1. How to measure of heavy metal concentration?

Answer:
A. Heavy metals in water
1. Water samples were collected randomly from Saft-El-Henna drainage
canal, it is about 10-km South-East Zagazig city, Egypt.
2. Water sample was taken below the water surface in the river using one
(1) liter acid-leached polythene bottles and keep in ice while being
transported to the laboratory.
3. Synthetic mixtures of heavy metal were prepared using different salts of
the heavy metals (Merck & Co Inc., NJ, USA), lead nitrate (Pb(NO3)2),
cadmium chloride (CdCl2.2.5H2O), zinc sulfate (ZnSO4), copper sulfate
(CuSO4.5H2O), which were dissolved in distilled water and then added to
the aquarium.
4. The samples were filtered via 0.8 m Millipore acetylcellulose filter
paper (Millipore, Bedford, MA, USA), digested with 10 mL concentrated
H2SO4 on a hot plate at 70C, concentrated to 50 mL, and transferred to a
volumetric flask. Samples were adjusted up to 100 mL with redistilled
water.
B. Heavy metals in fish body
1. Samples (flesh) were oven-dried at 85 C until constant weight and 1.0 g
dry weight was ashed in a muffle furnace for 6 h.
2. Ash was digested with 5 mL concentrated H2SO4 and kept at 130C on a
hot plate until complete dryness. Then, the digests were diluted with 2 N
HCl and transferred to 25 ml volumetric flask and fill up to the level with
de-ionized water.

Heavy metals concentrations were determined with an atomic absorption

spectrophotometer (AAS) (Thermo 6600, Thermo Electron Corporation, Cambridge,
UK), which was calibrated using standard solutions. Atomic absorption spectrometric
(AAS) methods are very suitable methods for monitoring the levels of heavy metals in
natural waters.
 Water Fish

Water put into the bottle (1 L) Samples were dried with oven
(85oC)

Water keep in ice while being

transported to the laboratory. 1.0 g dry weight was ashed in a
muffle furnace for 6 h.

Ash was digested with 5 mL

Add Synthetic of heavy metal
concentrated H2SO4 and kept at
mixtures (lead nitrate (Pb(NO3)2),
130C on a hot plate.
cadmium chloride (CdCl2.2.5H2O),
zinc sulfate (ZnSO4), and copper
sulfate (CuSO4.5H2O) The digests were diluted with 2 N
HCl and transferred to 25 ml
volumetric flask and fill up to the
level with deionized water.
Filtered with 0.8 m Millipore
acetylcellulose filter
paper

Dingested with 10 mL concentrated

H2SO4 on a hot plate at 70C,
concentrated to 50 mL.

Ttransferred to a volumetric flask

Samples were adjusted up to 100

mL with redistilled water.

Heavy metals concentrations were

determined with an atomic
absorption spectrophotometer
(AAS)

Figure 1. Procedure of heavy metal analysis in water and fish body

2. What is the effect of activated charcoal (AC) on glucose level and biochemical
parameters?
Answer:
A. Glucose
Blood glucose is an important factor for the organism because it is a source of
energy and can be used as a biomarker against the physiological conditions
of fish, especially in fish that experience stress. Blood glucose is very
sensitive to hormones that regulated stress such as cortisol. Cortisol has a
metabolic effect that increases blood glucose concentration by using protein
and fat stores.
Heavy metal content in water can cause stress in fish. The occurrence of
stress can increase glucocorticoids resulting in elevated blood glucose levels
to meet high energy requirements. If the glucose level fish blood is abnormal
then it will affect the growth of fish even cause death.
The addition of activated charcoal to the fish feed helps reduce blood glucose
levels in fish under stressful conditions. The adsorption of glucose by charcoal
occurring in the bowel has resulted in decreased dosage of insulin or oral
sugar-reducing agents.

B. ALT and AST

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are
enzymes found mainly in the liver, but also found in red blood cells, heart
cells, muscle tissue and other organs, such as the pancreas and kidneys.
The mechanism of organic solvent induced liver injury is known to be
increased oxidative stress. Thus, when the liver is exposed to chemicals
compounds, the liver could be damaged additively by the same mechanism of
oxidative stress.
The high ALT and AST concentration in heavy metal exposure are indicate to
liver dysfunction. These enzymes are normally present in liver cells, and there
is a certain normal level of these enzymes circulating in the bloodstream.
When the liver becomes inflamed or damaged, these enzymes are released
into the bloodstream in abnormal amounts by the damaged cells. This results
in elevated levels in the bloodstream.
The level ALT and AST decrease in the AC-rich diet. The addition of activated
charcoal to fish feed can help the process of detoxification of heavy metals in
the body fish because of its capability to absorb gases especially nitrogen
 and ammonia, activate the intestinal function, and eliminate the poisons and
impurities from the gastrointestinal tract of animals (Banner et al., 2000)

C. Uric acid and creatinine

Uric acid, a degradation product of purine nucleotides and protein catabolism,
produced primarily in the liver and white muscle of fish is generally converted
to urea for excretion. Creatinine is a breakdown product of a specific type of
muscle protein. It is formed in certain muscles and is carried via the
bloodstream to be eliminated by the kidneys. Level of uric acid and creatinine
in the blood provide a very accurate estimation of how well the kidneys are
working.
Uric acid and creatinine are screening indices for kidney function and renal
structural integrity. Therefore, the elevated uric acid and creatinine
concentrations in HM-exposed fish are indicative of kidney dysfunction, which
led to the leakage of these molecules from the specific organs into the
bloodstream.
Activated charcoal is one of the most common medicine in clinic renal failure
treatment for lowering creatinine. The main ingredients in medicine are
polymer carbide which can absorb the creatinine, uric acid and other toxins in
gastrointestinal tract quickly, lightening the burden of kidney excreting toxins,
so that the process of renal fibrosis can be delayed.

D. Total Protein and Total lipid

The low protein and lipid in plasma of HM-exposed fish may be due to the
breakdown of those molecules as energetic substrates to cope with stress
metabolically and because there is an improvement taking energy from
protein to meet energy needed during stress.
The increase in total protein and total fat content after heavy metal exposure
caused by the influence of activated charcoal on fish feed. The activated
charcoal supplementation could elevate the absorptive function of intestinal
villi, epithelial cells in the intestine and increased the height of intestinal villi,
where the longer villi result in an increased surface area that is capable of
greater absorption of available nutrients.