by mid-July, and then increased to over

3.1.3. Heavy metals in water and sediments In water, the concentrations of heavy metals in both the reclaimed water reservoir and the freshwater fish pond were below the detection level of the analytical methods used, and below the recommended concentrations in drinking water and in reclaimed water for unrestricted irrigation (Table 3).

Table 4 Field experiment Element

Arsenic

Sampling time: start or end of experiment

Start End Cadmium Start End Lead Start End Mercury Start End

Concentrations found in the sediments of freshwater fishpond

Concentrations found in the sediments of reclaimed water reservoir

Maximum concentrations in sediments that have no effect on organismsa

Minimal concentrations in sediments that do have a negative effect on organismsa

(mg/kg dry weight)

(mg/kg dry weight)

(mg/kg dry weight)

(mg/kg dry weight)

1.16 b 0.2 b 0.003 b 0.003 1.45 3 b 0.05 b 0.05

1.5 b0.2 b0.003 b0.003 1.1 9 b0.05 b0.05

7.24 0.68 30.2 0.13

41.6 4.21 112 0.7

Heavy metals found in the sediments of both the reclaimed water reservoir and the fish pond, compared with minimal concentrations that do or maximal concentrations that do not have negative effects on organisms. a Effects on aquatic organisms (mainly algae, crustaceans and fish) determined by bioassays MacDonald et al. (1996), ARMCANZ (2000, vol 2, part 2, p.348).

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3.3. Laboratory ‘feed’ experiment

Table 6 Field measurements Fish

Metal

Flesh

Liver

Bone

Food standard

Carp (n = 5)

As Cd Pb Hg As Cd Pb Hg As Cd Pb Hg

b0.2 b0.05 b0.2 b0.5 b0.2 b0.05 b0.2 b0.5 0.4 b0.05 b0.2 b0.5

b0.2 0.8 b0.2 b0.5 b0.2 b0.05 b0.2 b0.5 b0.2 1.3 b0.2 b0.5

b 0.2 0.06 1.05 b 0.5 b 0.2 b 0.05 b 0.2 b 0.5 b 0.2 0.08 b 0.2 b 0.5

1 0.05 0.2 0.5 1 0.05 0.2 0.5 1 0.05 0.2 0.5

H. Chinese carp (n = 1)

Tilapia (n = 2)

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Heavy metals found in fish reared for 2 years in a reservoir receiving secondary treated effluents, compared with the maximum concentrations allowed in the food standard in Israel. Values in bold were above the allowed food standard. All values in mg/kg dry weight.

In sediments, some detectable concentrations of arsenic and lead were measured (Table 4). At the beginning of the experiment arsenic was detected in the sediments of both the reclaimed water reservoir and the fish pond, but no traces were found 6 months later. In the same period the lead concentration doubled in the fish pond sediments and increased almost nine fold in those in the reclaimed water reservoir. Cadmium and mercury were not detected at all. All concentrations were much lower than those that might have a negative effect on aquatic organisms. 3.1.4. Heavy metals in fish flesh In all the flesh samples of fish reared in both freshwater and reclaimed waste water, the concentration of the four analyzed metals were below the detection limits and much below the maximum concentrations allowed in food (Table 5). 3.2. Field measurements (fish reared for 2 years in a reclaimed water reservoir) After 2 years of growing in secondary treated reclaimed water, no heavy metals were found in the flesh of any of the eight fish analyzed (concentrations below the detectable limits and below the food standard). However, concentrations of Cd and Pb above the food standard were found in the liver and bones of some of the fish (Table 6).

After 4 months feeding on pelleted feed enriched with cadmium and lead, common carp increased about 30% its individual weight and the other species almost doubled it, without significant differences among treatments (P N 0.10). Survival in all species widely varied within treatments, thus no significant differences were found (P N 0.10). Without considering one container in which all fish died before the experiment was finished, the survival rates of carp and hybrid Chinese carp were 60% and that of tilapia was 80% on the average. Concentrations of cadmium and lead in bone, liver and flesh of the three fish species were under detectable limits before the beginning of the experiment. After 4 months, both heavy metals were detected in the different organs of the fish (Table 7). The ANOVA model applied explained most of the variability of Cd in each tissue (85–90%, see model r2), accounted for less of Pb variability in bone and liver (61 and 76%) and was not significant for Pb in flesh. 3.3.1. Cadmium Most of the explained Cd variability (87, 85 and 70% respectively for liver, bone and flesh, see %SS) was accounted for by its concentration in the pelleted feed (treatment), about 10% by the fish species, and about a further 10% by the fish⁎treatment interaction (not significant for liver). The treatment main effects (Table 7) show that Cd in liver, bone and flesh increased with Cd concentration in feed. The mean multicomparisons by species show that common carp concentrated significantly more Cd in liver and flesh than the other species (about double), while tilapia concentrated significantly more Cd in bone than the other species (about 25% more). On average, Cd concentration was over the allowed food standard in liver in all species-dose combinations and in bone above 9-fold the standard in all species and in tilapia also at the lowest concentration. Cd in flesh was not detected in any fish receiving pelleted feed with 8-fold the food standard; at the other doses, it was higher in common carp than in the other species, and over the allowed standard for food (Fig. 1). Cd in liver was over 20 times higher than in bone when its concentration in feed was up to 10-fold the standard, and almost 50 times higher when its concentration in feed was 18-fold the standard.

Table 7 Lab ‘feed’ experiment Liver ANOVA models Significance r2 Variance source Fish species Treatment Fish species⁎treatment

Bone

Flesh

Cd

Pb

Cd

Pb

Cd

Pb

⁎⁎⁎ 0.85 Sign. %SS ⁎8 ⁎⁎⁎ 87 ns 5

⁎⁎⁎ 0.76 Sign. %SS ⁎⁎ 24 ⁎⁎⁎ 45 ⁎ 31

⁎⁎⁎ 0.90 Sign. %SS ⁎⁎ 6 ⁎⁎⁎ 85 ⁎9

⁎ 0.61 Sign. %SS ⁎⁎ 42 ⁎ 34 ns 24

⁎⁎⁎ 0.88 Sign. %SS ⁎⁎⁎ 17 ⁎⁎⁎ 70 ⁎ 13

ns 0.27 Sign. %SS ns . ns . ns .

0.29 a_ 0.13 _b 0.04 _b

0.33 _b 0.28 _b 0.44 a_

2.35 a_ 1.68 a_ 0.55 _b

0.11 a_ 0.04 _b 0.05 _b

0.02 a 0.06 a 0.00 a

0.03 __c 0.29 _b_ 0.39 _b_ 0.70 a__

0.58 _b 1.60 ab 1.54 ab 2.40 a_

0 _b 0.03 _b 0.06 _b 0.18 a_

0.00 a 0.03 a 0.00 a 0.07 a

Mean multicomparisons by fish species (n) (mg/kg tissue dry weight) C. carp (11) 19.1 a_ H. Chinese carp (11) 10.7 _b Tilapia (11) 10.0 _b

Mean multicomparisons by treatment (heavy metals in pelleted feed) (n)(mg/kg tissue dry weight) Cd: 8FStd Pb: 0.5FStd (9) 0.8 __c 0. _b Cd: 9FStd Pb: 1FStd (9) 6.8 _bc 0.12 _b Cd: 10FStd Pb: 2FStd (6) 12.2 _b_ 0.11 _b Cd: 18FStd Pb: 10FStd (9) 33.1 a__ 0.38 a_

Results of ANOVA and Scheffe mean multicomparisons of Cd and Pb (mg/kg dry weight). Sign. =Significance levels: * b 0.05, **b 0.01, ***b 0.001, ns= not significant. %SS = percentage of total sums of squares. r2 = coefficient of determination. Mean multicomparisons: same letters in each column indicate no significant differences at the 0.05 level. a N bN …. Values of means are given untransformed.

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allowed food standard in all species-dose combinations except for tilapia at half standard dose. In all species Pb in liver was undetected when fish were fed half standard doses; for the standard dose the accumulation was detectable in all species but was under the allowed food standard; in the double dose it was only detected in common carp liver; and in the 10-fold dose it was twice higher in the carp than in the herbivorous Chinese carp, both of them over the allowed food standard, and several folds higher than in tilapia (Fig. 2). 3.4. Laboratory ‘water’ experiment After 5.5 months in water enriched with heavy metals, common carp and tilapia doubled their individual weight and hybrid Chinese carp did not grow. Survival of common carp and tilapia widely varied, with overall means of 71 and 82% respectively. Survival of tilapia was not significantly different among treatments (P N 0.30), while that of common carp was significantly lower in the treatment without sediments (P b 0.02). Survival of hybrid Chinese carp was very variable in the 1WStd and 2WStd treatments (from 0 to 78%), and did not survive at all in the 10WStd and 10WStd-no-sed treatments.

Fig. 1. ‘Feed’ experiment. Fish⁎treatment cross effect for Cd in liver, bone and flesh. Thick horizontal line marks the Cd concentration allowed in the food standard. Initial Cd concentrations in each organ were below detectable limits.

3.3.2. Lead Lead accumulation shows different patterns than those shown by Cd. In liver, the main variability source was treatment (45%) followed by the fish⁎treatment cross effect (31%) and by fish species main effect (24%). In bone, most (42%) of the accounted variability of Pb was due to fish species followed by treatment (34%). In flesh, differences were not significant (no fish, treatment or fish⁎treatment effects on Pb accumulation in flesh). The species main effects (Table 7) show that both cyprinid species concentrated significantly more Pb in bone than tilapia (about four times more), while common carp concentrated significantly more Pb in liver than the other species (about triple). The treatment main effects show that in liver Pb was significantly higher when fish were fed 10-fold standard Pb dose, while in bone Pb was significantly higher when fish were fed 10-fold standard doses than half standard doses, with the other two treatments in an intermediary position not significantly different from either. In all species-dose combinations Pb in flesh was either undetectable or under the allowed food standard. In bone, Pb was over the

Fig. 2. ‘Feed’ experiment. Fish⁎treatment cross effect for Pb in liver, bone and flesh. Thick horizontal line marks the Cd concentration allowed in the food standard. Initial Pb concentrations in each organ were below detectable limits.

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Table 8 Lab ‘water’ experiment Liver ANOVA models Significance r2 Variance source Fish species Treatment Fish species⁎treatment

Bone Hg

Pb

Cd

Hg

Pb

⁎⁎ 0.69 Sign. %SS ns 10 ⁎⁎ 67 ns 23

⁎⁎⁎ 0.81 Sign. %SS ns 1 ⁎⁎⁎ 93 ns 6

ns 0.40 Sign. %SS ⁎. ns . ns .

ns 0.41 Sign. %SS ns . ns . ns .

⁎ 0.62 Sign. %SS ns 6 ⁎⁎ 83 ns 11

ns 0.27 Sign. %SS ns . ns . ns .

0.03 a 0.12 a

1.18 a 0.80 a

0.38 a 1.24 a

0.35 _b 0.11 _b 1.92 a_ 1.68 a_

0.89 a 0.54 a 1.22 a 0.64 a

Mean multicomparisons by fish species (n) (mg HM/kg tissue dry weight) Common carp (11) 0.70 _b 10.85 a 1.16 a_ Tilapia (12) 1.18 a_ 15.28 a 0.06 _b Mean multicomparisons 1WStd 2WStd 10WStd 10WStd-no-sed

Flesh

Cd

by treatment (heavy metals in water) (n) (mg HM/kg tissue dry weight) (8) 0.56 _b 6.55 __c 0.26 a 0.00 a (9) 0.57 _b 3.07 __c 0.43 a 0.05 a (6) 1.10 ab 17.0 _b_ 0.86 a 0.09 a (5) 1.72 a_ 28.6 a__ 0.84 a 0.21 a

Cd

Hg

Pb

⁎⁎⁎ 0.86 Sign. %SS ns 0 ⁎⁎⁎ 97 ns 3

ns 0.30 Sign. %SS ns . ns . ns .

0 0

5.54 a 5.08 a

0.01 a 0.00 a

0 0 0 0

1.26 _b 1.03 _b 10.76 a_ 8.70 a_

0.03 a 0a 0a 0a

Results of ANOVA and Scheffe mean multicomparisons of Cd, Hg and Pb (mg/kg dry weight). Significance levels: * = 0.05, ** = 0.01, *** = 0.001, ns = not significant. %SS = percentage of total sums of squares. r2 = coefficient of determination. Mean multicomparisons: same letters in each column indicate no significant differences at the 0.05 level. a N bN.

As, Cd, Hg and Pb concentrations in liver, bone and flesh of the three fish species was under detectable limits before the beginning of the experiment. At harvest, three heavy metals were detected in fish while arsenic was under detectable limits in all samples except one. For the three detected metals, results of 2-way-ANOVA analyses by fish species and treatment are presented in Table 8 for liver, bone and flesh. Due to the strong imbalance caused by the total mortality of hybrid Chinese carp in two treatments, this fish was not included in the ANOVA analyses. Figs. 3, 4 and 5 present the mean values of Cd, Hg and Pb in each tissue by species and treatment at harvest, including hybrid Chinese carp. The ANOVA models applied were significant for Hg in the three tissues and for Cd in liver, but were not significant for Cd in bone and flesh. The models were not significant for Pb in any tissue. The significant models explained over 80% of the variability of Hg in liver and flesh, and 62 and 69% of the variability of Hg in bone and Cd in liver respectively. 3.4.1. Mercury Most of the explained Hg variability (93, 83 and 97% respectively for liver, bone and flesh) was accounted for by its concentration in the water (treatment), with no significant effects of fish species and fish⁎treatment interaction. Hg in liver, bone and flesh was significantly higher when Hg concentration in the water was 10-fold the standard dose than when the dose in the water was 1 or 2 standards. This was similar in all fish species (Fig. 3). In liver it was especially so when no sediments were present in the aquaria. For each Hg dose in water, its concentration in liver was an order of magnitude higher than in bone, and by far over the allowed standard in food (0.5 mg/kg). At 10-fold Hg concentration in water, the Hg concentration in bone was 3–4 fold over the standard allowed in food, while at 1 or 2 standards in the water Hg concentration in the bone was under the standard allowed in food. At 10-fold Hg concentration in water, the Hg concentration in flesh was more than 15-fold over the standard allowed in food, while at 1 or 2 standards in the water Hg concentration in the flesh was over twice the standard allowed in food. 3.4.2. Cadmium Cd was under detectable limits in all flesh samples, thus ANOVA was not run. Mean Cd concentrations in bone were under the allowed standard in food when its concentration in water was low (1WStd) and were above the standard at the 10-fold standard treatments, mainly in tilapia (Fig. 4). However, due to the large variability of the data this trend was not significant (Table 8). Almost 70% of the explained Cd variability in liver was accounted for by its concentration in the water. Cd in liver was significantly

higher when Cd concentration in the water was 10-fold the standard dose, mainly when no sediments were in the aquaria, than when the dose in the water was 1 or 2 standards. In all cases Cd concentration in liver was at least one order of magnitude higher than the standard allowed in food (Fig. 4). 3.4.3. Lead There were no significant differences in Pb concentrations in any tissue due to treatment and/or fish species. Pb levels were over the allowed in food standards (a) in liver of carp in all treatments, (b) in bone of all species and treatments, and (c) in the flesh of hybrid Chinese carp that received 1WStd and 2WStd doses in the water. No Pb was found in hybrid Chinese carp liver at the low Pb doses and at any Pb concentration in the water in tilapia flesh (Fig. 5). 3.5. Regression of heavy metals in fish on heavy metals in the environment (‘feed’ and ‘water’ experiments) The relationships between concentrations in bone, flesh and liver and environmental concentrations were analyzed through linear regression combining data of both lab experiments (Table 9). Arsenic in fish was always under detection limits in spite of the increase of As in the environment. Pb in fish did not significantly increase as Pb in the environment increased. Cd in fish did increase as Cd in the environment increased. The regressions were highly significant with moderate correlations (r2 in the range 0.50–0.75). As expected, all intercepts were not significantly different from zero (no Cd in fish when there was no Cd in the environment). The slopes of the regressions indicate that for each increase of 1 mg/kg in the concentration of Cd in the water or feed, the concentration of Cd in flesh of fish increased by 0.17 mg/kg, in bone by 0.66 mg/kg and in liver by 32 mg/kg (more in common carp than in the other two species for the three organs). Also Hg in fish did increase as Hg in the environment increased. The Hg regressions were highly significant for each organ, with high correlations for flesh (r2 range 0.75– 0.94) and moderate for bone and liver, but the number of observations available were rather low to consider these results more than a trend (only data from the ‘Water’ experiment available). 4. Discussion 4.1. Differences between fish species Of the three species studied the filter feeder hybrid Chinese carp was the most sensitive to environmental conditions and heavy metals.

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4.2. “Food path” versus “water path” When the heavy metals were added in the laboratory experiments, the “water path” led to lower levels and fewer differences in heavy metal accumulation among species than the “feed path”. This is in agreement with the findings of Farkas et al. (2002), who in a study of Cd, Cu, Hg, Pb and Zn in the benthivorous fish bream Abramis brama of Lake Balaton, Hungary, concluded that the main route for the uptake of heavy metals for adult fish was by feeds and that this route was affected by the contents in heavy metals of the sediments and its biota rather than the ambient water. Also Liang et al. (1999) studying Cd, Cr, Cu, Ni, Pb and Zn in various fish species that were raised in secondary treated wastewater, found that heavy metal concentrations in the fish were more affected by their concentrations in the food than by those in the water. Metal accumulation in the food chain may explain the stronger effect of the food path than the ambient water: absorption of heavy metals from swallowed water and through gills exposes fish to the concentrations occurring in the ambient water, while feeding on organisms that already concentrated the heavy metals present in the environment increases amounts of metals ingested by fish. Whether heavy metals enter the fish via the “food path” or the “water path”, the relationship between the increase in environmental concentration and increase in body concentration was different for each metal and organ (Table 9). 4.3. Heavy metals in fish organs Flesh (muscle) is the most important fish organ regarding accumulation of toxic compounds because it is the most consumed one. Jezierska and Witeska (2001:80) in a comprehensive review of heavy metals in fish state that “Fish liver and kidneys are the most burdened organs. Muscles, compared with other tissues, usually contain low levels of metals. Sometimes, even when concentration in liver is high, muscle levels may be undetectable. Hg is probably the only exception where flesh concentration may exceed other organs.”

Fig. 3. ‘Water’ experiment. Fish⁎treatment cross effect for Hg in liver, bone and flesh. Thick horizontal line marks the Hg concentration allowed in the food standard. Empty columns indicate undetectable Hg level, except for hybrid Chinese carp in the 10WStd and 10WStd-no-sed treatments in which no fish survived. Initial Hg concentrations in each organ were below detectable limits.

It was the only species with significantly lower survival in reclaimed water than in the fish pond, and at the highest heavy metal concentrations in the water it did not survive at all. Tilapia was the strongest species, with relatively high survival in both field and laboratory experiments. The expected differences in heavy metal accumulation in fish fed with pelleted feed and fish feeding on the natural food web were not observed, since no heavy metals were detected in the flesh of any fish in the field experiment. Reviewing a large amount of literature, Jezierska and Witeska (2001:93) showed that various species of fish from the same water body may accumulate different amounts of metals, and that interspecific differences may be due to feeding habits. Thus, more Hg accumulates in predators, while more Cd and Pb accumulate in bentophagous than in pelagic fish. This trend can be seen in our ‘feed experiment’ data. While the three fish species concentrated most of the ingested Cd in liver and most of the ingested Pb in bone, the bentophagous common carp concentrated more Cd and Pb in liver and more Cd in flesh than the omnivorous tilapia and the pelagic grazer hybrid Chinese carp. However, tilapia concentrated more Cd and less Pb in bone than the other two species.

Fig. 4. ‘Water’ experiment. Fish⁎treatment cross effect for Cd in liver and bone. Thick horizontal line marks the Hg concentration allowed in the food standard. Empty columns indicate undetectable Cd level, except for hybrid Chinese carp in the 10WStd and 10WStd-no-sed treatments in which no fish survived. Initial Cd concentrations in each organ were below detectable limits.

M. Feldlite et al. / Aquaculture 284 (2008) 115–126

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this experiment and in fish reported to grow in natural pristine waters (Table 5). Other researchers have also reported very low concentrations of heavy metals in the flesh of fish raised in ponds with a range of reclaimed water types. Fattal and Shuval (1997) studied Hg, Cd, Cu and Zn in tilapia and mullet raised in freshwater fish ponds in Israel (there was no reclaimed water in the fishponds). The concentrations found in the fish were one to two orders of magnitude lower than the values allowed by Israeli food standards. Easa et al. (1995), Shereif et al. (1995) and Shereif and Mancy (1995) studied Pb, Cu, Zn and Cd in tilapia raised in fish ponds that received wastewater, in a research station in Egypt. The concentration of heavy metals in the fish increased during the culture period, but after 4 months they were still lower than those allowed by the food standards. Khalil and Hussein (1997) reported levels of Pb, Zn and Fe that were within international food standards in the flesh of Nile tilapia (O. niloticus) raised during 9 months in secondary treated waste effluents. Liang et al. (1999) studied Zn, Cu, Cd, Cr, Pb and Ni in six fish species including those herein studied, that were raised in secondary treated wastewater during over 1 year; they found heavy metal accumulation in the pond sediments and in the food web (phytoand zooplankton), but the concentrations in the fish flesh were within the range allowed by food standards. Although our field measurements have to be considered as preliminary due to the few samples obtained in only one reservoir, they also support the conclusion that heavy metals do not accumulate in flesh in fish reared in good quality reclaimed water for a long time (two years in our case). When heavy metals were purposely added to pelleted feed (“feed experiment”, only Cd and Pb tested) Cd in flesh was undetected in the three fish species even when its concentration in the feed was 8-fold the allowed in the food standard, and was over the allowed standard only when its concentration in the feed was over 9–10 fold this standard. Pb in flesh was always below the food standard. Thus, the “feed Table 9 Combined data of ‘feed’ and ‘water’ lab experiments Heavy metal

Fish species

Organ

n

Cd

All together All together

All together Bone Flesh Liver Bone Flesh Liver Bone Flesh Liver Bone Flesh Liver All together Each of them Each of them Each of them Each of them All together Bone Flesh Liver Bone Flesh Liver Bone Flesh Liver Bone Flesh Liver

204 68

Significance

C. carp

Fig. 5. ‘Water’ experiment. Fish⁎treatment cross effect for Pb in liver, bone and flesh. Thick horizontal line marks the Hg concentration allowed in the food standard. Empty columns indicate undetectable Pb level, except for hybrid Chinese carp in the 10WStd and 10WStd-no-sed treatments in which no fish survived. Initial Pb concentrations in each organ were below detectable limits.

H. Chin. carp

Tilapia

Numerous recent studies have confirmed that liver, kidneys, gills and bones in fish have much higher concentrations of heavy metals than muscle and skin, with Hg as an exception (e.g., Hamza-Chaffai et al., 1996; Zhou et al., 1998; Storelli and Marcotrigiano, 2001; Wong et al., 2001; Coetzee et al., 2002; Farkas et al., 2002). The findings of the experiments and measurements herein presented also confirm this fact: with the exception of Hg, the concentration of heavy metals in flesh was usually lower than in liver and bones. 4.4. Heavy metals in fish flesh To attain a safe standard for heavy metals in reclaimed water used for fish aquaculture, the length of the culture season should be considered. The results of the “field experiment” indicate that fish reared during a short growing season (5 months) in good quality reclaimed water do not accumulate heavy metals in their flesh (“good quality reclaimed water” herein means treated municipal wastewater where the concentration of heavy metals was below the detection level as listed in Table 3). The concentration of heavy metals observed in the flesh of fish reared in reclaimed water was similar to that found in fish reared in freshwater in

Pb

Hgb

Regression

All together All together C. carp H. Chin. carp Tilapia All together All together

C. carp

H. Chin. carp

Tilapia

24

19

25

204 68 24 19 25 96 32

12

7

13

ns ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ns ns ns ns ns ns ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ ns ⁎ ⁎⁎⁎ ⁎⁎

r2 0.60 0.51 0.64 0.72 0.73 0.75 0.60 0.37 0.66 0.56 0.63 0.64

Slopea 0.66 0.17 32.1 0.74 0.27 44.3 0.59 0.09 28.7 0.66 0.13 23.7

0.53 0.83 0.56 0.70 0.91 0.66 0.77 0.94

88 492 987 129 550 969 55 434

0.40 0.75 0.61

59 464 1066

Linear regressions of heavy metal concentrations in fish on heavy metal concentrations in environment. Significance levels: * b 0.05, ** b 0.01, *** b 0.001, ns = not significant. a All intercepts were not significantly different from zero. b For Hg only data from ‘Water’ experiment.

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experiment” indicates that the concentration of Cd and Pb in the fish food sources (pelleted feed, sediments, natural food web) should be at least 10-fold higher than the accepted food standard in order to find a significant amount of heavy metals in the flesh of fish. Zhou et al. (1998) and Wong et al. (2001) studied the level of Cd and Pb in different tissues respectively of inland and marine fish cultured in different polluted sites of Hong Kong. These fish were fed mainly with trash fish from fisheries, small fish and benthic organisms trawled from severely polluted areas and they also ingest sediments. The Cd and Pb concentrations found in fish flesh were below the maximum permitted in seafood by the Hong Kong Government, but some values were higher than the allowed Israeli standard for food consumption. However, the concentration of these metals in the water and sediments of the Hong Kong sites where the fish were reared were orders of magnitude higher than those in our experiments. When heavy metals were purposely added to water (“water experiment”) in concentrations up to 10-fold the wastewater standard for unrestricted irrigation, As and Cd in flesh of the three fish species and Pb in tilapia were undetected, and Pb in flesh of common carp was under the allowed Israeli standard for food consumption. Khalil and Hussein (1997) obtained different results regarding Pb: they reared Nile tilapia during 9 months in secondary effluents with Pb = 1 mg/l (10 fold the Israel wastewater standard for unrestricted irrigation) and found that the concentration of Pb in flesh was 10 fold the food standard. These findings by Khalil & Hussein contradict those found in the present study, but they still confirm the safety of the Israel wastewater standard regarding Pb. When heavy metals were purposely added to water, only Hg in flesh was found in higher concentrations than the allowed in the food standard, even when the added Hg was equivalent to the wastewater standard for unrestricted irrigation. High Hg in flesh was reported by several authors (e.g.: Jezierska and Witeska 2001, quote 66 such references in their Table 4.6; Farkas et al., 2002) and has been attributed to a higher affinity of methylmercury for binding to protein in fish muscle (Laarman et al., 1976).

kidneys – is the fish organ that most concentrates heavy metals (Jezierska and Witeska, 2001:68). Higher heavy metal levels in liver than in other organs were recorded by almost all the works discussed above in the section ‘Heavy metals in fish flesh’. Khalil and Hussein (1997) found that the Pb in the liver of Nile tilapia was 10% higher in fish reared in primary effluents than in fish reared in secondary effluents and more than twice higher than in fish reared in groundwater. The findings herein presented confirm the higher heavy metal accumulation in liver: fish reared during two years in good quality reclaimed water and fish exposed to heavy metal either in feed or in the water (at all levels tested) presented Cd in liver above the allowed food standard. Also common carp exposed to Pb either in feed or in the water and all species exposed to Hg in water accumulated those metals in the liver above the food standard. The ‘food’ and ‘water’ experiments showed that while Cd and Pb efficiently entered into the fish body through feed and water, the detoxification and filtering mechanisms that ultimately prevented their accumulation in the fish flesh were different. Cd was retained in the liver of all species in all concentrations tested. At the highest Cd concentration in feed and water the liver could not retain all of it and build up in the bones began. Differently, dietary Pb was retained in the bones of all species even when its concentration in the feed was rather low (half of the allowed standard dose), and the liver acted as a further filter mainly in both carp species. For waterborn Pb, bone was the primary filter of tilapia and liver the primary filter of common carp. Hg presented another pattern. Waterborn Hg was retained in the liver of common carp and tilapia in all concentrations tested. The presence of sediments in the aquaria should have trapped part of the Hg reducing its availability to the fish, leading to lower Hg accumulation in the liver. At the highest Hg concentration in water the liver could not retain all of it and build up mainly in flesh and also in bones began.

4.5. Heavy metals in bone

The concentrations of heavy metals found in reclaimed water in the present study were below the Israeli standards for drinking water and for reclaimed wastewater for unrestricted irrigation. The lack of heavy metal accumulation in the fish flesh after 5 months in reclaimed water indicates that, from the point of view of heavy metals, this water resource could be safely utilized for fish culture for periods of about half a year, which matches quite well the length of the fish culture season in Israel. The preliminary results of the “field measurement” and literature data indicate that even a much longer growing season of two years should be safe concerning the level of heavy metals in the flesh. However, levels of heavy metals in the liver and bones could be unsafe for consumption. The “feed” and “water” laboratory experiments herein presented and the large amount of bibliography consulted and discussed, further confirms that fish reared in reclaimed water matching the proposed wastewater standard for unrestricted irrigation do not accumulate As, Cd and Pb in flesh above the food standard, but might accumulate Hg. Much higher concentrations of As, Cd and Pb in water are needed to actually increase the concentrations in the flesh of the fish beyond the food standard. Thus, human consumption of flesh of fish reared on reclaimed water matching the quality proposed in the Israel “Inbar Commission” standard for unrestricted irrigation is safe from the heavy metal point of view, except for Hg where a lower value is required. The data herein presented indicate that the concentration of heavy metals in liver of fish reared on good quality reclaimed water may be higher than the food standard for human consumption. Also Wong et al. (2001) found that “The concentrations of Cd, Cr, and Pb in muscle and skin of fish, are well within acceptable limits for human consumption. However, consumption of internal organs of fish, such as liver and gonad, is probably not advisable”. A controversial aspect of

Fish bones or carcasses are almost not used for direct human consumption, except in the preparation of fish soups and gelatins. Fish bones are not separated from flesh in the preparation of fishmeal, which is an important ingredient of fish and livestock industrial pelleted feeds. Although fishmeal is not produced in Israel nowadays, the subject is relevant for future production and for imported fishmeal produced from fishes reared in reclaimed water. Jezierska and Witeska (2001:83–84) state that Cd and Pb may accumulate in bone in higher concentrations than in the rest of the body. The findings herein presented confirm this statement: fish reared during two years in good quality reclaimed water presented Cd and Pb in bones above the food standard. The “Feed experiment” indicates that Cd and Pb in bones are a function of their concentration in the feed. Experiments with the Atlantic salmon have demonstrated that only 2%–3% of dietary Cd is retained in the carcasses of fish fed with elevated dietary levels (Lie, 2001) but the experiments herein presented show that this may be enough to exceed the Israeli food standard. The “Water experiment” indicates that both Cd and Pb accumulate in fish bone even at the concentration allowed by the reclaimed water standard for irrigation. Differently, Hg in bones is a function of Hg in water, but a concentration in water 10-fold the reclaimed water standard for irrigation is required to reach the Hg food standard in bones. 4.6. Heavy metals in liver The fish liver is much less consumed than fish flesh, but still its consumption may be significant. Liver – together with gills and

4.7. Towards a standard for heavy metals in reclaimed water for fish aquaculture

M. Feldlite et al. / Aquaculture 284 (2008) 115–126

this problem is that the consumption of liver is much limited than the consumption of flesh, and thus it is arguable whether the food standard, that is based on a consumption-uptake basis, should be applicable to both liver and flesh on an equal basis. Alternative approaches to overcome this problem are: • The experts commission on food standards may reevaluate the food standard for the specific case of fish liver. • The experts commission on food standards may set a standard for Hg, Cd and Pb in reclaimed water stricter than the one proposed in the Israel “Inbar Commission” for unrestricted irrigation. It must be noted that the Inbar Commission also approved a set of values for the release of reclaimed water to rivers with stricter values for Hg, Cd and Pb. But, the standard for release of reclaimed water to rivers has less applicability than the one for unrestricted irrigation because, as quoted in the introduction, water for irrigation is accumulated in reservoirs where fish can be reared. • The liver from fish reared in reclaimed water will be discharged (only flesh will be consumed). The idea to consume only gutted fish in order to overcome the problem of heavy metals in the viscera has already been proposed by van den Heever and Frey (1996). Similar considerations apply to bone of fish reared on good quality reclaimed water. Although bone is almost not used for direct human consumption, it might be important in fish and livestock industrial feeds. In any case, monitoring of HM in fish pelleted feed is required because concentrations may be surprisingly high. 5. Conclusions The standards for unrestricted irrigation with reclaimed water are a good basis for the development of a water quality standard for growing fish in reclaimed water, because fish can be grown in the reservoirs used to storage the reclaimed water and/or this is the reclaimed water than can be used to fill fish ponds. Except for Hg, the proposed Israel requirements for heavy metals in reclaimed water used for unrestricted irrigation (As = 0.1 mg/l, Cd = 0.01 mg/l, Pb = 0.1 mg/l, Hg = 0.002 mg/l) are safe regarding the consumption of flesh from fish reared in reclaimed water, even if the growing season is long (up to two years). These requirements are not equally safe regarding the consumption of liver (and probably other viscera). Alternatives to overcome this problem may be a reevaluation of the food standard for liver, a stricter standard for heavy metals in reclaimed water for irrigation, or the discharge of the liver (and other viscera) of fish reared in reclaimed water. The concentration of heavy metals in fish pelleted feed may be surprisingly high. The active monitoring of heavy metals in all fish feed should be routinely carried out to assure the safety of the public. Acknowledgements This research was funded by a grant of the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development, and the Israel Water Commission. References ARMCANZ — Agriculture and Resource Management Council of Australia and New Zealand, 2000. National water quality management strategy: Australian and New Zealand guidelines for fresh and marine water quality. Prepared under the auspices of Australia and New Zealand Environment and Conservation Council and Agriculture and Resource Management Council of Australia and New Zealand. 6 volumes. Blumenthal, U., Peasey, A., Ruiz, G., Mara, D., 2000. Guidelines for wastewater reuse in agriculture and aquaculture: recommended revisions based on new research evidence. WELL Resource Centre, report to DFID — Department for International Development-UK. 67 pp. Buras, N., Duek, L., Niv, S., Hepher, B., Sandbank, E., 1987. Microbiological aspects of fish grown in treated wastewater. Water Res. 21 (1), 1–10.

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