ARTÍCULOS ORIGINALES
Chlorination: Phytotoxicity and effects on the production and quality of Lactuca sativa var. Mantecosa grown in a closed, soil-less system (With 5 Figures & 1 Table)
Cloración: fitotoxicidad y efectos sobre la producción y calidad de Lactuca sativa var. Mantecosa creciendo en un sistema de producción cerrado, sin suelo (With 5 Figures & 1 Table)
Premuzic1 Z, HE Palmucci1, J Tamborenea1 , M Nakama2
1 Facultad de Agronomía de la Universidad de Buenos Aires (FAUBA). Av. San Martín
4453. Capital Federal (1416). Address Correspondence to: Zdenka Premuzic, e-mail: premuzic@agro.uba.ar
2 Instituto de Floricultura INTA-CASTELAR. Buenos Aires, Argentina.
Recibido/Received 8.08.2006. Aceptado/Accepted 17.07.2007.
Abstract. The addition of chlorine constitutes an economical disinfection method for closed, soil-less systems. Three quantities of sodium hypochlorite (0.55, 5.5 and 11 ppm) were applied to closed, soil-less greenhouse- grown lettuce (Lactuca sativa), to study the effect on its production (fresh weight and dry matter) and some commercial and nutritional quality factors (phytotoxicity, Vitamin C, nitrates). Sodium hypochlorite was weekly added within the nutrient solution, and the chemical properties (pH, EC and chlorides) of the recycled solution were measured. Damage to leaves was evaluated 15 days before harvest. Chlorination produced different effects regarding both production and quality. All treatments presented plants with excellent commercial weight, although quantities of 0.55 and 5.5 ppm presented a 17% greater weight. However, this improvement was statistically not significant. Three different groups were observed for the phytotoxicity effects: the 11 ppm dose showed 42% of damage to leaves, while leaf damage was 22% for the 0.55 and 5.5 ppm quantities, and 15% for the control. The addition of sodium hypochlorite did not affect (significantly) Vitamin C and dry matter production. All treatments presented nitrate contents exceeding the allowed values; this fact was related with the nitrate composition of the nutrient solution. The chemical properties of the nutrient solution were adequate for the species. This study suggests the 0.55 and 5.5 ppm quantities as a positive option regarding plant weight. Further research should be developed to adjust fertilization, and to diminish the phytotoxicity symptoms.
Key words: Chlorination; Lactuca sativa; Nitrates; Phytotoxicity; Vitamin C.
Resumen. En los cultivos sin suelo con recirculación la adición de cloro constituye un método económico de desinfección. Tres dosis de hipoclorito de sodio (0,55; 5,5 y 11 ppm) se agregaron semanalmente a la solución nutritiva, con la finalidad de estudiar el efecto sobre la producción (peso fresco y seco) y algunos factores de calidad comercial y nutricional (fitotoxicidad, vitamina C y nitratos) de un cultivo de lechuga (Lactuca sativa). Se evaluaron las propiedades químicas pH, conductividad y cloruros. La cloración produjo diferentes efectos en la producción y calidad. Todos los tratamientos presentaron plantas con excelente tamaño comercial. Las dosis de 0,5 y 5,5 ppm presentaron un peso fresco un 17% mayor que las otras dosis, aunque la diferencia no fue estadísticamente significativa. Para la fitotoxicidad se observaron tres diferentes grupos. La dosis de 11 ppm mostró un 42 % de daño en hojas, mientras que el daño producido por las dosis de 0,55 y 5,5 ppm sólo fue de un 22 %, y en el control un 15 %. La adición de hipoclorito de sodio no afectó (significativamente) la vitamina C y la producción de materia seca. Todos los tratamientos presentaron valores de nitratos que excedieron los permitidos, lo que se atribuyó a la composición de nitratos de la solución nutritiva. Las propiedades químicas de la solución nutritiva fueron adecuadas para la especie. Este estudio sugiere que dosis de 0,55 y 5,5 ppm constituyen una opción positiva para lograr mejores pesos frescos en las plantas, siendo necesario nuevos ensayos a fin de ajustar la fertilización y disminuir los síntomas de fitotoxicidad.
Palabras clave: Cloración; Lactuca sativa; Nitratos; Fitotoxicidad; Vitamina C.
INTRODUCTION
Soil-less growing systems present advantages in greenhouse plant production over conventional systems. These systems can provide water and nutrients to the plant without restricting root development. Therefore, they can promote plant growth and achieve higher yields. Recently, the demand for environmentally friendly growing systems has boosted in many agricultural and horticultural crops, both in developed and developing countries. Sustainable production and development would enable an appropriate use of the resources and the environment (Ikeda et al., 2002). Closed soil-less systems seem to be an adequate technique to reduce the environmental impact caused by draining away the nutrient solution, and for saving water, fertilizers and then economical resources. When recirculation is used, this system has high water and fertilization use efficiency and low environmental impact (Gonnella et al., 2003). However, the risk of dispersing root infecting pathogens with a recycled nutrient solution needs to be avoided (Jarvis, 1992; Stanghellini & Rasmussen, 1994; Hong et al., 2003; Schmitzler, 2004). Propagation of diseases in these systems is favoured by the more appropriate environmental conditions to pathogens and the presence in the recycled solution of pathogens morphologically more adapted (waterborne pathogens).
Lactuca sativa L. is, among the vegetables, one of the major leafy vegetables cultivated in soil-less systems in Argentina.
Within the disinfection methods (Garibaldi et al., 2004), chlorination
is a chemical method used to prevent, reduce or eliminate the pathogens
present in the nutrient solution (Braun & Supkoff, 1994; Van Os, 2000;
Poncet et al., 2001; Newman, 2004). Some pathogens usually affect root
systems and progress to minimal foliar symptoms until the plant dies. In
these cases, prevention is the best strategy for eliminating or minimizing the
initial inoculum (Kong et al., 2004). This method of low cost and easy
application is based on the oxidation property of hypochlorous acid, and
takes into account the chlorine demand to reduce the pathogens, the residual
chlorine (chlorine in the solution after the reaction) and the time of
contact (time for the application effectivity) (http://www.ext.nodak.edu/extpubs/hòqual/watsys/ae1046whtm; Newman, 2004). In general, chlorine has
been used for killing microorganisms in drinking water. Chlorine at a concentration
of 1-2 ppm (residue chlorine) could kill zoospores of Pytaceae
fungi within 1 minute; concentrations of 100 ppm could kill the mycelia
(Smith, 1979). However the residue chlorine at a concentration of 10 ppm
has affected plant growth, and could result in root damage when used for
any extended time period (Ikeda et al., 2002). Experimental works inform
that different quantities and application times were selected for (1) their
effectiveness to control pathogens, (2) its phytotoxicity and (3) effects on productivity
(Frink & Bugbee, 1987; Pierna Inistta & Diaz Gil, 2001; Escobar et
al., 2003). The chemical instability of the chlorinated compounds constitutes
a major problem of chlorination, resulting in a loss and subsequent change of
concentration. One of the main forms of chlorine used includes sodium
hypochlorite (NaOCl). This compound, when mixed with water, forms sodium
hydroxide (NaOH) and hypochlorous acid (HCLO). The last one constitutes
the available and active chlorine. The hypochlorous acid is what kills pathogens.
In high pH solutions, most of the hypochlorous acid dissociates to form
hypochlorite ion (OCl-) which is not an effective sanitizer. A pH around 6.5-
7.5 will maintain about 80% of Chlorine as active chlorine or HCLO
(http://edis.ifas.ufl.edu/HS336; Newman, 2004).
The phytotoxicity of chlorine makes it necessary to quantify the
effects on the quality of edible plant species.
The ingestion of nitrates through either food or water produces
health problems in humans due to the formation of toxic compounds: nitrites
(as Methahemoglobinemia) and nitrosamines (Carcinogenic) (Wright &
Davidson, 1964; Maynard et al., 1976; Walters & Smith, 1981; Craddock,
1983; Vajagic et al., 1986; Preussmann, 1988; Somogyi, 1988).
The importance of nitrate content in vegetables is underlined in the European
Commission Regulation Nº 563/2002 (http://www.food/gov.uk/food_surv.htm).
It sets the maximum nitrate levels for lettuce and spinach in the different
seasons and cultivation methods. The JECFA (Joint FAO/WHO Expert
Committee on Food Additives) has set the Acceptable Daily Intake (ADI) of
nitrate between 0 and 3.65 mg/kg of body weight (WHO, 1995).
According to this ADI, only 100g of raw vegetables (with a NO3-
concentration of 2500 mg/kg f.w.) accounts for an intake of 250 mg NO3-.
Hence, if a 60-kg person consumes this amount alone, the ADI for NO3- will
be exceeded by 14%.
Therefore, an important quality feature of leaf vegetables is their
low nitrate content, even more for ready-to-use vegetables. Product cutting
(Caponigro et al., 1997) and packaging, especially airtight packages, can
cause an increase of nitrite content. This is even more dangerous than
nitrate (Achtzehn & Hawat, 1970).
The carcinogenic effects of nitrosamine compounds could not be
proved in humans exposed to diets with large nitrate contents (fish, lettuce,
spinach) (Van Manen et al., 1998). The absence of a relation between
the presence of these compounds and the risk of cancer in humans
(Gangoli et al., 1994) could be related to the fact that vegetables have
Vitamin C. Therefore, the intake of nitrates through water (no Vitamin C)
constitutes a greater risk than its intake from vegetables (Hotchkiss et
al., 1992).
Vitamin C is one of the most important vitamins present in vegetables
and fruits, contributing more than 90% of human vitamin C requirements.
Therefore, this vitamin results one of the most important nutritional
quality factors. Many biological processes, like the inhibition of
the nitrosamine synthesis and the reduction of cardiovascular and carcinogenic
diseases are attributed to Vitamin C (Harris, 1996).
Lactuca sativa is a species with great ability to accumulate nitrates
(Richardson & Hardgrave, 1992). There exists an antagonism between
the effects of nitrates and vitamin C (Graifenberg et al., 1993). Then,
low contents of nitrates and high levels of vitamin C are selected as parameters
for good quality foods.
Chlorination is an economical disinfection method. However, the
chemical changes of the associated recycled nutrient solution are complex,
and differ with seasonal and location factors. Soil-less cultures are
widespread, but there is a lack of information on the effects of chlorination
on some crops.
Our objective was to characterize the production and quality of
Lactuca sativa cultivar Mantecosa growing in a closed soil-less system
after different quantities of sodium hypochlorite were added to the
nutrient solution, as a disinfection method.
MATERIALS AND METHODS
Mantecosa, Butterhead or loose-head type of lettuce (Lactuca sativa) was grown during the spring (October) period, in a 240 m2 greenhouse of metallic structure, covered with polyethylene and provided with lateral ventilation. It was located in the research area of the Floriculture Institute (INTA-CASTELAR), Buenos Aires, Argentina.
Pots were filled with perlite and received individual drip irrigation with a nutrient solution, following the plant needs. They were placed on black plastic- covered wood benches with an inclination of 5 % to enable draining of the nutrient solution to a storage tank. One lettuce plant was placed in each pot.
Irrigation and fertilization were performed adding the Enshi solution (CETTEFFHO), which provides macronutrients (252 mg/l N ( 9% as NH4 + ), 312 mg/l K, 54 mg/l P, 48 mg/l Mg, 65 mg/l S and 169 mg/l Ca) and micronutrients (0.26 mg/l Co, 0.54 mg/l Zn, 0.48 mg/l Fe, 0.35 mg/l Mo, 0.78 mg/l Mn, 0.32 mg/l Cu and 0.2 mg/l B). This solution was prepared with water previously subjected to reverse osmosis (pH: 6.58; EC: 0.016 dS/m2 and Cl- : 19.56 mg/l).
The Chlorine stock solution was prepared with 100 ml of commercial sodium hypochlorite (55.5%) in 10 liters of water, previously subjected to reverse osmosis. The quantities for each treatment were prepared by dilution of the stock solution. Three different quantities of sodium hypochlorite, and a control without sodium hypochlorite, were applied to four treatments. The corresponding quantities for each treatment were: 0.55 ppm (0.015 meq /l) with 1 ml stock/liter solution; 5.5 ppm (0.15 meq/l) with 10 ml/l, and 11ppm (0.30 meq/l) with 20 ml/l. One week after placing the plants in the pots, the first chlorination was applied. It continued throughout all the culture, and within the fertilization and irrigation solution. To improve the collection of the lixiviated material, a 5% slope was allowed to conduct it towards the storage tank of the nutrient solution.
Controls of the electrical conductivity (conductimeter), pH (potenciometry) and chlorides (volumetric) of the re-circulating nutrient solution were weekly performed. This allowed to keep the water volume and the chlorine and fertilizer quantities at the initial levels for each treatment.
Fifteen days before harvest, symptoms of phytotoxicity were monitored evaluating the relation: number of leaves with necrosis/ total number of leaves per plant affected by chlorine x 100.
At harvest (45 days after sowing), plant fresh and dry matter were determined using a thermo-ventilated oven at 70 ºC until constant weight was reached. Determinations of Vitamin C content in the fresh material (AOAC, 1980) and nitrate content in the dry material (Cataldo et al., 1975) evaluated effects on plant quality.
A completely randomized experimental design with four treatments was used with 20 replicates/treatment, to examine the effects of the independent variable treatment (four treatments: 1- control; 2- 0.55 mg/l; 3- 5.5 mg/l and 4- 11 mg/l) on dependent variables. One-way ANOVA was performed on fresh weight, dry matter, vitamin C, nitrates, pH, EC and chlorides, and significant differences were examined using the LSD test (p < 0.05). With this purpose, SPSS software was used (Field, 2000).
RESULTS
Phytotoxicity. Damage to leaves caused by the addition of sodium hypochlorite showed three different groups, presenting values from 15.7 to 42.7 %. Lowest (p < 0.05) leaf damage was found in the control treatment, while it was intermediate at the 0.55 and 5.5 mg/l quantities (22%) (Fig.1). The third group, with 11mg/l of sodium hypochlorite, showed the greatest (p < 0.05) damage to leaves (42%, Fig. 1).
Production of Lactuca sativa, cultivar Mantecosa
Fresh weight. The 0.55 and 5.5 mg/l treatments presented larger values of
fresh weight (mean=17%) than the other treatments. However, this increase
was not statistically significant (p > 0.05) (Fig. 2).
Production of dry matter. Dry matter production was 5.52 - 6.27 % of fresh weight, and there was no significant differences (p > 0.05) among treatments (Fig. 3).
Quality of Mantecosa
Nitrate contents. On a fresh weight (f.w.) basis, there were no significant differences (p > 0.05) in nitrate content among treatments (Fig. 4).
Vitamin C contents. There were no significant differences (p > 0.05) among treatments for vitamin C contents (Fig. 5).
Chemical properties of the recycled nutrient solution
Ph of the solution. The 11 ppm treatment showed a significantly higher (p < 0.05) pH than the other treatments (Table 1).
Electrical conductivity of the solution. Electrical conductivity was higher (p < 0.05) in the 5.5 than in the 11 ppm treatment (Table 1) Contents of Chlorides in the solution. The highest (p < 0.05) chloride content was observed in the 11 ppm treatment (Table 1).
DISCUSSION
The chlorine dose of 11 ppm caused a higher amount of chlorotical and
necrotic tissues on leaves, affecting the commercial quality of plants. Symptoms
of chlorosis and necrosis were observed on lower leaves in the plant. Chlorosis
was developed first at the leaf tips, and as toxicity progressed, it moved towards
the leaf edges. This is a typical response to phytotoxicity by chlorine (http://www.geocities.com/ pbarrosvanc/tema01.html). Our results were similar to those of
Fink & Bugbee (1987). They found a general lack of vigor and foliage chlorosis
on vegetable seedlings when residual chlorine reached 18 mg/l.
No symptoms of disease by natural infections were observed during the
period of the experiment.
The different sodium hypochlorite quantities did not affect plant
weight production. Values of plant fresh weight were similar to those cited
by Fernandes et al. (2002). There was a negative correlation between fresh
weight and dry matter production.
All the nitrate values exceeded those allowed (3500 mg/kg) by the
European legislation for spring, greenhouse-grown lettuce (European
Commission Regulation Nº.563/2002 (http://food/gov.uk/food_surv.htm)).
These values could be caused by a high dose of nitrogen fertilization,
since there is a direct relationship between the quantities of nitrogen supplied
by the fertilizer and the leaf nitrate content (Saez Sironi et al., 2002).
Also, the nitrate -ammonium (NO3
-/ NH4
+) relation in the nutrient solution
could have affected the levels of leaf nitrates. The solution presented 9 %
of nitrogen as ammonium. Other experiments showed that contents of 20 %
nitrogen as ammonium in the solution diminished the amount of leaf
nitrates (Van Der Boon et al., 1990).
The range of values reported for vitamin C contents is very wide.
Geigy (1975) observed values of 31 mg/100 g of f.w., while Souci (1981) and
Comenge (1962) mentioned 8-21 mg/100 g of f.w. Watt & Merril (1975) also
reported values of 18 mg/100 g of f.w.
Mantecosa variety presented vitamin C values (15-20 mg/100 g of
f.w.) similar to those cited by Watt & Merril (1975). The vitamin C content
showed a negative correlation with fresh weight (Drews et al., 1997),
and a positive correlation with dry matter. This could be related to the
incidence of light on the vitamin C contents and fresh weight production.
Light presence is related to a greater photosynthesis and carbohydrate
production, and therefore, to a greater production of dry matter. Vitamin
C synthesis is also favoured by the presence of light (Blom- Zandstra &
Lampe, 1985).
Although there was an increase of pH in the recycled solution before
the addition of increasing chlorine quantities, values did not surpass the
range recommended for the species by Van Der Boon et al. (1990), and they
were suitable for chlorination. The electrical conductivity maintained adequate
range values (Reed, 1999), and they were similar to those presented
by Magnani & Oggiano (1997). The chloride content showed values from
0.90-1.29 meq/l. The largest contents corresponded to the largest sodium
hypochlorite quantities added, although they did not exceed the adequate
values for the species (Reed, 1999).
ACKNOWLEDGEMENTS
This work was supported by the Project UBACYT G076. We acknowledge the technical staff from the Floriculture Institute (INTA-CASTELAR), who prepared the necessary infrastructure for developing this study.
REFERENCES
1. Achtzehn, M.K. & H. Hawat (1970). Zur Nitritbilddung in Gemüse und Gemüsezubereitungen. 1. Mitt. Rohspinat. Die Nahrung 14: 383-394.
2. AOAC. Association of Analytical Chemists (1980). Official methods of analysis of the Association of Analytical Chemists. Washington.D.C.
3. Blom-Zandra, M. & J. Lampe (1985). The role of nitrate in the osmoregulation of lettuce (Lactuca sativa) grown at different light intensities. Journal of Experimental Botany 39: 1043-1052.
4. Braun, A. & D. Supkoff (1994). Options to methyl bromide for the control of soil-borne diseases and pests in California with reference to the Netherlands. A. Braun and D. Supkoff. Pest Management Analysis and Planning Program, State of California. PM 94-02.
5. Caponigro, V., G. Cafiero, A. Tonini, C. Perrella & F. Piro (1997). Microbiologia superficiale della rucola confezionata pronta all'uso. Italus Hortus 4:58-62.
6. Cataldo, D., M. Haroon, L. Schrader & V. Young (1975). Rapid Colorimetric determination of nitrate in plant by nitration of salicylic acid. Comunications in Soil Science and Plant Analysis 6:71-80.
7. Comenge, M. (1962). Análisis de Alimentos. II. 3. Ed. Escellicer, S. A. Madrid. Disease 76: 457-461.
8. Craddock, V. M. (1983). Nitrosamines and human cancer: proof of an association Nature (London) 306: 638.
9. Drews, M., I. Schonhof & A. Krumbein (1997). Contents of minerals, vitamins and sugars in Iceberg lettuce (Lactuca sativa var capitat L.) grown in greenhouse, dependent on cultivar and development stage. Gartenbauwissenschaft 62:65-72.
10. Escobar I, J.J Berenguer, M García, J.Gómez & A. Alvarez (2003). Control de Phytophthora spp. en cultivos sin suelo de pepino y tomate cereza. Actas de Horticultura Nº 39. X Congreso Nacional de Ciencias Hortícolas, Pontevedra, Protección de Cultivos. p. 540-542.
11. Fernandes, A. A., H.E.P. Martinez, P.R.G. Pereira & M.C.M Fonseca (2002). Productividade, acúmulo de nitrato e estado nutriciona de cultivares de alface, em hidroponia, em funcao de fontes de nutrientes. Horticultura Brasileira 20:195-200.
12. Field, A. (2000). SPSS C8057 Research Methods Contrast and post Hoc Tests.
13. Frink, C. R. & G. J. Bugbee (1987). Response of potted plants and vegetable seedlings to chlorinated water. Horticultural Science 22: 581-583.
14. Gangoli, S., P. Van den Brandt, V. Feron., J. Janzowsky, J. Koemen, G. Speijers, B. Spiegelhalder, R. Walker & J. Wishnok (1994). Nitrate, Nitrite and N- nitroso compounds. European Journal of Pharmacology Environmental Toxicology and Pharmacology Section 4, N 1, 1-38.
15. Garibaldi A., A. Minuto & D. Salvi (2004). Disinfection of nutrient solution in closed soilless systems in Italy. Acta Horticulturae (ISHS) 644:557-562.
16. Geigy, J. R. (1975). Tablas Cientificas. 7ª ed.. Ed. Geigy División farmacéutica, Barcelona.
17. Gonnella, M., G. Conversa, P. Santamarina & F. Serio (2003). Yield and Quality of Lettuce Grown in Floating System Using Different Sowing Density and Plant Spatial Arrangements. Acta Horticulturae 614:687-692.
18. Graifenberg, A., L. Barsanti, L. Botrini & O. Temperini (1993). La problemática dei nitrati. L'informatore Agrario 6: 43-48.
19. Harris, J. (1996). Subcelullar biochemistry, Ascorbic Acid: Biochemistry and biomedical. Cell biology, vol. 25. Plenum, New York.
20. Hong C. X., P. A. Richardson, P. Kong & E. A. Bush (2003). Efficacy of Chlorine on Multiple Species of Phytophthora in Recycled Nursery Irrigation Water. Plant Disease 1183-1189
21. Hotchkiss, J., M. Helser, C. Maragos & Y. Weng (1992). Nitrate, nitrite and nitrosocompounds food safety and biological implications. In: ACS (American Chemical Society) Symposium series 484 Food safety Assesment; 200th National Meeting of the American Chemical Society, Washington, D.C, USA, American Chemical Society 1992. Finley and Amstrong 400-418 Washington, D.C USA. American Chemical Society.
22. http://www.ext.nodak.edu/extpubs/h2oqual/watsys/ae1046w.htm. Bergsrud F., B. Seelig & R. Derickson (1996). Treatment Systems for household water supplied. Chlorination.
23. http://www.food/gov.uk/food_surv.htm European Commission Regulation Nº 563/2002.
24. http://www.geocities.com/pbarrosvanc/tema01.html. Barros, P. La hidroponia, pero si es muy fácil.
25. http// edis.ifas.ufl.edu/HS336. Ritenour, M.A., S.A Sargent, J.A. Bartz & E.E. Lon Kan. Uso del cloro en las líneas de empacado de productos cosechados frescos. University of Florida IFAS, Extensión.
26. Ikeda, H., K. Prommart & J. Tanimnun (2002). Problems and countermeasures in the re-use of the nutrient solution in soiless production. Acta Horticulturae 578: 213-219.
27. Jarvis, W.R. (1992). Managing diseases in greenhouse crops. American Phytopathological Society Press, St. Paul, MN, USA. 288 p.
28. JECFA/FAO/ WHO (The Joint Expert Committee on Food Additives in cooperation with Food and Agricultural Organization and World Health Organization) (Commission European, 1997).
29. Kong, P., P.A. Richardson & C. Hong (2004). Characterization and control of Pythium Pathogens in Recycled Irrigation Water. Research Report F.2004-2 April 2004.
30. Magnani, G. & N. Oggiano (1997). Reducing the level of nitrates in hydroponic lettuce. Colture Protette 26:57-61.
31. Maynard, D. N., A.V. Barker, P.L Minotti & N.H. Peck (1976). Nitrate accumulation in vegetables. Advances in Agronomy 28: 71-118.
32. Newman, S. E. (2004). Disinfection Irrigation Water for Disease Management. 20th Annual Conference on Pest Management on Ornamentals. Society of American Florist, February 20-22, 2004, San José, California. p. 2-10.
33. Pierna Inistta E. & R.V. Díaz Gil (2001). Recirculación de soluciones nutritivas en cultivos sin suelo. Revista Agricultura 826:246-256.
34. Poncet C., M. Offroy & G. Bonnet (2001). Disinfection of recycling water in rose cultures. Proc. III IS Rose Research (eds). N. Zieslin & H. Agbaria. Acta Horticulturae 547:121-126.
35. Preussmann, R. (1988). Nitrite und Nitrate in der Nahrung und in Wasser. Bibliotheca Nutritio et Dieta (Basel) 41: 66-75.
36. Reed, D. (1999). Agua, sustratos y nutrición. Ed. Ball Pub. 335, North River St., Batavia, Illinois, USA, & Ed. Horti Tecnia, Bogotá, Colombia, p. 31-40.
37. Richardson, S .J. & M. Hardgrave (1992). Effect of temperature, carbon dioxide enrichment, nitrogen form and rate of nitrogen fertiliser on the yield and nitrate content of two varieties of glasshouse lettuce. Journal of Science Food Agriculture 59: 345-349.
38. Saez Sironi, J. , L. Rincón Sánchez, J. A. Pérez Crespo, C. Pellicer Botía & A. Abadía Sánchez (2002). Influencia de la fertilización nitrogenada en la absorción de nitrógeno y acumulación de nitratos en la lechuga iceberg. Investigación Agraria. Producción y Protección Vegetales 17:303-318.
39. Schmitzler, W. H. (2004). Pest and Disease Management of Soil-less Culture. Acta Horticulturae 648:191-196
40. Smith, P. M. (1979) A study of the effects of fungitoxic compounds on the Phytophtora cinnamomi in water. Annals of Applied Biology 93:149-157.
41. Somogyi, J.C. (1988). Belastung der Nahrung durch di uUmwelt Gefahr für die Gesundheit? Einführung in die Thematik. Bibliotheca Nutritio et Dieta (Basel) 41: 26-33.
42. Souci, S., W. Fackmann & H. Kraut (1981). La composition des aliments. Wissenschaftliche Verlagsgesellschaft, M.B.H Stuttgart.
43. Stanghellini, M.E. & S.L Rasmussen (1994). Hydroponics, a Solution for Zoosporic Pathogens. Plant Disease 78: 1129-1138.
44. Vajagic, L., B. Lazic, M. Kristoforic & M. Popovic (1986). Znacaj kontaminacie namirnica biljnog porekla azotnim jedinjenjima iz zivotne sredine. Hrana I Ishrana. 4: 239- 242.
45. Van Der Boon, J., J. W Steenhuizen & E. G. Steingrover (1990). Growth and nitrate concentration of lettuce as affected by total nitrogen and chloride concentration, NH4 + / NO3 - ratio and temperature of the recirculating nutrient solution. Journal of Horticultural Science 65: 309-321.
46. Van Os, E. A. (2000). New Developments in recirculation systems and disinfection methods for greenhouse crops. The 15th Workshop on Agricultural Structures and ACESYS (Automation, Culture, Environment & System). IV Conference. December 4-5. Tsukuba, Ibaraki, Japan. p. 81-91.
47. Van Maanen, J., D. Pachen, J. Dallinga & J. Kleinjans (1998). Formation of nitrosamines during consumption of nitrate and amine rich foods and the influence of the use of mouthwashes. Cancer detection and Prevention 22:204-212.
48. Walters, C. & P. Smith (1981). The effect of water-borne nitrate on salivary nitrite. Food and cosmetic toxicology 19:297-302.
49. Watt, B. K & A. L. Merrill (1975). Composition of food. United States Department of Agriculture, Washington.
50. WHO (1995). Nitrite and Nitrate. In: Toxicological evaluation of certain food additives and contaminants in food, Prepared by the forty-fourth report of the joint FAO/WHO experts committee on food additives, Food add. Series 35:269-360.
51. Wright, M. G. & K. L. Davidson (1964). Nitrate accumulation in crops and nitrate poisoning of animals. Advances in Agronomy 16:197- 247.