Toxicity of animal bone charcoal from pig and cattle to aquatic bioassays: Vibrio fischeri, Daphnia Magna and
Selenastrum capricornutum
Abschlussarbeit
im Rahmen des Postgradualstudiums “Toxikologie und Umweltschutz”
der Universität Leipzig
Noah Ngandwe
Leipzig, November 2007
Contents Abbreviations
1. Introduction 1
2. Materials and method 4
2.1. Used materials and Instruments 4
2.2. Used materials and methods 4
2.2.1. Animal bone charcoal. 4
2.2.2. Elutriate extraction 7
3. Bioassays 9
3.1. Bioassays with Vibrio fischeri 9
3.2. Immobilization of Daphnia Magna 12
3.3. Bioassays with Selenastrum capricornutum (algae) 14
4. Results 16
4.1. Vibrio fischeri bioassays 17
4.2. Daphnia Magna bioassays 19
4.3. Selenastrum capricornutum bioassays 21
5. Conclusion 23
6. References 25
7. Tables 30
List of Abbreviations and Chemical Formulae ABC animal bone charcoal or carbon
ADaM Aachener Daphnia medium
ADP Adenosine diphosphate
ATP adenosintriphosphat
BSE Bovine Spongiforme Enzephalopathie
BBodSchV German Federal Soil Protection and Contaminated Site Ordinance.
CaCl2 calcuim chloride
DO dissolved oxygen (concentration) EC50 median effective concentration
FMN Flavin mononucleotide
FMNH2 reduced Flavinprotein
g gram
h hour
NaCl sodium chloride
NADH Reduced form of nicotinamid adenin dinucleotide
NADPH Reduced Nicotinamide adenine dinucleotide phosphate NaHCO3 sodium dihydrogen Carbonate
PAHs polycyclic aromatic hydrocarbons
MBM meat and bone meal
ml millilitre
min minute
SeO2 Selenium dioxide
SRM specified risk material
< Less than
> Greater than
1. Introduction
Meat and bone meal (MBM) has traditionally been used as a feed supplement for animals but has been banned for such use in the European Union to address public and animal health concerns. The ban governed by EU legislation, “EC Directive 1774/200/EC on Animal By-products“, has led to the accumulation of MBM awaiting disposal. Feeding MBM to cattle is thought to have been responsible for the spread of BSE. Currently there is little that can be legally done with MBM, other than
combustion for energy generation, land filling or incineration. Several countries outside the EU also adopted same measures of protection by banning the feeding of MBM to animals and for fertilizer usage. In Japan the ministry of agriculture, forestry and fisheries instructed fertilizer industries to incinerate fertilizer made of MBM whose use was suspended after the out Break of mad and cow disease or BSE (Kamisato, 2005). Identification of alternative means of safe disposal is therefore inevitable. One way of achieving this goal is by burning animal bones into charcoal that could be used in agriculture as a fertilizer or soil additive. Unlike MBM animal bone charcoal is activated by subjecting it to very high temperatures making it virtually sterile.
Historically, bone charcoal has been used for a variety of purposes for several
thousand years. For agricultural purposes, it has been used principally for its fertilizer and its medicinal values in veterinary medicine. It has thus been considered as a universal antidote (NOSB TAP, 2002) probably because of its ability to removing toxic substances in human and animals. The Amazonian Dark Earth also called
"Terra Preta do Indio" for example is well known for its highly fertile dark soils rich in C, N and P. Investigations conducted by international researchers (Glaser 1999;
Glaser et al. 2000, 2001a) found that the soils contained tremendous amounts of charring residues containing high amounts of nutrients that have persisted in the environment over centuries. Scientists have now accepted and concluded that these soils were a product of indigenous soil management. Activated carbon also known as activated charcoal is used in used in our daily life without us noticing. It is used to remove impurities that cause objectionable color test, taste, odor or toxic substances from drinking water, waste water, food and beverages (e.g. white sugar production), to control air pollution (e.g. gas masks) and to separate or purify pharmaceuticals and chemicals. Zackrisson and Nilsson (1992) observed reduction of phytotoxins when
activated carbon was added to water extracts in laboratory experiments and to the soil surface in field experiments. Some decades ago green keepers in England applied charcoal fines to golf courses as a top dressing during maintenance to eliminate excess fertilizer and chemicals (Hurdzan, 2004). This practice used to be encouraged to gain the benefit of deep rooting, and improved soil capillarity.
However, today charcoal has been substituted by sharp sand probably because of lack of supply and difficulties in handling of charcoal fines. Addition of charcoal to agricultural soils has frequently been proven by many authors to have increased soil fertility and reconditioned the soil. After adding charcoal Iswaran (1980) reported enhanced nutrient availability, removal of pesticide residue (Mc Carty, 2002), reduced nutrient leachates and increased crops growth (Lehman et al., 2003). Charcoal has the potential to support microbial communities as its porous structure according to Zackrisson et al. (1996) could shelter microbes against predators and giving them improved environment for nutrient cycling. However it must be acknowledged that, the source of charcoal material strongly influences the direct effects of charcoal amendments on nutrients contents and availability (Glaser B, 2002). Addition of large amounts of charcoal on the other hand may also have negative effects of crops growth. Kishimoto and Sugiura (1985) for example observed decline in yield of soyerbeans and maize after addition of 5 Mg charcoal probably due to increased pH values. The safe and responsible disposal of animal waste today is an important element of animal agriculture and public health. Recycled animal by products such as ABC contain chemical elements like phosphor and calcium that can provide nutrients to agricultural soils but may also contain compounds that can be harmful to the environment. Potentially hazardous constituents of ABC such as heavy metals and PAH`s might be taken up by plants and ingested by humans or animals in quantities that could be harmful to health. Additional concern about the use of ABC may include risks to farmers and their families, damage to soil fertility, dispersion into groundwater and air. Therefore, any decision on safety of ABC must be based on scientific risk assessment. An essential requisite of using a risk assessment framework is to ensure that human health; animal health and the environment are protected. The objective of this study is therefore to access the toxicity of elutriate from pig and cattle ABC by exposing it to a battery of bioassays, chosen according to the row they play in the environment. The three biotests consisted of acute and chronic endpoints in Daphnia
magna (immobilization test), Vibro fischeri (biolumenscence test) and Selestrum capricornutum (inhibition of reproduction test). All these tests are DIN standardized test procedures, which are employed for characterization of possible waste
ecotoxicity (CEN, 2002).
The luminescent bacteria test using Vibrio fischeri is the oldest standardized screening tool that is used for measuring toxicity of environmental samples. Its
advantage lies in its speed, simplicity, and relatively low cost. Its strongest attribute is its usefulness as a primary screening test for a broad spectrum of toxicants and its monitoring capacity over a long time. The test has been standardized in several national Standards as an environmental early warning system and other research purposes (Zieseniss, K. al. 1994). In Germany the test was adopted in 1999 as a DIN EN ISO (11348) method for testing water, waste water and dissolved chemicals.
Immobilization test uses Daphnia magna that is an excellent organism used in bioassays because of their sensitivity to changes in water chemistry and are simple and inexpensive to grow. Their maturation is short (a few days) and being clones, genetically identical individuals can be compared. Much is known about their life history, physiology, population dynamics, genetics and evolution. In this study clonally reproduced Daphnia magna was used.
The Algal inhibition test is used to determine the effects of water soluble substances on the growth of a unicellular green algal species. In this study S. capricornutum was used. The test can assess effects over 1 generation of 24 h. Several other unicellular algal species can also be used. Chemical analysis was conducted to measure the contents of heavy metals and PAHs in ABC.
2. Materials and method 2.1. Used materials and method
Chemicals
CaCl2 ADaM Merck, Darmstadt, German
NaHCO3 ADaM Merck, Darmstadt, German
SeO2 ADaM Merck, Darmstadt, German
Sea Salt ADaM Wiegandt, Krefeld, German
NaCl V. fischeri Test Merck, Darmstadt, German 7% NaCl positive control Merck, Darmstadt, German
ABC Pig test substance Thermal Desorption Technology Group, Hungary ABC Pig test substance Thermal Desorption Technology Group, Hungary Activated Carbon negative control Merck, Darmstadt, German
Materials
pH-Meter 765 Calimatic Knick, Berlin, German Dissolved Oxygen meter WTW, Weilheim, German CASY 1-Cell Counter and Analyzer
Systems
Schärfer, Reutlingen, German
Luminometer LUMISTOX Dr. Bruno Lange, Düsseldorf, German Incubation Block LUMIStherm Dr. Bruno Lange, Düsseldorf, German Magnetic stirrer IKA Labortechnik, Staufen, German Weighing scale BL 150 Sartorius, Göttingen, German
Centifuge Megafuge 2.0 R Heraeus Instruments, Gera, German Eppendorf-Pipettes Eppendorf, Wesseling-Benzdorf, German
Pyrex glases Normag Labor & Prozesstechnik GmbH, German volumetric flasks Brand, Wertheim/Hirschmann, Eberstadt, German measuring cylinder Brand, Wertheim/Hirschmann, Eberstadt, German LUMIStox Glass cuvettes Dr. Bruno Lange, Düsseldorf, German
2.2.1. Animal bone charcoal
Any carbon material such as animal meal can be used to make activated carbon or charcoal. Different activation methods are employed. Preparations often include dehydration, carbonization and activation. Dehydration and carbonization involve slow heating of the carbon source in anaerobic condition. Activated carbon used in this study was produced by carbonization of animal bones (meal) from pigs and cattle. Animal bone charcoal is traditionally used for color removal in the sugar refining industry. During carbonization, most of the non-carbon material and a large amount of CO2 are volatilized. This process leads to a great loss of weight usually ranging from 60 to 70 % (Sun et. al., 1997, Diaz- Teran, 2001). In gas activation, an oxidizing gas such as CO2 is used at very high temperatures to erode pores into the char. In chemical activation, the char is impregnated with chemicals and then fired to high temperatures ranging from 800oC to 1000oC. The activating chemical corrodes the carbon to form the pore structure. Chemical activation also alters the carbon surface. Activation chemicals are usually strong acids, bases or corrosives (phosphoric acid, sulfuric acid, KOH, Zinc chloride, potassium sulfide or P-
thiocynate). After activation the chemicals are washed out for reuse. The final pore structure depends on the starting material and the activation process (Mozammel et.
al., 2002). Activated carbon is one of the best tools that are used in the industry in the preparation of many products. Its use ranges from removing of impurities that cause an objective color, test, and odor or health hazard in drinking water, food and beverages. Different sources carbon material will produce activated carbon with different properties. Activated carbon has an extraordinary large area and pore volume that gives it a unique adsorption capacity (Baker, et al., 1992). The fine pore structure is formed during the activation process. These provide a large surface area relative to the size of the actual particle and its visible exterior surface. An
approximate ratio is 1 gram = 100 m2 of surface area (Hoehn, 1999).
Chemical and physical effects of activated carbon on substances it’s exposed to are adsorption, mechanical filtration, ion exchange and surface oxidation. Adsorption is the collection of a substance onto the surface of adsorbent solids. It is a removal process where certain particles are bound to an adsorbent particle surface by either chemical or physical attraction. Adsorption should not be confused with absorption, where the
substance being collected or removed actually penetrates into the other solid
(Reynolds, 1996). ABC was supplied by „Thermal Desorption Technology Group“ of companies whose biotechnological recycling projects aim is the upgrading of high phosphorus containing organic waste of non SRM origin, into safe crops protection and nutrition products for environmentally friendly crops production. The EU Project Protector (FOOD-2005-510482, “STREP“) was financed by the European Union.
Animal bones used for carbonization were all of European origin. ABC was supplied in granular form (<4mm) and no further grinding was required. Safety measures must be taken when handling animal bone charcoal as it can cause respiratory problems especially as particle size decreases. Inhalation causes cough, dyspnea, black sputum, and fibrosis (Patnaik, 1999). There is also a potential danger for it to
spontaneously combust and carbon monoxide poisoning may occur (Cheremisinoff, 1999). Activated carbon which was used as for negative control testing (Merck, GmbH) was in powdered form. It is a commercial standardized product that is mainly used in laboratories.
2.2.2 Elutriate extraction
The toxicity of ABC was determined by means of chronic algae toxicity test, luminescent bacteria test and D. magna immobilization test. ABC was sterilized before use at 160o C for 3 h. Different sample preparations methods were followed.
A - ABC elutriates were prepared according to Dr. Lange acute toxicity test procedure (Dr. Lange, GmbH, Düsseldorf, Germany). 40 ml 2% NaCl solution was added to 20g ABC. The suspension was then rotated on an overhead shaker for 24 h at room temperature. After standing for 1 h the suspension was centrifuged at 3300 g for 20 min and filtered on a 0.45 µm pore diameter syringe filter. Elutriates were stored in the refrigerator at 4o C pending V. fischeri toxicity testing. PH values were determined before use. Measured pH values of both test substances were highly alkaline (pH 9 to pH 12). Values were adjusted to pH 7 ± 0.2 to avoid interference in test conditions. A number of variables besides the toxicity of the test substance can interfere with readings of light production of V. fischeri surviving in the filtrate of each test concentration (Ringwood et al., 1997). Variables, which can interfere with the light production of V. fischeri test filtration, include temperature, pH values and salinity content (Krebs, 1983).
B - The second elutriate extraction was prepared following the procedure in above (A), without adding NaCl. 40 ml demonized water was added to 20g ABC and rotated for 24h. Extracted test samples were used for acute Daphnia magna immobilization test and chronic algae toxicity tests.
C- Another sample was prepared which was used for negative control testing using the commercially available activated carbon (Merck, Darmstadt). 40 ml deionized water was added to 10g activated carbon (ratio 4:1) and prepared like above.
Chemical analysis
Trace element analysis was done at the department of analytical chemistry at
Helmholzt center for environmental research-UFZ. PAHs and heavy metal contents in ABC were investigated. Results will be presented and discussed in another chapter.
ABC was analyzed for heavy metal contents by ICP-AES (Inductivity Coupled Plasma-Atomic Emission Spectroscopy) using the Jobin Yvon model JY70 Plus analyzer and pH values, oxygen content and electrical conductivity were also measured.
The soil heavy metal contents were analyzed by ICP-OES (Inductively Coupled Plasma-Potical Emission Spectroscopy).
3. Bioassays
3.1. Bioassays with Vibrio fischeri
Vibro fischeri (formerly called Photobacteruim phosphoreum)is a rod shaped
bacterium found globally in marine environments. In nature Vibrio fischeri survives on decaying organism matter and lives mostly in symbiosis with various marine animals.
The bacteria move by means of flagella and have bioluminescence properties (light emittion from living organisms). This phenomenon takes place in many other species of bacteria, fungi and animals (invertebrates and vertebrates). The mechanisms of luminescence in all of these groups of organisms are generally similar. However, substrates for the chemical reaction may vary considerably among different species.
Interestingly, Bioluminescence seems to have appeared several times independently during the evolution of life forms (Rees et al., 1998). Light emitting bacteria are the most abundant and widespread of luminescent organisms (Meighen 1994). Vibrio fischeri and Vibrio harveyi are the most intensively studied bacteria species that are able to emit light. Vibrio fischeri is symbiotic bacterium living in the light organs of fish of the family Monocentridae and of the cephalopods Sepiola and Euprymna
(Fitzgerald 1977). Vibrio harveyi is a free-living bacterium that may also be found on the surface of marine animals or their gut (Baumann et al. 1973).
Bioluminescence is a result of a chemical reaction within an organism, during which chemical energy is converted to light energy. At least two chemicals are required.
The one that produces light (luciferin) and the one that drives or catalyzes the
reaction (luciferase), which is composed of two subunits known as a and b (Belas et al. 1982). The bacterial luciferin substrate is a NADH - reduced riboflavin phosphate (FMNH2), and a long chain fatty aldehyde, are oxidized in the presence of oxygen and the enzyme. The resulting complex interacts with aldehyde as a monooxygenase to form an excited but highly stable intermediate, which decays slowly, resulting in emission of light as a product of this reaction (Meighen, E. A., 1991). This reaction product is summarized as follows:
RCHO + FMNH2 + O2 RCOOH + FMN + H2O + hv
The fatty acids produced in the reaction catalyzed by luciferase are subsequently reduced to aldehydes by a specific reductase. In the same reaction, NADPH + H+ is
converted to NADP+ and ATP is hydrolyzed to ADP (Ziegler & Baldwin 1981). This is necessary for luminescence reaction, is generated from FMN by NAD (P) H-FMN oxidoreductase (Jablonski &DeLuca 1978). In accordance to the description of light emission, bioluminescence is an energy consuming reaction, which may use up to 20% of the total cellular energy (Bassler & Silverman 1995). Sometimes the Luciferin and luciferase (as well as co-factors such as oxygen) are bound together in a unit called photoprotein. This molecule can be triggered to produce light when a particular type of ion is added to the system (frequently calcium).
Test description
Freshly prepared Vibrio fischeri was exposed to a concentration series of ABC test substances in accordance to DIN 38412 L34. Diluents for the bacterium comprised of 2 % NaCl. Each test substance was diluted with the same diluent. Diluent was
prepared by dissolving 2.0 g NaCl in 100 mL of deionized water (DIN 38412 L34, 1991). According to DIN 38412 L34 conserved or freshly prepared organisms can be used for testing. Conserved organisms are commercially obtainable as standardized culture of freeze-dried bacteria or in a liquid form. Organisms used in this test are standardized culture (strain NNRLB-11177), from Dr. Bruno Lange GmbH & Co. KG, Düsseldorf. V. fischeri was reactivated by adding 9.45 mL of 2% NaCl deionized water to 1050 ml bacteria solution. The equipment used for performing the toxicity test was the Lumistox apparatus (Dr. Lange GmbH) that consisted of the Lumistox model analyzer block and the LUMIStherm block (incubator). The LUMIStherm block is for incubating test tubes containing concentrations of test material and V. fischeri pending testing. It consists of three rows (A - C), which can accommodate 10 test tubes each. Each column is numbered 1 to 10, and are referred to as A1 to C10. Test tubes in the first row (A) consist of 8 concentrations of the test substance, a negative control (2%NaCl water solution) and a positive control. To each test tube in rows B and C, 0.5 mL reconstituted bacterial solution is added. Each test sample (including reference substances) inoculated with bacteria was incubated for 20 min at 15o C.
This temperature control is according to Krebs (1983) based on an agreement for standardized control. Using the LUMIStox System from Dr. Bruno Lange GmbH of German, bioluminescence was measured according to ISO 11348 (1998) in a saline Medium (2% NaCl) at pH 7 and 15o C.
The toxicity was evaluated after 15 and 30 min of exposure. The light output readings were recorded and used to calculate an EC50 for each sample. The EC50 is defined as the medium effective concentration, and is a calculated toxicity value representing the sample concentration (%) estimated to cause a 50 % response by the exposed test organisms. The EC50 was then compared against a control to evaluate relative toxicity.
3.2. Immobilization of Daphnia magna
Daphnia are planktonic crustaceans that belong to the phyllopoda (Brachiopoda), which are characterized by flattened leaf-like legs used to produce water current for the filtering apparatus. Within the branchiopods, Daphnia belong to the Cladocera, whose body is enclosed by an uncalcified shell, known as the carapace (Remane, et al., 1997) Adults range from less than 1 mm to 5 mm in size. The body is divided into segments, which are nearly invisible. The head is fused, and is generally bent down towards the body with a visible notch separating the two. Daphnia (water fleas) are small in size and live in freshwater found in ponds throughout the temperate regions of the world. They are an important source of food for fish and other aquatic
organisms. Daphnia feed on small, suspended particles (< 50 mm) in the water usually planktonic algae. Given good environmental conditions, most daphnia are female and will reproduce clonally. When reproducing without breeding, the eggs do not get fertilized and the young ones (clones) are exact copies of their mothers. The unfertilized eggs develop into live embryos inside the female’s body, and the young are released into the environment within 2 to 3 days. When exposed to environmental stressors, Daphnia adapt to producing male and female embryos which when
matured breed and produce fertilized eggs (ephippia) encased in tough protective shells. By so doing they can survive hash conditions where survival is otherwise not possible. Daphnia typically live 40 to 56 days depending on species and
environmental conditions. Each brood typically holds 6-10 eggs, which turn into embryos and are released within a few days. For the acute toxicity test only 3 and 4 weeks old neonates were used. Daphnia are quite sensitive to the condition and chemistry of the water in which they live. The culture can become stressed if the population density gets too high or if there is shortage, poor water quality, or extreme temperatures. Used ADaM was prepared according to the original recipe of Klüttgen et al. (1994).
Compound Concentration Concentration (g/L)
Sea Salt 0,333 g / L
CaCl2–Lsg. 0,4mol/L
(58,8g CaCl2 . 2 H2O / L
1,84 mmol / L*
(4,6 mL SL / L)
NaHCO2–Lsg. 0,3 mol / L
(25,2 NaHCO3 / L)
0,66 mmol / L*
(2,2 mL SL / L)
SeO2–Lsg. 0,13 mmol / L
(14,4 mg SeO2 /L)
0,013 µmol / L (0,1 mL SL / L)
Tab.1 Artificial Daphnia medium: ADaM (Aachener Daphnia medium)
All compounds in Tab. 1 were mixed with deionized water, reaching the specified end concentrations of ions (Wiegand, Krefeld, Germany) (CaCl2 * 2H20 1.84 mM,
NaHCO3 0.66 mM, SeO2 0.013 mM, marine salt 0,33 g.L-1). Determined pH was not adjusted like prescribed by the guidelines for daphnia test. To increase dissolved oxygen concentration, air was bubbled into ADaM, for at least 24 h.
Test description
The Daphnia immobilization test was performed in accordance to DIN 38412 L 30 (1989) using Daphnia magna from Aachen, Germany. Neonates were collected from parent animals cultured individually in glass vessels containing 100 ml media to equilibrate the condition of Daphnia and were fed on green algae. All experiments were carried out using the 3rd and 4th brood neonates (max. 24 h old).
Concentration series were prepared by mixing ADaM and ABC samples on the magnet stirrer in defined geometric series .The concentration series was widely spaced in a dosage of 3.125%; 6.25%, 12.5%, 25%, 50%, and 100%. Four replicates per test concentration and control were used in which 15 ml of the test substance was added. 5 randomized neonates were exposed to each 15-ml airtight and closed Pyrex glass tubes with Teflon sealing caps containing the test substance in defined concentration series. Test samples were then kept at 20o C in the climate chamber for 48 h pending observations. DO and pH values were recorded at 0 and 48 h (start and end of test). Immobilization (death) recordings of daphnia neonates were done after 24 and 48 h.
3.3. Bioassays with Selenastrum capricornutum (Green Algae)
Green algae belong to the most diverse group of algae species (>7000 species) growing in a variety of habitats. It belongs to the paraphyletic group because it excludes the Plantae. Like the plants, the green algae contain two forms of
Chlorophylls a and b, that is used to collect light energy for manufacturing of sugars, but unlike plants green algae are primarily aquatic. Because they are aquatic and manufacture their own food, these organisms are called algae, along with certain members of the Chromista, the Rhodophyta, and photosynthetic bacteria, even though they do not share a close relationship with any of these groups
(ucmp.Berkeley.edu).
The green algae include unicellular and colonial flagellates, usually but not always with two flagella per cell, as well as various colonial, coccoid, and filamentous forms.
In the Charales, the closest relatives of higher plants, full differentiation of tissues occurs.
Chlorophyll gives green algae a bright green color as well as the accessory pigments beta-carotene and xanthophlls. Green algae have stacked thylakoids. They are bound by a double membrane, so presumably were acquired by direct
endosymbiosis of cyanobacteria. Green algae probably share a common origin with the red algae; the two are grouped as the Archaeplastid or Plantae sensu lato. All green algae have mitochondria with flat cristae. A cross-shaped system of
microtubules anchors present flagella, which are absent among higher plants and charophytes. They usually have cell walls containing Cellulose, and undergo open mitosis without centrioles. Sexual reproduction varies from fusion of identical cells (isogamy) to fertilization of a large non-motile cell by a smaller motile one (oogamy).
Some organisms rely on green algae to conduct photosynthesis for them. The chloroplasts in euglenids and chlorarachniophytes were presumably acquired from ingested green algae, and in the latter retain a vestigial nucleus (nucleomorph).
Some species of green algae, particularly of genera Trebouxia or Pseudotrebouxia (Trebouxiophyceae), can be found in symbiotic associations with fungi to form lichens. In general the fungi species that partner in lichens cannot live on their own, while the algal species is often found living in nature without the fungus. The first plants probably evolved from green algae.
S. capricornutum is a non-motile, unicellular, and crescent shaped (40 - 60 µm3) green alga found in most fresh waters. Its advantages as bioassays organism are well documented for example: small size, access to large populations, ease of culture media, use of simple inorganic culture media and rapid growth rate (Walsh, 1988). Its uniform morphology with the structures that does not make chains makes it ideal for enumeration with an electronic cell counter. Growth is sufficiently rapid and cells can be counted after 72 h and it’s known to be moderately sensitive to toxic substances.
The test, which was developed in the early 1970, as eutrophication assessment tool was later, adapted and standardized for toxicological testing (ASTM, 1993a).
Test description
Chronic algae toxicity was measured as inhibition of reproduction of S. capricornutum exposed to ABC test samples integrated in a micro plate system. The toxicity
endpoint was the inhibition of the cellular reproduction of the S. capricornutum during one-generation cycle lasting 24h following the procedure described by Altenburg et al. (1990). After 24 h cell number and cell volume distribution were measured and analyzed using a cell counter CASY II from Schärfer System, Germany.
4. Results
To determine the toxicity of ABC a biotest battery consisting of acute and chronic endpoints in Daphnia magna, Vibrio fischeri and Selenastrum capricornutum was used. Chronic algae toxicity was measured as inhibition of reproduction of the unicellular green algae Selenatrum capricornutum after a generation cycle of 24 h.
Acute bacterial toxicity was measured as inhibition of energy metabolism or
bioluminescence in the marine bacterium Vibrio fischeri after an incubation time of 15 min and 30 min. Finally, the acute Daphnia toxicity was also measured as
immobilization of movement (death) of the platonic marine Daphnia magna neonates.
Obtained results were then used to quantify the EC50. Commercially available activated carbon from Merck (German) was used as negative control that did not show any significant toxic effects on bioassays (Figure 2). 7% sodium chloride was used as positive control and was highly toxic to Vibrio fischeri. Both ABC samples were exposed to bioassays in a defined test concentration series that exhibited significant toxicity in all bioassays. Using the computer program SigmaPlot, dose- response relationships were established. Individual results concerning the effect of both ABC samples on biotest battery and chemical analysis results are discussed separately. Data sheets are attached at the end this work. To ensure that
confounding valuables did not interfere with test results, pH values and DO were monitored.
Elutriate (%)
0,1 1 10 100
Inhibition of bacterial luminescence (%)
0 20 40 60 80
100 Inhibition after 30 min
(Fig. 2) Dose-response relationship between the inhibition of bacteria luminescence of
V. fischeri and increasing concentration of negative control activated carbon (Merck).
4.1 Effects of ABC Elutriate on Vibrio fischeri
Elutriate (%)
1 10 100
Inhibition of bacterial luminescence (%)
-40 -20 0 20 40 60 80
100 After 15 min exposure After 30 min exposure
(Fig.3) Dose-response relationship between the inhibition of bacteria luminescence of V. fischeri and increasing concentration of ABC elutriate from pig.
eluate (%)
10 100
Inhibition of bacterial luminescence (%)
-20 0 20 40 60 80
100 Inhibition after 30 min Inhibition after 30 min B-Probe Inhibition after 15 min Inhibition after 15 min B-Probe
(Fig.4) Dose-response relationship between the inhibition of bacteria luminescence of V. fischeri and increasing concentration of ABC (cattle).
To determine the inhibition of bioluminescence, Vibrio fischeri was exposed to a defined series of ABC test substances. After the incubation time of 15 min and 30 min the light production of Vibrio fischeri was measured by means of the LUMIStox- 400 luminometer. Significant inhibition of light production was observed that
increased substantially with increasing ABC sample test concentrations and
incubation time. Inhibition of bacterial luminescence was however completed after 30 min exposure time as no reduction in light production was observed. Dose-response relationships for both activated carbons were established. The highest concentration of activated bone charcoal (ABC)
samples (100 %) from pig inhibited bacterial luminescence at 90 % and from cattle at 70%. The EC50 values determined by a sigmoidal fit were ranging from 60% to 70%
in both ABC samples.
4.2 Effects of ABC Elutriate on Daphnia magna bioassays
Elutriate (%)
0,1 1 10 100
Daphnia immobilisation (%)
0 20 40 60 80
100 Immobilisation after 48 h Immobilisation after 24 h x column 1 vs y column 1
(Fig.5) Dose-response relationship between the immobilization of D. magna neonates and increasing concentration of ABC elutriate from pig
Elutriate (%)
1 10 100
Daphnia immobilisation (%)
0 20 40 60 80 100
Immobilisation after 48 h Immobilisation after 24 h x column 1 vs y column 1
(Fig.6) Dose-response relationship between the immobilization of D. magna neonates and increasing concentration of ABC elutriate from cattle
The 48 h chronic toxicity on Daphnia was performed by means of inhibition of immobilization of Daphnia neonates exposed to a series of ABC test substances.
Recordings were done by observation of immobility (death) after 24 h and 48 h exposure time. Significant immobility (death) were observed and recorded after 24 h that increased with increasing ABC sample concentration and incubation time. The highest immobilisation rates (rates causing 100% death) were obtained within 24 h incubation time with ABC sample concentrations of 50 % in both ABC. At test end (after 48 h) there was an increment in immobilization (death) of up to 40%.
Throughout the Daphnia tests measured DO concentrations were above 50%
suggesting that immobilization could not have been caused by lack of oxygen.
Measured pH values ranging from 6.8 to 7.7 were also in good agreement with test conditions. Obtained results were used to establish the dose response relationships.
The EC50 values determined by a sigmoidal fit were ranging from 10% to 15% in both ABC samples with no significant difference between them. No immobilization was observed in glass vessels containing ADaM negative control medium 48 h exposure.
4.3 Effects of ABC Elutriate on Selenastrum capricornutum
Elutriate (%)
0,1 1 10 100
Inhibition of reproduction (%)
-40 -20 0 20 40 60 80 100
Inhibition after 24 h
(Fig.7) Dose-response relationship between the inhibition of growth of S.
capricornutum and increasing concentration of ABC elutriate from pig
Elutriate (%)
0,1 1 10 100
Inhibition of reproduction (%)
-20 0 20 40 60 80
100 After 24 h exposure Col 13 vs y column
(Fig. 8) Dose-response relationship between the inhibition of growth of S.
capricornutum and increasing concentration of ABC elutriate from cattle
Chronic algae toxicity was measured as inhibition of reproduction of Selenastrum capricornutum exposed to a concentration series of ABC test samples integrated in a micro plate system. The toxicity endpoint was the inhibition of the cellular
reproduction of the S. capricornutum during one-generation cycle lasting 24h
following the procedure as described by Altenburg (1990). After 24 h exposure, cells were measured and analyzed using a cell counter CASY II from Schärfer System, German.
Measured properties showed a reduction in cell quantity and distribution that increased with the increasing ABC sample concentration. The EC50 values
determined by a sigmoidal fit were ranging from 9% to 12% in both ABC samples.
Heavy metals
For land application, animal bone charcoal has benefits and risks. Rich in calcium and phosphorous, the product has value as a fertilizing and soil amendment agent.
Being a by product of carbonization, ABC contain traces of metals and PAHs that can potentially be hazardous to the environment and human health. Identification of heavy metals and PAHs suspected of causing observed toxicity was done through chemical analysis at the Department of Analytical Sciences-UFZ. Identified heavy metals like Cd, As and Cr (Tab. 2) that are known for their toxicological impact in the environment and in human health were found in low concentrations. These metals did not exceed concentration limits stipulated by BbodSchV legislations in German.
Therefore it can be assumed that these metals did not contribute to the exhibited toxic effects in bioassays.
PAHs
Unlike heavy metal contents, some identified PAHs like naphthalene, phenanthrene and anthracene (Tab. 3) were found in high concentrations that exceeded allowed limits for ground water. These PAHs were consiquently suspected to have caused the observed toxicity in used bioassays. This assumption can be supported by the nature of PAHs in the environment of posing significant impact on human and animal health. PAHs are known to effect toxicity in organisms after ingestion. PAHs are a group of chemicals that are found naturally in all forms of organic materials and formed during incomplete combustion.
5.0. Conclusion
This study favors the use of ABC in agriculture as a fertilizer and as a soil
amendment agent. It also saves as a best alternative means of disposal for the still banned meat and bone meal traditionally used as an animal feed and fertilizer.
Obtained results however show potential environmental risk in the implementation of such products like ABC in agriculture for the intended usage. Both ABC samples caused light production inhibition in V. fischeri of more than 70%, immobilized movement or caused death of Daphnia magna of more than 90% and inhibited reproduction of alga S. capricornutum of more than 80%. Performed negative controls using activated carbon from Merck on the hand showed no significant toxic effects on used organisms. Chemical analysis results show high amounts of certain PAHs and low amounts of heavy metal concentrations in both samples. The sum of PAHs in water leachates (16 without naphthalene) amounted to 77µg/l upsets the 0.2µg/l limit set by the German environmental legislation authority (BBodSch). A concentration of naphthalene in both ABC leachates was 10µg/l upsetting its
limitation of 2µg/l in ground water. Naphthalene is known to be harmful to aquatic life in very low concentrations. The EC50 in Daphnia magna ranges from 2.16 to 8.60 mg/l (US EPA 1980). Naphthalene has been observed to inhibit oxygen consumption of Daphnia magna (CRIDER et al. 1982). Based on chemical analysis and the data mentioned above, identified PAHs were made responsible for causing the observed toxicity. Heavy metals were in good agreement with stipulated limitation. Thus, it can be assumed that they did not contribute significantly to the observed toxic effects.
However, it is fundamental to point out that lots of substances can still be toxic to living organisms at levels that are below chemical detection limits. Petersen et al., (1998); O’Conner and Paul (2000) attribute their work to not measured toxicants not reflected in chemical analysis but affected toxicity of sediments. Since detected compounds were not tested separately it can be concluded that toxicity might was caused by a mixture of these PAHs. PAHs that were found in high concentrations are naphthalene, phenanthrene and anthracene (Tab 2). Brack et al (1999) also made PAHs in the concentration range 10-100 µg/l responsible for the exhibited toxicity in green algae S. vacuolatus. Although not the objective of this work, reduction in PAHs concentration may help reduce toxicity in bioassays. Other PAHs not mentioned above but listed in Tab 2 were in good agreement with German environmental
legislations. Activated carbon from Merck that was used as negative control did not show any significant toxic response with V. fischeri (Fig.2). Results obtained in this study were somehow unexpected since activated carbon is generally considered as non -toxic (NOSB TAP, 2002). For luminescence inhibition EC50 values of 70% in both pig and cattle ABC elutriates were estimated. For immobilization of Daphnia neonates EC50 values of 12% in both ABC were estimated. For inhibition of reproduction of S. capricornutum EC50 values of 10% in both ABC elutriates were estimated. There was no significant difference in toxicity between the 2 ABC samples.
The fact that animal waste in meat industry is unavoidable and its disposal faces many obstacles and restrictions worsened after the discovery of BSE, much attention should be focused on finding alternative ways of disposal. Based on the available data, it can be concluded that ABC if added to agricultural soils may have immediate and most likely long-term harmful effect on the environment or its biological diversity.
Thus, it can be concluded that both animal bone charcoal used in this study from the Project (FOOD-2005-510482, “STREP“) can be considered as “toxic” and that
evaluation of options- to reduce toxicity must be considered.
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8. Tables
Tab. 1 Results of solid samples and cl/HNO3- extracts
Element Solid sample (XRF) Mean [mg/kg]
Eluates: Hcl/HNO3-extracts Mean [mg/kg]
Precautionary values for metals
c,
Aqua regia decomposition, [mg/kg]
P 133000 151000a --
Ca 318000 351000a --
Na 6650 6890a --
Mg 6290 5710a --
K 813 790a --
Zn 115 106a 150
Ni <5 -- 50
Hg <2 -- 0.5
Cr <10 0.9b 60
Pb 53 17.7b 70
Cd <2 0.03b 1
Cu 3.8 1.5b 40
As <2 0.45b --
. aICP-AES, bICP-MS cGerman Federal Soil Protection and Contaminated Ordinance
Tab.2 Results of PAH analysis
Charcoal Charcoal Charcoal Charcoal ABC ABC ABC ABC
pH7 PH4 ethylacetate toluene pH7 pH4 ethylacetate toluene
PAHs
C in Ng/
5g
C in Ng/
5g
C in Ng/
5g
C in Ng/
5g
C in Ng/
5g
C in Ng/
5g
C in Ng/
5g
C in Ng/
5g
naphthalene <0.2 23,2 6,9 479,7 8,2 1,7 70000,0 230000,0
acenaphthalene n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q.
fluorene 3,6 1,1 0,5 2,0 1,2 <0.2 <0.2
phenathrene 20,6 8,8 1,5 227,5 15,2 11,3 2138 17948,8
anthracene <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 416,2 2555,8
fluoranthene 8,4 2,6 <0.2 1,3 3,6 1,8 167,2 785,7
pyrene 68,3 18,7 <1.0 <1.0 35,8 3,7 123,3 590,1
benzo(a)anthracene <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 7,5 14,3
chrysene <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 24,6 35,6
benzo(b)fluoranthene <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 4,1 4,5
benzo(k)fluoranthene <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0,7 1,3
benzo(a)pyrene <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 2,9 2,2
dibenzo(a,h)anthrace ne
<0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2
benzo(g,h,i)perylene 8,7 1,0 <0.3 <0.3 2,6 1,0 1,4 1,3
