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Studies on the effects of the insecticide aldrin on aquatic microbial populations
International Biodeterioration & Biodegradation 50 (2002) 83 – 87
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Studies on the e"ects of the insecticide aldrin on aquatic microbial populations L. L'opez, C. Pozo, M.A. G'omez, C. Calvo, J. Gonz'alez L'opez ∗ Environmental Microbiology Group, Department of Microbiology, Faculty of Pharmacy and Institute of Water Research, University of Granada E-18071 Granada, Spain Received 2 April 2000; received in revised form 1 May 2001; accepted 1 September 2001
Abstract The e"ects of aldrin at rates 10 and 50 mg ml−1 on microbial function were studied under aerobic conditions. Parameters monitored included total culturable population of heterotrophic bacteria, number of culturable phosphate-solubilizing micro6ora, number of bacteria which decompose protein with liberation of H2 S, nitrifying bacteria, denitrifying bacteria and denitri8cation activity. Culturable heterotrophic bacteria, phosphate-solubilizing bacteria, bacteria which decompose proteins with liberation of H2 S and denitrifying bacteria were signi8cantly increased at dose rates of 10 and 50 mg ml−1 of aldrin. However, the presence of aldrin decreased denitri8cation activity at concentration of 50 mg ml−1 . Nitrifying population were not a"ected as a consequence of the addition of aldrin, showing that these microorganisms can tolerate the concentrations of aldrin tested. ? 2002 Elsevier Science Ltd. All rights reserved. Keywords: Insecticides; Aldrin; Aquatic micro6ora; Xenobiotics
1. Introduction Modern agriculture and industry depend on a wide variety of synthetic chemicals, including insecticides, fungicides, herbicides and others pesticides. Continual wide-spread use and release of such synthetics has become an everyday occurrence, resulting in environmental pollution. In this context, the in6uence of pesticides on the microbial activity of aquatic microorganisms has been studied by some investigators both in pure culture and in a mixed populations (Brockway et al., 1984; Mirgain et al., 1993; Ramanand et al., 1993). However, it is not possible to reach a general conclusion regarding the e"ect of these substances on aquatic microbial activity because a number of factors (e.g., climate, water type, chemical structure and the composition of the microbial community) in6uence the e"ects of those xenobiotics. Aldrin (1,2,3,4,10,10a-hexachloro-1,4,4a,5,8,8a hexahydro-1,4,endo-exo 5,8-dimethanonaphthalene) is a commonly used insecticide in intensive agriculture, applied at concentrations of 2.0 –10:0 kg ha−1 for pest control such as ∗
Corresponding author. Tel.: +34-58-243-870; fax: +34-58-200-962.
leatherjacket. However, because of their persistence in the environment, susceptibility to biomagni8cation, and toxicity to higher animals, these organochlorine insecticides have been banned in technologically advanced countries (Lal, 1982). Organochlorine insecticides can enter into aquatic environments by direct or indirect routes. The two primary sources for the direct entry of insecticides into aquatic environments are the huge amounts added to water for effective pest control and the enormous quantities discharged into water along with industrial and domestic sewage waters (Foulkes, 1991). These agrochemicals indirectly enter into aquatic environments by drift from aerial or ground application and through water soil erosion, which include run-o", wash-o" and leaching from treated lands. In this sense, it is important to evaluate how the aquatic micro6ora could be a"ected by some chemical substances such as organochlorine insecticides applied to soil and water for pest control. The purpose of the present study was to examine how the insecticide aldrin at normal 8eld concentrations a"ects the aquatic micro6ora of a protected wetland located in the southeast of Spain. To our knowledge, this wetland has not previously been microbially characterized. Microbial parameters examined in this study were selected because of their
0964-8305/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 4 - 8 3 0 5 ( 0 1 ) 0 0 1 2 8 - 7
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L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87
relation to overall microbial activity and the microbially mediated cycling of carbon, phosphorous, sulphur and nitrogen. 2. Materials and methods 2.1. Water samples Samples were collected from a wetland located on the Atlantic Mediterranean hydrographic divide (southeast Spain) with no previous aldrin applications (Benavente et al., 1993). Water samples were collected from the top 15 cm according to Rodina (1972). Water samples were collected in sterile bottles (1 l), shipped to the laboratory and re◦ frigerated (4 C). Chemical and physical characteristics of the water samples were measured by techniques describe previously (Anon, 1985) and are presented in Table 1. 2.2. Treatment and water incubation Laboratory studies were performed on 8ve replicates. Five hundred milliliter samples were placed in a 1 l Erlenmeyer 6ask and amended with 5 ml of appropriate concentrations of sterile aldrin in acetone to give concentrations of 10 and 50 mg ml−1 (equivalent to 2 and 10 Kg ha−1 ) and then in◦ cubated at 20 C with continuous shaking (100 rpm:). After 1, 7, 14 and 28 days, microbial populations and biological activities were determined. Control samples received equal amounts of acetone for comparison. In the present investigation, technically pure grades (98%) of the insecticide (Eurolab, Granada, Spain) were used to determine their e"ect on the aquatic micro6ora. 2.3. Determination of microbial populations Total culturable heterotrophic bacteria were counted by dilution plate technique using Trypticase Soy Agar (TSA, Difco). The inoculated agar plates (three replicates) were ◦ incubated at 28 C for 3 days for mesophilic bacteria or at Table 1 Chemical and physical characteristics of water samples∗ ◦
Temperature ( C) pH ◦ Conductivity (mmhos=cm at 25 C) Dissolved oxygen (mg=l) Cl− (mg=l) CO3 H− (mg=l) CO2− (mg=l) 3 SO2− (mg=l) 4 SiO2 (mg=l) Ca2+ (mg=l) Mg2+ (mg=l) Na+ (mg=l) K + (mg=l) ∗ Data
◦
4 C for 7 days for psychrophilic bacteria before colonies were counted. Culturable phosphate-solubilizing bacteria were estimated by a standard dilution series in sterile saline solution, followed by plating on insoluble phosphate agar medium. The composition of insoluble phosphate medium was: glucose 5:0 g, K2 HPO4 0:8 g, NaCl 0:2 g, MgSO4 ·7H2 O 0:2 g, CaSO4 ·2H2 O 0:05 g, MaMoO4 ·2H2 O 0:01 g, FeSO4 0:003 g, Ca3 (PO4 )2 8:0 g, agar 15:0 g and distilled water 1 l. The pH was adjusted to 7.0 with sterile 0.1M NaOH. ◦ Inoculated plates (three replicates) were incubated at 28 C for 5 days. The most probable number (MPN) technique according to Rodina (1972) was used to count bacteria which decompose proteins with liberation of H2 S. The formations of H2 S by bacteria was detected in test tubes containing 10 ml of beef-peptone broth with suspended indicator paper soaked in a saturated solution of lead acetate. Inoculated test tubes ◦ (three replicates) were incubated at 22–23 C for 6 days. The dilution tube technique (MPN) was employed for enumeration of nitrifying bacteria. A separate analysis was performed for nitrifying phase I bacteria, which oxidize ammonia salts to nitrite, and for nitrifying phase II bacteria, which oxidize nitrite to nitrate. An ammonium sulphate medium was used for nitrifying phase I bacteria and a sodium nitrite medium for nitrifying phase II bacteria, according to Rodina (1972). Inoculated 6asks (three repli◦ cates for water samples) were incubated at 28 C and the presence of nitrite-N and nitrate-N in these 6asks was determined once a week for up to 4 weeks as described by Rodina (1972). Numbers of denitrifying bacteria were determined by a most probable number (MPN) technique according to Rodina (1972). The liquid enrichment medium contained per liter of distilled water: sucrose, 30:0 g, NaNO3 2:0 g, K2 HPO4 1:0 g, MgSO4 ·7H2 O 0:5 g, KCl 0:5 g, FeSO4 ·7H2 O trace, pH 7.0. Inoculated test tubes (three replicates) were ◦ incubated at 28 C for 7 days and denitri8cation was detected by the liberation of gas in the test tubes served as an indicator of denitrifying activity. 2.4. Determination of denitri8cation activity
18:4 ± 2:4 7:4 ± 0:2 4:5 ± 0:4 9:0 ± 0:5 430 ± 12:3 95:0 ± 7:6 5:5 ± 0:3 2660:0 ± 91:3 5:2 ± 0:5 52:5 ± 4:5 210:0 ± 9:1 261:0 ± 8:3 10:0 ± 1:1
are means ± SE of three separate determinations.
N2 O production, as indicators of the potential for denitri8cation, was determined by the acetylene inhibition method (Yoshinari and Knowles, 1976). Four milliliter samples of water amended or unamended with aldrin were placed in 10 ml tubes containing 1 ml of culture medium for denitri8cation and sealed with rubber stoppers. The composition of culture medium for denitri8cation was: sucrose 30 g, NaNO3 10:0 g, K2 HPO4 5:0 g, MgSO4 ·7H2 O 2:5 g, KCl 2:5 g, FeSO4 ·7H2 O 0:03 g and distilled water to 1 l. The pH was adjusted to 7.0 with 0.1M NaOH. After 100% of the atmosphere had been replaced by He, the tubes were inoc◦ ulated at 28 C and 0:25 ml gas were assayed for N2 O after
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87
1, 4, 7, 9 and 15 days by injection into a Varian Star 3400 gas chromatograph equipped with a thermal conductivity detector. 2.5. Statistical analysis Student’s “t” test was used to measure the di"erence between control without insecticide and water samples treated with 10 and 50 mg ml−1 aldrin. An ANOVA test was used to assess homogeneity of variance. Where the variance was heterogeneous, the data were suitably transformed before the analysis. A signi8cance level of 5% (p ¡ 0:05) was selected. 3. Results and discussion Microbial transformation is recognized as a critical factor a"ecting the fate and behaviour of pesticides in soil and aquatic ecosystems (Cook, 1987). Aquatic micro6ora comprises a diversity of species and is a dynamic population able to degrade a wide variety of chemically complex compounds. In this context, the biodegradation of complex halogenated compounds such as aldrin is considered to be a cometabolic process (Salmer'on et al., 1991; Saxena et al., 1987). There have been constant pressures, on authorities controlling the registration and approval of agricultural chemical, to include measurements of side e"ects on the soil and water micro6ora in their requirements. The Environmental Protection Agency in the United States, for
85
example, in guidelines for registering pesticides (Barbera, 1989) required data from “Studies of e"ects on microbial function...”. Nitri8cation is one of the functions speci8ed. To encourage uniformity of presented data and aid interpretation and comparison, the guidelines recommended the use of standard methods, usually a single sample temperature of ◦ 20 –30 C. The plate-count data indicated that culturable heterotrophic bacteria (mesophilic and psychrophilic micro6ora) in freshwater amended with aldrin were signi8cantly stimulated at doses of 10 and 50 mg ml−1 . This positive e"ect was evident in water samples after 1, 7, 14 and 28 days incubation with the insecticide (Table 2). In the absence of data on the mineralization or biotransformation of aldrin in water samples, the proliferation of bacterial 6ora in treated waters may be associated with the transformation of aldrin by the bacterial in water. However, it is likely that the e"ect obtained in our studies can be in6uenced by the species of bacteria present in the water. The presence of aldrin at concentrations of 10 and 50 mg ml−1 in the water positively a"ected phosphatesolubilizing bacteria and H2 S liberating bacterial population. The plate-count data indicated that those microorganisms were signi8cantly increased under our experimental conditions (Table 2), suggesting that phosphate-solubilizing and H2 S liberating bacterial, can tolerate high amounts of the insecticide aldrin without inhibition to their biological activities. The degree of stimulation was related to the concentration of aldrin. The presence of aldrin in the water positively a"ects the populations of bacteria which decompose protein with liberation of H2 S (Table 2).
Table 2 Number of culturable heterotrophic bacteria (mesophilic and psychrophilic), culturable phosphate-solubilizing bacteria, H2 S liberating and denitrifying bacteria in water samples in the absence and presence of aldrin∗
Aldrin (mg=ml)
Incubation time (d)
Mesophilic bacteria (104 =ml)
Psychrophilic bacteria (103 =ml)
Phosphate-solubilizing bacteria (103 =ml)
H2 S liberating bacteria (103 =ml)
Denitrifying bacteria (ml)
0
0 1 7 14 28
6:1 ± 0:15 6:1 ± 0:1 4:2 ± 0:4 0:6 ± 0:1 0:1 ± 0:02
9:1 ± 0:4 31:3 ± 5:0 6:3 ± 0:5 0:1 ± 0:01 0:1 ± 0:01
1:7 ± 0:17 7:5 ± 0:4 0:1 ± 0:01 0:2 ± 0:02 ¡ 0:1
35:0 ± 3:0 22:0 ± 3:0 17:0 ± 2:0 3:4 ± 0:3 0:7 ± 0:1
220 ± 1:0 170 ± 2:0 64 ± 2:0 33 ± 0:6 27 ± 0:4
10
1 7 14 28
47.7 ±1:4a 20:5 ± 0:1a 5:7 ± 0:2a 1:0 ± 0:04a
211:7 ± 10:0a 8:2 ± 0:4a 0:9 ± 0:7a 1:2 ± 0:4a
75:2 ± 4:5a 0:8 ± 0:04a 1:0 ± 0:1a 0:2 ± 0:02a
28:0 ± 3:0 35:1 ± 2:0a 17:0 ± 4:0a 1:2 ± 0:3a
280 ± 3:0a 170 ± 2:0 110 ± 0:2a 90 ± 1:0a
50
1 7 14 28
58:8 ± 1:0a 21:5 ± 0:4a 5:3 ± 0:5a 5:8 ± 0:02a; b
178:7 ± 18:0a 40:0 ± 2:0a; b 1:0 ± 0:1a 0:7 ± 0:04a
78:0 ± 3:6a 3:1 ± 2:7a; b 4:1 ± 1:0a; b 0:6 ± 0:05a; b
∗ Data
are means ± SE of 8ve samples. signi8cant di"erence (p ¡ 0:05) from control. b Represents signi8cant di"erence (p ¡ 0:05) between 10 and 50 g aldrin. a Represents
35:0 ± 5:0a 160:0 ± 6:0a; b 28:3 ± 2:0a; b 14:0 ± 1:0a; b
900 ± 7:0a; b 900 ± 6:0a; b 355 ± 4:0a; b 280 ± 6:0a; b
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87
Table 3 Number of nitrifying (phase I and II) bacterial per milliliter in water samples in the absence and presence of aldrin∗
Aldrin (g=ml) 0
10
50
∗ Data
Incubation time (d)
Phase I
Phase II
0 1 7 14 28
263 ± 35 269 ± 36 281 ± 24 266 ± 38 299 ± 32
416 ± 36 471 ± 42 466 ± 21 416 ± 16 451 ± 40
1 7 14 28
247 ± 39 266 ± 51 281 ± 37 269 ± 30
436 ± 23 436 ± 50 439 ± 34 464 ± 44
1 7 14 28
273 ± 32 284 ± 41 275 ± 32 300 ± 29
471 ± 26 473 ± 33 439 ± 34 464 ± 27
are means ± SE of 8ve samples.
The number of ammonia-oxidizing bacteria 50 mg ml−1 and nitrite-oxidizing bacteria in water samples amended with 10 and 50 mg ml−1 of aldrin was not a"ected during 28 days of incubation. The mean (MPN) ammonium oxidizers per ml for amended (50 mg ml−1 ) and unamended water samples were 299 ± 32 and 300 ± 29 respectively. The variation in the count of nitrite oxidizer (MPN) per ml for amended 50 mg ml−1 and unamended water samples were 451 ± 40 and 464 ± 275 respectively. During 28 days of incubation the number of ammonium oxidizers and nitrite oxidizer bacteria in amended water samples with aldrin at concentrations of 10 and 50 mg ml−1 was similar to unamended water sample (Table 3). The results obtained in our study on nitrifying bacteria (phase I and II) was not a"ected as a consequence of the incubation time (1, 7, 14 and 28 days), showing that these microorganisms (usually the most sensitive to pollutants) can tolerate 10 and 50 mg ml−1 of aldrin in aquatic environment. Our results show that aldrin, applied at agricultural concentration, positively a"ected number of denitrifying bacteria, an e"ect that is strongly correlated with dose rate of insecticide (Table 2). However, the presence of aldrin a"ected the potential denitri8cation activity in the water samples when the insecticide was applied at the rate of 50 mg ml−1 (Fig. 1). Since denitri8cation activity could be considered as a growth-linked process, it was expected that as the insecticide positively a"ect growth of denitrifying bacteria it would positively a"ect denitri8cation activity. However, di"erent result have been obtained with denitrifying bacteria and aerobic diazotrophs. Thus, it was reported (G'omez et al., 1998) that these biological activities in the presence of bromopropylate are processes independent of the microbial growth. In this context, microbial respiration and ATP generation are substantially di"erent processes in denitrifying bacteria and consequently, the e"ect of the
200 N2O production (nmol/ml)
86
• •
150 • •
100
* •
50 0
•• •
0
•
*
•*
•
5
10 Incubation time (d)
15
20
Fig. 1. N2 O production (nmol=ml) in water samples in the presence of 0 ( ), 10 (·-·) and 50 mg ml−1 (· · · ·) of aldrin. Bars represent SE of 8ve samples. Means marked with ∗ are signi8cant (p ¡ 0:05) versus control.
aldrin on certain microbial activities such as denitri8cation activity could be very di"erent. Among various microbial activities in water, denitri8cation contributes signi8cantly to the overall nitrogen economy of aquatic environments. Denitri8cation activity is often used for testing the e"ects of the pesticides on the nitrogen cycle because of their sensitivity to environmental toxicants (Svensson and Leonardson, 1992; Yeomans and Breuner, 1985). In this context numerous applications of aldrin may a"ect the nitrogen economy of an aquatic environment and thus the delicate balance established between nitrogen 8xation, nitri8cation and denitri8cation. Our data show that aldrin a"ected denitri8cation and number of denitrifying bacteria in the water samples used when the insecticide was added at agricultural doses. In conclusion, application of aldrin may a"ect the composition of the microbial communities in aquatic environments and thus disturb the ecology and biological activity of these microorganisms. In this context, rotational use and rational use of pesticides could be suggested as an important way to preventing environmental hazard. However, the degree to which the e"ects obtained in our studies can be extrapolated, for example, to natural conditions deserves more attention, because other factor (water types, climates, etc.) also in6uence the e"ects of these agrochemicals. Acknowledgements This study has been carried out within the framework of the research project AMB94-611 supported by the CICYT (Comisi'on Interministerial de Ciencia y Tecnologia, Spain). References Anon., 1985. Standard Methods For The Examination of Water and Wastewater, 16th Edition, APHA, AWWA, WPLF, Washington. Barbera, C., 1989. Pesticidas Agricolas. Omega, Barcelona, pp. 259 –265. Benavente, J., Carrasco, F., Almecija, C., Rodriguez-Jim'enez, P.D., Cruz-San Julian, J., 1993. La zona de transici'on agua dulce-salmuera
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87 bajo el borde norte de la laguna salada de Fuente Piedra. Geogaceta 14, 6–8. Brockway, D.L., Smith, P.D., Staneil, F.J., 1984. Fate and e"ects of atracine in small aquatic microcosms. Bulletin of Environmental Contamination and Toxicology 32, 345–353. Cook, A.M., 1987. Biodegradation of S-atracine xenobiotics. FEMS Microbiological Reviews 46, 93–116. Foulkes, D.M., 1991. Agrochemicals in the environments. In: Richardson, M.L. (Ed.), Chemistry, Agriculture and the Environments. Bookcraft Ltd., UK, pp. 3–10. G'omez, F., Salmer'on, V., Rodelas, B., Martinez-Toledo, M.V., Gonz'alez-L'opez, J., 1998. Response of Azospirillum brasilense to the pesticides bromopropylate and methidation on chemically de8ned media and dialysed-soil media. Ecotoxicology 7, 43–47. Lal, R., 1982. Accumulation, metabolism and e"ects of organophosphorous insecticides on microorganisms. Advances in Applied Microbiology 28, 149–200. Mirgain, I., Green, G.A., Monteil, H., 1993. Degradation of atracine in laboratory microcosm: isolation and identi8cation of the biodegrading bacteria. Environmental and Toxicological Chemistry 12, 1627–1634. Ramanand, K., Nagarajan, A., Su6ita, J.M., 1993. Reductive dechlorination of the nitrogen heterocyclic herbicide picloram. Applied Environmental Microbiology 59, 2252–2256.
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Rodina, A.G., 1972. Methods in Aquatics Microbiology. University Park Press, Baltimore. Salmer'on, V., Martinez-Toledo, M.V., Gonz'alez-L'opez, J., 1991. E"ects of alachlor and metolachlor on the biological activity of Azospirillum brasilense grown in chemically de8ned and dialysed-soil media. Environmental and Toxicological Chemistry 10, 493–499. Saxena, A.R., Zhang, R., Bollag, J.M., 1987. Microorganisms capable of metabolizing the herbicide metalachlor. Applied Environmental Microbiology 53, 390–396. Svensson, J., Leonardson, L., 1992. E"ects of agricultural pesticides on denitri8cation in lake sediments. In: Anderson, J.E.P., Arnolld, D.J., Lewis, L. Torstensson, L. (Eds.), Proceedings of the International Symposium of Environmental Aspects of Pesticide Microbiology. Swedish University of Agricultural Sciences, Uppsala, Sweden. Yeomans, J.C., Breuner, J.M., 1985. Denitri8cation in soil: e"ects of herbicides. Soil Biology & Biochemistry 17, 447–452. Yoshinari, T., Knowles, K., 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochemical and Biophysical Research Communications 69, 705–710.
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Studies on the e"ects of the insecticide aldrin on aquatic microbial populations L. L'opez, C. Pozo, M.A. G'omez, C. Calvo, J. Gonz'alez L'opez ∗ Environmental Microbiology Group, Department of Microbiology, Faculty of Pharmacy and Institute of Water Research, University of Granada E-18071 Granada, Spain Received 2 April 2000; received in revised form 1 May 2001; accepted 1 September 2001
Abstract The e"ects of aldrin at rates 10 and 50 mg ml−1 on microbial function were studied under aerobic conditions. Parameters monitored included total culturable population of heterotrophic bacteria, number of culturable phosphate-solubilizing micro6ora, number of bacteria which decompose protein with liberation of H2 S, nitrifying bacteria, denitrifying bacteria and denitri8cation activity. Culturable heterotrophic bacteria, phosphate-solubilizing bacteria, bacteria which decompose proteins with liberation of H2 S and denitrifying bacteria were signi8cantly increased at dose rates of 10 and 50 mg ml−1 of aldrin. However, the presence of aldrin decreased denitri8cation activity at concentration of 50 mg ml−1 . Nitrifying population were not a"ected as a consequence of the addition of aldrin, showing that these microorganisms can tolerate the concentrations of aldrin tested. ? 2002 Elsevier Science Ltd. All rights reserved. Keywords: Insecticides; Aldrin; Aquatic micro6ora; Xenobiotics
1. Introduction Modern agriculture and industry depend on a wide variety of synthetic chemicals, including insecticides, fungicides, herbicides and others pesticides. Continual wide-spread use and release of such synthetics has become an everyday occurrence, resulting in environmental pollution. In this context, the in6uence of pesticides on the microbial activity of aquatic microorganisms has been studied by some investigators both in pure culture and in a mixed populations (Brockway et al., 1984; Mirgain et al., 1993; Ramanand et al., 1993). However, it is not possible to reach a general conclusion regarding the e"ect of these substances on aquatic microbial activity because a number of factors (e.g., climate, water type, chemical structure and the composition of the microbial community) in6uence the e"ects of those xenobiotics. Aldrin (1,2,3,4,10,10a-hexachloro-1,4,4a,5,8,8a hexahydro-1,4,endo-exo 5,8-dimethanonaphthalene) is a commonly used insecticide in intensive agriculture, applied at concentrations of 2.0 –10:0 kg ha−1 for pest control such as ∗
Corresponding author. Tel.: +34-58-243-870; fax: +34-58-200-962.
leatherjacket. However, because of their persistence in the environment, susceptibility to biomagni8cation, and toxicity to higher animals, these organochlorine insecticides have been banned in technologically advanced countries (Lal, 1982). Organochlorine insecticides can enter into aquatic environments by direct or indirect routes. The two primary sources for the direct entry of insecticides into aquatic environments are the huge amounts added to water for effective pest control and the enormous quantities discharged into water along with industrial and domestic sewage waters (Foulkes, 1991). These agrochemicals indirectly enter into aquatic environments by drift from aerial or ground application and through water soil erosion, which include run-o", wash-o" and leaching from treated lands. In this sense, it is important to evaluate how the aquatic micro6ora could be a"ected by some chemical substances such as organochlorine insecticides applied to soil and water for pest control. The purpose of the present study was to examine how the insecticide aldrin at normal 8eld concentrations a"ects the aquatic micro6ora of a protected wetland located in the southeast of Spain. To our knowledge, this wetland has not previously been microbially characterized. Microbial parameters examined in this study were selected because of their
0964-8305/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 4 - 8 3 0 5 ( 0 1 ) 0 0 1 2 8 - 7
84
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87
relation to overall microbial activity and the microbially mediated cycling of carbon, phosphorous, sulphur and nitrogen. 2. Materials and methods 2.1. Water samples Samples were collected from a wetland located on the Atlantic Mediterranean hydrographic divide (southeast Spain) with no previous aldrin applications (Benavente et al., 1993). Water samples were collected from the top 15 cm according to Rodina (1972). Water samples were collected in sterile bottles (1 l), shipped to the laboratory and re◦ frigerated (4 C). Chemical and physical characteristics of the water samples were measured by techniques describe previously (Anon, 1985) and are presented in Table 1. 2.2. Treatment and water incubation Laboratory studies were performed on 8ve replicates. Five hundred milliliter samples were placed in a 1 l Erlenmeyer 6ask and amended with 5 ml of appropriate concentrations of sterile aldrin in acetone to give concentrations of 10 and 50 mg ml−1 (equivalent to 2 and 10 Kg ha−1 ) and then in◦ cubated at 20 C with continuous shaking (100 rpm:). After 1, 7, 14 and 28 days, microbial populations and biological activities were determined. Control samples received equal amounts of acetone for comparison. In the present investigation, technically pure grades (98%) of the insecticide (Eurolab, Granada, Spain) were used to determine their e"ect on the aquatic micro6ora. 2.3. Determination of microbial populations Total culturable heterotrophic bacteria were counted by dilution plate technique using Trypticase Soy Agar (TSA, Difco). The inoculated agar plates (three replicates) were ◦ incubated at 28 C for 3 days for mesophilic bacteria or at Table 1 Chemical and physical characteristics of water samples∗ ◦
Temperature ( C) pH ◦ Conductivity (mmhos=cm at 25 C) Dissolved oxygen (mg=l) Cl− (mg=l) CO3 H− (mg=l) CO2− (mg=l) 3 SO2− (mg=l) 4 SiO2 (mg=l) Ca2+ (mg=l) Mg2+ (mg=l) Na+ (mg=l) K + (mg=l) ∗ Data
◦
4 C for 7 days for psychrophilic bacteria before colonies were counted. Culturable phosphate-solubilizing bacteria were estimated by a standard dilution series in sterile saline solution, followed by plating on insoluble phosphate agar medium. The composition of insoluble phosphate medium was: glucose 5:0 g, K2 HPO4 0:8 g, NaCl 0:2 g, MgSO4 ·7H2 O 0:2 g, CaSO4 ·2H2 O 0:05 g, MaMoO4 ·2H2 O 0:01 g, FeSO4 0:003 g, Ca3 (PO4 )2 8:0 g, agar 15:0 g and distilled water 1 l. The pH was adjusted to 7.0 with sterile 0.1M NaOH. ◦ Inoculated plates (three replicates) were incubated at 28 C for 5 days. The most probable number (MPN) technique according to Rodina (1972) was used to count bacteria which decompose proteins with liberation of H2 S. The formations of H2 S by bacteria was detected in test tubes containing 10 ml of beef-peptone broth with suspended indicator paper soaked in a saturated solution of lead acetate. Inoculated test tubes ◦ (three replicates) were incubated at 22–23 C for 6 days. The dilution tube technique (MPN) was employed for enumeration of nitrifying bacteria. A separate analysis was performed for nitrifying phase I bacteria, which oxidize ammonia salts to nitrite, and for nitrifying phase II bacteria, which oxidize nitrite to nitrate. An ammonium sulphate medium was used for nitrifying phase I bacteria and a sodium nitrite medium for nitrifying phase II bacteria, according to Rodina (1972). Inoculated 6asks (three repli◦ cates for water samples) were incubated at 28 C and the presence of nitrite-N and nitrate-N in these 6asks was determined once a week for up to 4 weeks as described by Rodina (1972). Numbers of denitrifying bacteria were determined by a most probable number (MPN) technique according to Rodina (1972). The liquid enrichment medium contained per liter of distilled water: sucrose, 30:0 g, NaNO3 2:0 g, K2 HPO4 1:0 g, MgSO4 ·7H2 O 0:5 g, KCl 0:5 g, FeSO4 ·7H2 O trace, pH 7.0. Inoculated test tubes (three replicates) were ◦ incubated at 28 C for 7 days and denitri8cation was detected by the liberation of gas in the test tubes served as an indicator of denitrifying activity. 2.4. Determination of denitri8cation activity
18:4 ± 2:4 7:4 ± 0:2 4:5 ± 0:4 9:0 ± 0:5 430 ± 12:3 95:0 ± 7:6 5:5 ± 0:3 2660:0 ± 91:3 5:2 ± 0:5 52:5 ± 4:5 210:0 ± 9:1 261:0 ± 8:3 10:0 ± 1:1
are means ± SE of three separate determinations.
N2 O production, as indicators of the potential for denitri8cation, was determined by the acetylene inhibition method (Yoshinari and Knowles, 1976). Four milliliter samples of water amended or unamended with aldrin were placed in 10 ml tubes containing 1 ml of culture medium for denitri8cation and sealed with rubber stoppers. The composition of culture medium for denitri8cation was: sucrose 30 g, NaNO3 10:0 g, K2 HPO4 5:0 g, MgSO4 ·7H2 O 2:5 g, KCl 2:5 g, FeSO4 ·7H2 O 0:03 g and distilled water to 1 l. The pH was adjusted to 7.0 with 0.1M NaOH. After 100% of the atmosphere had been replaced by He, the tubes were inoc◦ ulated at 28 C and 0:25 ml gas were assayed for N2 O after
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87
1, 4, 7, 9 and 15 days by injection into a Varian Star 3400 gas chromatograph equipped with a thermal conductivity detector. 2.5. Statistical analysis Student’s “t” test was used to measure the di"erence between control without insecticide and water samples treated with 10 and 50 mg ml−1 aldrin. An ANOVA test was used to assess homogeneity of variance. Where the variance was heterogeneous, the data were suitably transformed before the analysis. A signi8cance level of 5% (p ¡ 0:05) was selected. 3. Results and discussion Microbial transformation is recognized as a critical factor a"ecting the fate and behaviour of pesticides in soil and aquatic ecosystems (Cook, 1987). Aquatic micro6ora comprises a diversity of species and is a dynamic population able to degrade a wide variety of chemically complex compounds. In this context, the biodegradation of complex halogenated compounds such as aldrin is considered to be a cometabolic process (Salmer'on et al., 1991; Saxena et al., 1987). There have been constant pressures, on authorities controlling the registration and approval of agricultural chemical, to include measurements of side e"ects on the soil and water micro6ora in their requirements. The Environmental Protection Agency in the United States, for
85
example, in guidelines for registering pesticides (Barbera, 1989) required data from “Studies of e"ects on microbial function...”. Nitri8cation is one of the functions speci8ed. To encourage uniformity of presented data and aid interpretation and comparison, the guidelines recommended the use of standard methods, usually a single sample temperature of ◦ 20 –30 C. The plate-count data indicated that culturable heterotrophic bacteria (mesophilic and psychrophilic micro6ora) in freshwater amended with aldrin were signi8cantly stimulated at doses of 10 and 50 mg ml−1 . This positive e"ect was evident in water samples after 1, 7, 14 and 28 days incubation with the insecticide (Table 2). In the absence of data on the mineralization or biotransformation of aldrin in water samples, the proliferation of bacterial 6ora in treated waters may be associated with the transformation of aldrin by the bacterial in water. However, it is likely that the e"ect obtained in our studies can be in6uenced by the species of bacteria present in the water. The presence of aldrin at concentrations of 10 and 50 mg ml−1 in the water positively a"ected phosphatesolubilizing bacteria and H2 S liberating bacterial population. The plate-count data indicated that those microorganisms were signi8cantly increased under our experimental conditions (Table 2), suggesting that phosphate-solubilizing and H2 S liberating bacterial, can tolerate high amounts of the insecticide aldrin without inhibition to their biological activities. The degree of stimulation was related to the concentration of aldrin. The presence of aldrin in the water positively a"ects the populations of bacteria which decompose protein with liberation of H2 S (Table 2).
Table 2 Number of culturable heterotrophic bacteria (mesophilic and psychrophilic), culturable phosphate-solubilizing bacteria, H2 S liberating and denitrifying bacteria in water samples in the absence and presence of aldrin∗
Aldrin (mg=ml)
Incubation time (d)
Mesophilic bacteria (104 =ml)
Psychrophilic bacteria (103 =ml)
Phosphate-solubilizing bacteria (103 =ml)
H2 S liberating bacteria (103 =ml)
Denitrifying bacteria (ml)
0
0 1 7 14 28
6:1 ± 0:15 6:1 ± 0:1 4:2 ± 0:4 0:6 ± 0:1 0:1 ± 0:02
9:1 ± 0:4 31:3 ± 5:0 6:3 ± 0:5 0:1 ± 0:01 0:1 ± 0:01
1:7 ± 0:17 7:5 ± 0:4 0:1 ± 0:01 0:2 ± 0:02 ¡ 0:1
35:0 ± 3:0 22:0 ± 3:0 17:0 ± 2:0 3:4 ± 0:3 0:7 ± 0:1
220 ± 1:0 170 ± 2:0 64 ± 2:0 33 ± 0:6 27 ± 0:4
10
1 7 14 28
47.7 ±1:4a 20:5 ± 0:1a 5:7 ± 0:2a 1:0 ± 0:04a
211:7 ± 10:0a 8:2 ± 0:4a 0:9 ± 0:7a 1:2 ± 0:4a
75:2 ± 4:5a 0:8 ± 0:04a 1:0 ± 0:1a 0:2 ± 0:02a
28:0 ± 3:0 35:1 ± 2:0a 17:0 ± 4:0a 1:2 ± 0:3a
280 ± 3:0a 170 ± 2:0 110 ± 0:2a 90 ± 1:0a
50
1 7 14 28
58:8 ± 1:0a 21:5 ± 0:4a 5:3 ± 0:5a 5:8 ± 0:02a; b
178:7 ± 18:0a 40:0 ± 2:0a; b 1:0 ± 0:1a 0:7 ± 0:04a
78:0 ± 3:6a 3:1 ± 2:7a; b 4:1 ± 1:0a; b 0:6 ± 0:05a; b
∗ Data
are means ± SE of 8ve samples. signi8cant di"erence (p ¡ 0:05) from control. b Represents signi8cant di"erence (p ¡ 0:05) between 10 and 50 g aldrin. a Represents
35:0 ± 5:0a 160:0 ± 6:0a; b 28:3 ± 2:0a; b 14:0 ± 1:0a; b
900 ± 7:0a; b 900 ± 6:0a; b 355 ± 4:0a; b 280 ± 6:0a; b
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87
Table 3 Number of nitrifying (phase I and II) bacterial per milliliter in water samples in the absence and presence of aldrin∗
Aldrin (g=ml) 0
10
50
∗ Data
Incubation time (d)
Phase I
Phase II
0 1 7 14 28
263 ± 35 269 ± 36 281 ± 24 266 ± 38 299 ± 32
416 ± 36 471 ± 42 466 ± 21 416 ± 16 451 ± 40
1 7 14 28
247 ± 39 266 ± 51 281 ± 37 269 ± 30
436 ± 23 436 ± 50 439 ± 34 464 ± 44
1 7 14 28
273 ± 32 284 ± 41 275 ± 32 300 ± 29
471 ± 26 473 ± 33 439 ± 34 464 ± 27
are means ± SE of 8ve samples.
The number of ammonia-oxidizing bacteria 50 mg ml−1 and nitrite-oxidizing bacteria in water samples amended with 10 and 50 mg ml−1 of aldrin was not a"ected during 28 days of incubation. The mean (MPN) ammonium oxidizers per ml for amended (50 mg ml−1 ) and unamended water samples were 299 ± 32 and 300 ± 29 respectively. The variation in the count of nitrite oxidizer (MPN) per ml for amended 50 mg ml−1 and unamended water samples were 451 ± 40 and 464 ± 275 respectively. During 28 days of incubation the number of ammonium oxidizers and nitrite oxidizer bacteria in amended water samples with aldrin at concentrations of 10 and 50 mg ml−1 was similar to unamended water sample (Table 3). The results obtained in our study on nitrifying bacteria (phase I and II) was not a"ected as a consequence of the incubation time (1, 7, 14 and 28 days), showing that these microorganisms (usually the most sensitive to pollutants) can tolerate 10 and 50 mg ml−1 of aldrin in aquatic environment. Our results show that aldrin, applied at agricultural concentration, positively a"ected number of denitrifying bacteria, an e"ect that is strongly correlated with dose rate of insecticide (Table 2). However, the presence of aldrin a"ected the potential denitri8cation activity in the water samples when the insecticide was applied at the rate of 50 mg ml−1 (Fig. 1). Since denitri8cation activity could be considered as a growth-linked process, it was expected that as the insecticide positively a"ect growth of denitrifying bacteria it would positively a"ect denitri8cation activity. However, di"erent result have been obtained with denitrifying bacteria and aerobic diazotrophs. Thus, it was reported (G'omez et al., 1998) that these biological activities in the presence of bromopropylate are processes independent of the microbial growth. In this context, microbial respiration and ATP generation are substantially di"erent processes in denitrifying bacteria and consequently, the e"ect of the
200 N2O production (nmol/ml)
86
• •
150 • •
100
* •
50 0
•• •
0
•
*
•*
•
5
10 Incubation time (d)
15
20
Fig. 1. N2 O production (nmol=ml) in water samples in the presence of 0 ( ), 10 (·-·) and 50 mg ml−1 (· · · ·) of aldrin. Bars represent SE of 8ve samples. Means marked with ∗ are signi8cant (p ¡ 0:05) versus control.
aldrin on certain microbial activities such as denitri8cation activity could be very di"erent. Among various microbial activities in water, denitri8cation contributes signi8cantly to the overall nitrogen economy of aquatic environments. Denitri8cation activity is often used for testing the e"ects of the pesticides on the nitrogen cycle because of their sensitivity to environmental toxicants (Svensson and Leonardson, 1992; Yeomans and Breuner, 1985). In this context numerous applications of aldrin may a"ect the nitrogen economy of an aquatic environment and thus the delicate balance established between nitrogen 8xation, nitri8cation and denitri8cation. Our data show that aldrin a"ected denitri8cation and number of denitrifying bacteria in the water samples used when the insecticide was added at agricultural doses. In conclusion, application of aldrin may a"ect the composition of the microbial communities in aquatic environments and thus disturb the ecology and biological activity of these microorganisms. In this context, rotational use and rational use of pesticides could be suggested as an important way to preventing environmental hazard. However, the degree to which the e"ects obtained in our studies can be extrapolated, for example, to natural conditions deserves more attention, because other factor (water types, climates, etc.) also in6uence the e"ects of these agrochemicals. Acknowledgements This study has been carried out within the framework of the research project AMB94-611 supported by the CICYT (Comisi'on Interministerial de Ciencia y Tecnologia, Spain). References Anon., 1985. Standard Methods For The Examination of Water and Wastewater, 16th Edition, APHA, AWWA, WPLF, Washington. Barbera, C., 1989. Pesticidas Agricolas. Omega, Barcelona, pp. 259 –265. Benavente, J., Carrasco, F., Almecija, C., Rodriguez-Jim'enez, P.D., Cruz-San Julian, J., 1993. La zona de transici'on agua dulce-salmuera
L. L+opez et al. / International Biodeterioration & Biodegradation 50 (2002) 83 – 87 bajo el borde norte de la laguna salada de Fuente Piedra. Geogaceta 14, 6–8. Brockway, D.L., Smith, P.D., Staneil, F.J., 1984. Fate and e"ects of atracine in small aquatic microcosms. Bulletin of Environmental Contamination and Toxicology 32, 345–353. Cook, A.M., 1987. Biodegradation of S-atracine xenobiotics. FEMS Microbiological Reviews 46, 93–116. Foulkes, D.M., 1991. Agrochemicals in the environments. In: Richardson, M.L. (Ed.), Chemistry, Agriculture and the Environments. Bookcraft Ltd., UK, pp. 3–10. G'omez, F., Salmer'on, V., Rodelas, B., Martinez-Toledo, M.V., Gonz'alez-L'opez, J., 1998. Response of Azospirillum brasilense to the pesticides bromopropylate and methidation on chemically de8ned media and dialysed-soil media. Ecotoxicology 7, 43–47. Lal, R., 1982. Accumulation, metabolism and e"ects of organophosphorous insecticides on microorganisms. Advances in Applied Microbiology 28, 149–200. Mirgain, I., Green, G.A., Monteil, H., 1993. Degradation of atracine in laboratory microcosm: isolation and identi8cation of the biodegrading bacteria. Environmental and Toxicological Chemistry 12, 1627–1634. Ramanand, K., Nagarajan, A., Su6ita, J.M., 1993. Reductive dechlorination of the nitrogen heterocyclic herbicide picloram. Applied Environmental Microbiology 59, 2252–2256.
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Rodina, A.G., 1972. Methods in Aquatics Microbiology. University Park Press, Baltimore. Salmer'on, V., Martinez-Toledo, M.V., Gonz'alez-L'opez, J., 1991. E"ects of alachlor and metolachlor on the biological activity of Azospirillum brasilense grown in chemically de8ned and dialysed-soil media. Environmental and Toxicological Chemistry 10, 493–499. Saxena, A.R., Zhang, R., Bollag, J.M., 1987. Microorganisms capable of metabolizing the herbicide metalachlor. Applied Environmental Microbiology 53, 390–396. Svensson, J., Leonardson, L., 1992. E"ects of agricultural pesticides on denitri8cation in lake sediments. In: Anderson, J.E.P., Arnolld, D.J., Lewis, L. Torstensson, L. (Eds.), Proceedings of the International Symposium of Environmental Aspects of Pesticide Microbiology. Swedish University of Agricultural Sciences, Uppsala, Sweden. Yeomans, J.C., Breuner, J.M., 1985. Denitri8cation in soil: e"ects of herbicides. Soil Biology & Biochemistry 17, 447–452. Yoshinari, T., Knowles, K., 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochemical and Biophysical Research Communications 69, 705–710.