Ecotoxicity tests provide a direct measure of the bioavailability of toxicants and help to establish linkages between site
contamination and adverse ecological effects. There are more current laboratory test methods outlined, but it is very
important the major advantages/disadvantages of each of them. The present paper uses as research methodology the
OECD Guideline for testing of chemicals no. 207 which meets the most criteria expected for ecotoxicological testing.
Earthworms Eisenia foetida (Savigny, 1826) had been exposed for 14 days to a geometrical series range of
concentrations of the test substances (organic-mineral fertilizers L-200-Hum and SH-120). The performed study
showed that the fertilizer L-200-Hum determined lower earthworm mortality comparing to SH-120 for all tested
concentration levels and the fertilizer L-200-Hum positively influenced the earthworm rate of biomass accumulation.
DL 50 it was established to be the concentration 4 g for the SH-120 fertilizer at the end of the test (14 days)

Borza, Iacob

Dicu, Daniel

Gaica, Ioan

Iordache, Mădălina

ALSE Repository

      ECOTOXICOLOGICAL ASSESSMENT OF THE EFFECT OF SOME  NEW ORGANIC-MINERAL FERTILIZERS ON EISENIA FOETIDA EARTHWORMS  Mădălina IORDACHE 1, Ioan GAICA 1, Iacob BORZA 1, Daniel DICU 1  1 University of Agricultural Sciences and Veterinary Medicine of Banat, Timişoara   Abstract  Ecotoxicity tests provide a direct measure of the bioavailability of toxicants and help to establish linkages between sitecontamination and adverse ecological effects. There are more current laboratory test methods outlined, but it is veryimportant the major advantages/disadvantages of each of them. The present paper uses as research methodology the OECD Guideline for testing of chemicals no. 207 which meets the most criteria expected for ecotoxicological testing. Earthworms Eisenia foetida (Savigny, 1826) had been exposed for 14 days to a geometrical series range ofconcentrations of the test substances (organic-mineral fertilizers L-200-Hum and SH-120). The performed study showed that the fertilizer L-200-Hum determined lower earthworm mortality comparing to SH-120 for all tested concentration levels and the fertilizer L-200-Hum positively influenced the earthworm rate of biomass accumulation. DL 50 it was established to be the concentration 4 g for the SH-120 fertilizer at the end of the test (14 days). . Key words: earthworms, ecotoxicology test, organic-mineral fertilizers, mortality, reproduction   The concern regarding the effect of the toxic substances on the environment is growing rapidly in whole world and the efforts to develop more reliable methods for evaluating side-effects on non-target organisms are being increased. In Europe, there were elaborated several test methods which use a large variety of organisms, from aquatic or terrestrial environments, like algae, superior plants, fish and in the last time earthworms which became the most preferred organisms for such tests. Toxicity tests provide a direct measure of the bioavailability of toxicants, and, when combined with chemical analyses and field surveys, can help establish linkages between site contamination and adverse ecological effects. Toxicity tests evaluate acute, subchronic, and chronic exposures and measure biological endpoints such as mortality, reproductive performance, growth, and behavioural changes (Lee B.T. et al., 2007). Also by using toxicity tests, the relative toxicity of a mixture of chemicals can be assessed by taking into account synergistic or antagonistic interactions among chemicals. For the general screening of most chemicals, laboratory testing appears to be the most realistic option (Christensen O.M., Mather J.G., 1994). Earthworms are considered efficient test organisms for assessing ecotoxicological effects of chemicals, in order to elaborate recommendations and risk assessment schemes earthworms not only by considerations of reproducibility and practicability but also because of ecotoxicological relevance (Christensen O.M., Mather J.G., 1994). In order to test the ecotoxicity of some substances on earthworms, an essential importance is attributed to the test soil, because earthworms are organisms whose life environment is represented by soil. The differences appeared in the soil composition influence the bioavailability and the toxicity of the pollutants (Stephenson G.L. et al., 1998). Earthworms are often used as test organisms to assess soil processes because of they accomplish important function, e.g. as decomposer (Edwards C.A., Bohlen P.J., 1996), their high biomass in soil, between 10-200 g fresh weight m-2 (Hund-Rinke K., Wiechering H., 2001) and their sensitive reactions towards environmental influences.  MATERIAL AND METHOD  There are more current laboratory test methods outlined, but it is very important the major advantages/disadvantages of each of them. OECD (Organisation for Economic and Cooperation Development) founded in 1961 in Paris, France, provided the method of artificial soil test (Christensen O.M., Mather J.G., 1994) which meets the most criteria expected for ecotoxicological testing known as OECD Guideline for testing of chemicals 207 adopted in April 1981.  The equipment and the materials are those stipulated by OECD Guidelines 207/1984: earthworms from the species of Eisenia foetida 112Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi      (Savigny, 1826) (belonging to Phyllum Annelida, Class Oligochaeta, Family Lumbricidae) and artificial soil consisting of: 10 per cent sphagnum peat (as close to pH 5.5 to 6.0 as possible, no visible plant remains, finely ground, dried to measured moisture content), 20 per cent kaolin clay (kaolinite content preferably above 30 per cent), 70 per cent industrial sand (fine sand should be dominant with more than 50 per cent of the particles between 50 and 200 microns). The pH must be adjusted to 6.0 ± 0.5 by addition of calcium carbonate. The characteristics that make the species of Eisenia foetida (Savigny, 1826) suitable for such ecotoxicological tests are: its susceptibility to chemicals because it is a true soil-inhabiting species, it has a short life cycle, hatching from cocoons in 3 to 4 weeks, and reaching maturity in seven to eight weeks at 20°C. It is very prolific, each worm producing two to five cocoons per week from each of which emerge several worms. It is available commercially and can be bred readily in a wide range of organic waste materials. There were tested two types of organic-mineral fertilizers which had been coated with potassium humic acids, noted as L-200-Hum and SH-120. The chemical composition and the chemical and physical properties of these fertilizers are listed in the table 1.   Table 1Chemical composition and chemical and physical properties of the organic-mineral fertilizers tested  on earthworms No. crt. Chemical composition Organic-mineral fertilizers L-200-Hum SH-120 Chemical compositions 1 Humic acids 29.9% 26.4% 2 Nitrogen (Nt) 23.49% 10.47% 3 Phosphorous (P2O5) - 16.50% 4 Potassium (K2O) 0.255% 0.307% Chemical and physical properties  5 Cationic exchange capacity 96.3 me/100g 100.3 me/100g 6 pH in aqueous solution 1:2.5 7.3 7.0 7 Apparent density 0.823 g/cm3 0.852g/cm38 Granulation (1-5 mm) 89.9% 93.5%  RESULTS AND DISCUSSIONS  The earthworms Eisenia foetida had been exposed for 14 days to a geometrical series range of concentrations of the test substances (the fertilizers) as following: 1, 2, 4, 8, 16 g active substance/kg dry weight artificial soil). There were realized 4 replicates according to OECD methodology. All earthworms were adults, at least 2 months old and with weight ranging between 400 – 500 mg. At the test start and at the end there was established their biomass, and before starting the testing the worms were washed with tap water and incubated on wet paper for 24h to empty their guts. The monitoring of worms and data analyzing had been realized in the days 7 and 14 in order to establish the earthworm mortality, the reproduction capacity (number of cocoons) and the weight changes. In the tables 2 and 3 there are presented the data regarding the mortality and the reproduction activity of earthworms under exposure to the fertilizer L-200-Hum.    Table 2Earthworm mortality after exposure to the fertilizer L-200-Hum  Earthworms remained alive (mean values) Fertilizer concentration  (g/kg dry weight) Control1 2 4 8 16 After 7 days 10 9 8 8 7 10 After 14 days 10 9 8 7 7 10   Table 3Earthworm reproduction index after exposure to the fertilizer L-200-Hum Number of cocoons (mean values) Fertilizer concentration  (g/kg dry weight) Control1 2 4 8 16 After 7 days 38 33 26 28 22 47 After 14 days 42 36 27 25 22 41     113Lucrări Ştiinţifice - vol. 53, Nr. 1/2010, seria Agronomie     In the tables 4 and 5 there are shown the data regarding the mortality and the reproduction activity of earthworms under exposure to the fertilizer  SH-120. In the study there was also monitored the changes appeared in the earthworm weight after one week (7 days) and then after another week (14  days) of test. The results for the two tested fertilizer are shown in the tables 6 and 7. Comparing to the control variant, it can be observed that there is no mortality under L-200-Hum exposure for 1 g concentration, after 14 day of testing, after what the mortality start to increase as concentration level is higher (fig. 1, 2).  Instead, the reproduction rate of earthworms decreases under L-200-Hum action comparing to the control pots in the same time with concentration increasing level (fig. 3, 4). Regarding the fertilizer SH-120 it was observed that it manifests a negative influence on mortality and reproduction rate of earthworms, being found 50% mortality for 4 g concentration and 100% mortality for 8 and 16 g concentration (fig. 1,2,3,4). As well, the cocoon number drastically decreases after 14 days of SH-120 exposure to 1/2 comparing to L-200-Hum and to 1/3 related to the control pots for 2 g concentration and even to zero values (because of 100% mortality for 8 and 16 g concentration).     Table 4Earthworm mortality after exposure to the fertilizer SH-120 Earthworms remained alive (mean values) Fertilizer concentration  (g/kg dry weight) Control1 2 4 8 16 After 7 days 10 9 7 5 4 10 After 14 days 9 7 5 0 0 10  Table 5Earthworm reproduction index after exposure to the fertilizer SH-120   Number of cocoons (mean values) Fertilizer concentration  (g/kg dry weight) Control1 2 4 8 16 After 7 days 36 31 22 17 8 47 After 14 days 23 18 10 0 0 41  Table 6Earthworm weight after exposure to the fertilizer L-200-Hum  Earthworms weight (mg) Fertilizer concentration  (g/kg dry weight) Control1 2 4 8 16 After 7 days 471 439 394 353 311 457 After 14 days 462 427 379 347 324 431  Table 7Earthworm weight after exposure to the fertilizer SH-120 Earthworms weight (mg) Fertilizer concentration  (g/kg dry weight) Control1 2 4 8 16 After 7 days 467 426 327 238 178 457 After 14 days 449 294 216 0 0 431  0246810Number of earthworms remained aliveCon t r ol 1 2 4 8 16Concentration level (g)L-200-HumSH-120Figure 1 Earthworm mortality rate after 7 test days  0246810Number of earthworms remained aliveControl 1 2 4 8 16Concentration level (g)L-200-HumSH-120Figure 2 Earthworm mortality rate after 14 test days 114Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi      01020304050Number of earthworm cocoonsCon t r ol 1 2 4 8 16Concentration level (g)L-200-HumSH-120Figure 3 Earthworm reproduction rate  after 7 test days 01020304050Number of earthworm cocoonsControl 1 2 4 8 16Concentration level (g)L-200-HumSH-120Figure 4 Earthworm reproduction rate  after 14 test days  Referring to the earthworm weight evolution (biomass) it was noticed that it increased for 1 g concentration under L-200-Hum exposure comparing to the control pots. This fact is explained by the high content in nitrogen of the fertilizer L-200-Hum which is an essential element for earthworm surviving, because it means trophic resource and it can be found in large amount in the chemical composition of the mucus that is excreted by these organisms. The fertilizer SH-120 exerts in this case too a negative influence on earthworms, their weight decreasing with 50% for 4 g concentration related to the control pots (fig. 5; 6). 0100200300400500Earthworm weight after 7 test days (mg)Control 1 2 4 8 16Concentration level (g)L-200-HumSH-120Figure 5 Earthworm weight after 7 test days 0100200300400500Earthworm weight after 14 test days (mg)Con t r ol 1 2 4 8 16Concentration level (g)L-200-HumSH-120Figure 6 Earthworm weight after 14 test days  CONCLUSIONS  The performed study shows the following conclusions: The fertilizer L-200-Hum determined lower earthworm mortality comparing to SH-120 for all tested concentration levels. The fertilizer L-200-Hum positively influenced the earthworm rate of biomass accumulation, being found its increasing for 1 g concentration for the entire test period comparing to the control variants. The fertilizer SH-120 determined a 50% mortality of earthworms for 4 g concentration and 100% mortality for 8 and 16 g concentration, which requires a precaution attitude regarding its use in agriculture, and it is recommended the attentive establishment of the amounts applied into the field to not affect the earthworm populations. DL 50 it was established to be the concentration 4 g for the SH-120 fertilizer at the end of the test (14 days). The highest concentrations without causing mortality were: L-200-Hum, 1 g, 7 days; L-200-Hum, 1 g, 14 days; SH-120, 1 g, 7 days.  The lowest concentration causing 100% mortality was SH-120, 8 g, 14 days. Due to the limitative factors which appear in the laboratory tests it is recommended their completion with field tests in order to obtain more realistic results.  BIBLIOGRAPHY  Christensen, O.M., Mather, J.G., 1994 – Earthworms as ecotoxicological test-organisms, Danish Environmental Protection Agency, no. 5, p. 8-17. Edwards, C. A., Bohlen, P.J., 1996 – Biology and Ecology of Earthworms, Chapman & Hall Ltd., London, New York. Hund-Rinke, K., Wiechering, H., 2001 – Earthworm Avoidance Test for Soil Assessment, Journal of Soils and Sediments, Vol. 1, No. 1, pp. 15-20. Lee, B.T., Shin, K.H., Kim, J.Y., Ki,. K.W., 2007 – Progress in earthworm ecotoxicology, in Advanced Environmental Monitoring, Springer Netherlands, pp. 248-258. Stephenson, G.L., Kaushik, A., Kaushik, N.K., Solomon, K.R., Steele, T., Scroggins, R.P., 1998 – Use of an avoidance- response test to assess the toxicity of contaminated soil to earthworms, Setac Press, 67-81. ***, 1984 - OECD Guidelines for testing of chemicals-Earthworm, acute toxicity tests, no 207. ***http://www.icsu-scope.org. 115Lucrări Ştiinţifice - vol. 53, Nr. 1/2010, seria Agronomie

Ecotoxicological assessment of the effect of some new organic-mineral fertilizers on Eisenia foetida earthworms

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Ecotoxicological assessment of the effect of some new organic-mineral fertilizers on Eisenia foetida earthworms

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