Levels of lead and copper in plasma of dairy cows, pastures, soil and water from selected areas of Morogoro suburbs, Tanzania

P S Mlay and Y O Mgumia

Department of Physiology, Biochemistry, Pharmacology and Toxicology, P. O. Box 3017, Morogoro, Tanzania
panejo@yahoo.com

Abstract

Knowledge of trace and toxic metal concentrations in livestock, feeds, water and soil is important for assessing the effects of pollutants on domestic animals and contaminant intakes by human. A study was conducted in Morogoro municipality to determine the levels of lead and copper in plasma of dairy cattle, in pastures, soils and water from two areas, Kingorwila and Mzinga suspected to be polluted with these metals. Forty eight blood samples from cows, nine pastures, three soil and three water samples were used. Dry matter of soils and pastures was determined by drying in hot air oven to constant weight at 60^oC. Lead and copper assay were done using the standard procedures and read by the atomic absorption spectrophotometer. PCV, Hb, and blood cell counts were done by standard procedures involving the use of hematocrit tubes, Sahli haemometer and haemocytometer sets respectively.

Kingolwira cows had significantly (P<0.05) higher plasma lead levels and significantly lower (P<0.05) values of RBC counts compared to Mzinga cows. Plasma levels of lead and copper in the cows from both locations (0.27 and 0.98mg/l respectively for Mzinga and 0.45 and 0.93mg/l respectively for Kingorwila) were within the acceptable limits. Pasture, soil and water levels of lead and copper ranged between 3.5 - 3.6mg/kg, 11-31mg/kg and 0.0-0.01kg/l respectively; and all were within the acceptable levels.

It is hereby concluded that levels of lead and copper in pastures, soils and water in Mzinga and Kingorwila areas in Morogoro does not pose a threat to animals and man.

Key words: blood, blood cell counts, heavy metals, hemoglobin, packed cell volume

Introduction

Lead and copper are among the heavy metals implicated to cause toxicity in animals and man (Roberts 1999). This is due to wide spread environmental pollution by materials containing these metals e.g. paints, pipes, batteries, soldering rods, gunpowder, pesticides, fungicides, gasoline, engine oils, some antihelmintics, chemical fertilizers or when they occur in high amounts in air, soil, water, plants and other compounded animal feeds. 

While copper is a trace element required in small amounts in various metabolic functions in the body, lead and other heavy metals have no function in the body and can be highly toxic due to interference directly in the metabolic pathways or indirectly by causing deficiencies of other trace metals (Farr 2001; Farr 2004). Excessively higher levels of these metals in blood and tissues of animals suggest an exposure either from the air, soil, water or feeds or all of theses sources (Dupler 2001; Farr 2004). Animals can tolerate elevated levels of these metals though at certain levels clinical signs of toxicity manifest which can be acute (sudden exposure to very high levels) or chronic when there is low exposure for a long time since these metal tend to bio-accumulate in the body (Allcroft 1951; IARC 1997).

The major clinical signs in animals and man for lead and copper poisoning include among others deviations of the hematological parameters due to their direct effects on hematopoiesis, reduced integrity of red blood cells’ membrane leading to intravascular haemolysis and anaemia and dehydration (Radostits et al 1994). Thus hematological parameters are of diagnostic value in animals suspected of heavy metal toxicity.

Man becomes at risk by eating food and drinking fluids contaminated with heavy metals,  or through air, or direct contact with the metals in people working in industries/factories dealing with heavy metals and their derivatives (Anonymous 1999; Farr 2004).

Morogoro town is endowed with a lot of small to large factories (Anonymous 1997) dealing with metal works, car maintenance and repair, construction works, backyard farming that use chemicals (pesticides, fungicides), soap and leather factories that use a lot of chemicals  thereby posing a risk of contaminating the environment with hazardous substances including heavy metals. It was therefore the objective of this study to investigate the levels of lead and copper in the plasma of cows, pastures, soils and water in Kingolwira and Mzinga areas of   Morogoro where herbage and river water used by ruminants are obtained near factories and highway.

Materials and methods

Study area

The study area was carried out in Morogoro Region, in and around Morogoro Municipality.  Morogoro town is located along 6⁰ to 51⁰ South and Longitude 31⁰ to 41⁰ East at an altitude of 528.8M above the sea level. Blood samples from dairy cattle was taken from Kingolwira ward that is located few kilometers to the east of Morogoro Municipality along the high way to Dar es salaam City and from Mzinga, some 10km West  from the town centre.

Dairy cows

The dairy cattle used in this study were crosses between Boran/Short horn zebu with Ayrshire or Friesian. Samples were taken mature dairy cows above four years old weighing between300-400kg belonging to smallholder farmers who complied with our request to use their animals.

Sample collection

Blood samples

About 10 ml blood sample was taken from the jugular vein using a 20ml heparinised vacutainer tubes. The tubes with blood were preserved in ice packed cold box from the field and immediately transported to the laboratory of the Department of Physiology, Biochemistry Pharmacology and Toxicology for the various analyses as will be described later.

Pasture, soil and water sampling

Soil and pastures were collected in 3 different areas (Area 1 some 10m from factories dealing with metal works), Area 2 in the upper mountain zone, Area 3 in the lower mountain zone) where cattle grazed. The choice of the sampling areas was determined on the basis of where farmers obtain herbage for their dairy animals. At the selected points (3/area), grasses were cut v carefully at 15 cm height from the ground in an area of 1.5x1.5m². The grasses were put in a clean polythene bags and quickly transported to the laboratory where samples were sorted to separate the predominant species. Each bundle of a given species was divided into two. One half was used for DM determination while the remaining was rinsed with de-ionized water several times before drying and further analysis for lead and copper as will be described latter.

At the centre of the of 1.5x1.5m² area where grasses were taken, the topsoil was dug to 12 cm depth at an area of 24x24cm squire. The soil was carefully mixed and put in clean polythene bags, then transported to the laboratory, ground in a mortar to obtain small particles of uniform size, then DM, lead and copper levels determined as will be described latter.

About 50ml of water samples were taken in clean unused 50ml plastic bottles from three different points along a river passing through the grazing area: Upper, mid and lower river just before pouring into  Mindu dam.  Each sample was filtered through Whatman® filter paper grade 40 to remove debris before analysis for lead and copper using atomic absorption spectrophotometer.

Laboratory analyses

Determination of dry matter of soil and pastures

%DM of soil and pasture samples were determined by weighing 2g of samples in crucibles (in duplicates) and drying them in a hot air over to constant weight at 60^oC (AOAC 1990). Two hundred grams of pastures were weighed to clean aluminum pans (radius of 12cm) in duplicates and them dried to constant weight in a hot air over at 60^oC (AOAC 1990).

Determination of lead and copper levels in soils pastures and water

Conventional aqua regia digestion was performed in 250-mL glass beakers covered with watch glasses. A well-mixed sample of 0.5000 g soil or ground pastures was digested in 12 mL of aqua regia on a hotplate for 3 h at 110°C. After evaporation to near dryness, the sample was diluted with 20 mL of 2% (v/v with H₂O) nitric acid and transferred into a 100-mL volumetric flask after filtering through Whatman® filter paper grade 40 and diluted to 100 mL with dionized distilled water then analyzed for levels of lead and copper using the atomic absorption spectrophotometer.

Determination of hemoglobin concentration (Hb)

Haemoglobin was measured using the cyanomethaemoglobin method described by Baker and Silverton (1976). In this method Drabkin`s solution that contains potassium ferricyanide (K₃Fe(CN)₆) and potassium cyanide (KCN) was used as a diluent by mixing 5mls of the diluent with 20μl of the blood in a test tube. The mixture was thoroughly mixed and allowed to stand for 10 minutes before reading the absorbance by spectrophotometer at a wavelength of 540nm. . Potassium ferricyanide caused the oxidation of hemoglobin which then reacted with potassium cyanide to form cyanomethamoglobin.

Determination of PCV (%)

Blood sample was mixed by gentle shaking followed by immersing hematocrit capillary tubes (three per sample) into which blood was drawn by capillary attraction to three quarters. The tubes were centrifuged in a microfuge at 12000g for 5 minutes. The height of the column of red cells was read using a PCV reader.

White blood cell counts

White blood cell counts were done following a standard procedure as described by Baker and Silverton (1976). The blood was drawn from the test tube into white cell pipette to 0.5 marks and diluted with diluting fluid sucked to reach 11 mark. Then contents in the bulb were thoroughly mixed and the fluid up to mark 1 was discarded using a tissue paper before charging the improved Neubeaur chamber by touching the edge of the cover slip. WBCs were counted from the 4 big corner chambers (each 1x1x 0.1 mm dimensions) under the microscope using x 40 objective and x 10 eyepiece.

Red blood cell counts

Red blood cell count was done following a standard procedure as described by Baker and Silverton (1976). Briefly, after gentle shaking of the blood sample, blood drawn into red cell pipette (bulb type) to a mark of 0.5 and then add diluting fluid to 101 mark  followed by through mixing of the contents in the bulb. The excess diluent in the pipette just below the bulb was removed by using tissue paper before charging the improved Neubeaur counting chamber ready for counting using a microscope at magnification of x10 eyepiece and x40 objective lens using 4 small corner squares and one central small square each with 0.2x0.2x 0.1 dimensions. The calculation of the RBC counts was done using the formula:

Determination of lead and copper levels in plasma

The blood sample in test tube kept in centrifuge machine and the lid closed. The samples were centrifuged at 12000g for 5 minutes and then the plasma was harvested into clean test tubes. The plasma was mixed with 10% Trichloroacetic acid in a 1:1 ratio so as to precipitate proteins and the mixture was centrifuged at 12000g for 10 minutes after which the clear supernatant fluid was carefully decanted into a clean test tubes and submitted to the Department of Soil Science, Sokoine University of Agriculture for reading the levels of lead and copper using the atomic absorption spectrophotometer.

Water samples were filtered Whitman’s filter papers to remove any debris and submitted to the same laboratory for lead and copper analysis.

Statistical analyses

The data were analyzed using SAS (2000) statistical package to obtain the mean, standard deviation, standard error of the mean, range and student’s test on the measured parameters.

Results

Dry matter of pasture, soils and lead/copper levels

Indicated in Table 1 are the DM and levels of lead and copper in pastures from the area used in this study. The %DM ranged between 53.0-73.5 while the mean lead and copper levels were almost of similar order 3.5 and 3.6mg/kg respectively). Panicum spp obtain near factories had the highest lead and copper levels (8.8 and 9.0 mg/kg respectively) while Bothriochloa spp from mountain area had the lowest lead and copper levels (1.4 and 2.0 mg/kg respectively).

Lead and copper level in the water

Table 2 indicates the levels of lead and copper in the water used by animals in a river passing across the grazing area. In all sampled areas, the water had negligible amounts of lead and copper that ranged between 0-0.01mg/l.  

 %DM, lead and copper level in soils

Soil samples collected in three different areas in the study area had an average DM of 95.8 and mean lead and copper levels of 10.4 and 11.8mg/kg respectively (Table 3). Soil samples taken from area 1 had the highest levels of lead and copper (30.5 and 33.8mg/kg respectively).

Hematological parameters and plasma levels of lead and copper

Shown in Table 4 are the mean levels of PCV, Hb, RBC and WBC counts, plasma lead and copper levels in the dairy animals used in this study. Blood samples from Mzinga animals showed significantly higher (P<0.01) RBC counts compared to Kingolwira animals. However, Kingolwira animals had significantly higher (P<0.001) plasma lead levels compared to Mzinga animals. The other measured parameters were not significantly different between the two localities.

Discussion 

The major pasture species fed to animals in Mzinga and Kingolwira were similar to those reported by Mlay et al (2001). The DM of the pastures was fairly high (53-73.5%) for freshly cut forages. This was due to the fact that the forages were cut at advanced stage of maturity before the onset of the rainy season. The DM of the soils was also high (96%) because they were collected during the dry season when soil water content is normally low.

Lead and copper levels in pastures depend on intrinsic nature of the soil (its metal content) and extrinsic factors such as contamination through human activities leading to increased levels of lead and copper far above the background levels (Osweiler 1996). For example, lead levels of 10 – 390mg/kg were reported in pasture close to major roadways and railways (Motto et al 1970; Radostits et al 1994). Under such situations, the pastures become a potential source of heavy metal toxicity since unlike other metals that when their concentration increases cause early death to plants before the animals can eat the plants, many plants have an ability to accumulate lead and to a lesser extent; copper to levels that can cause problems in animals.

This study also proved that within the same growth conditions, plants differ in their ability to accumulate heavy metals. For example, Panicum spp form the same area (close to factories) with Pennisetum spp and Bothriochloa spp had almost twice and thrice the levels of lead and copper respectively. Also, it was evident that pastures of the same species growing in soils with high levels of heavy metals had higher levels of these metals compared those growing in soils with less amounts of heavy metals. The implication here is that care need to be taken in the selection of the type of pasture to grow in areas with heavy metal contamination so as to avoid passing the metals to the animals through the food chain (Pendias 1989; Farr 2004).

Generally the lead levels in soil range from 5-25mg/kg (Frank 1991). Thus, the levels observed in this study were far much lower compared to the minimum value in the normal range. The soil samples taken near factories showed a relatively high lead and copper levels that was almost three times the levels in the soil from other sources. This was expected due to pollution arising from the factories.

The observed water lead and copper levels (0.0 – 0.01mg/l) was far below the minimum acceptable levels of 0.015mg/l (ILO 1983; Fitzgerald 1998) thus the water is safe for use by animals in as far as the levels of lead and copper are concerned.

The findings in this study showed that the plasma lead levels in the cows were very high compared to those reported by Lopez et al (2000) in cows in Galicia, Spain (0.01mg/l) indicating that the cows were exposed to lead contaminants. The significantly higher plasma lead levels in Kingolwira compared to Mzinga cows was most likely caused by pollution through exhaust fumes from motor vehicles that use leaded gasoline and contaminations from car service and filling stations scattered along the very busy Dar es salaam – Tunduma highway that passes along Kingolwira (Anonymous 1997). The possibility for the cows to obtain extra lead through ingestion of feeds, soil and water cannot be ruled out due to the fact that these metals tend to bio-accumulate in the body. The threat of heavy traffic as a source of lead that can affect animals and humans has been reported in other parts of the world (Motto et al 1970; Radostits et al 1994). The levels of plasma copper in the cows from Mzinga and Kingolwira (0.98 and 0.93mg/l respectively) were slightly higher than the minimum acceptable levels of 0.6mg/l (range 0.6 – 1.47/l) recommended by the environmental Protection Agency but similar to those reported by Lopez et al (2000) (0.89mg/l) in cows from Galicia Spain. The major factor causing elevated copper levels was that in addition to environmental and feed sources, farmers give mineral supplements rich in copper (Mlay et al 2001).

The significantly higher RBC counts in Mzinga cows compared to Kingolwira cows proves that high lead levels can suppress the RBC synthesis possibly by inhibiting the delta-aminolevulinic acid dehydratase (ALA-D) enzyme involved in the heme synthesis (Radostits et al 1994). Thus, the low lead levels in Mzinga cows were congruent to higher RBC counts observed in this study.

Conclusion 

• Despite the presence of potential sources of environmental contaminants containing lead and copper, the levels of these metals in the blood of cattle, pastures, water and soils in Morogoro suburbs have not reached alarming level.
  
  
• There is a need to continuously monitor the status of heavy metals in animals, soils and feeds so as to avoid toxicity to animals and man.

Acknowledgement 

The authors of this article would like to express their sincere thanks to Mr. P Jingu, Mr. Kibirige W and Ms Juliana Jerome for their technical assistance.

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Received 13 May 2007; Accepted 18 January 2008; Published 7 April 2008

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