JCBPS; Section D; May 2015 – July 2015, Vol. 5, No. 3; 3182-3192. E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences Available online atwww.jcbsc.org

Section D:Environmental Sciences

CODEN (USA): JCBPAT Research Article

Density and Distribution of Fejervarya limnocharis (Anura: Ranidae) in Spentwash sprayed agro- ecosystems in Karnataka

Mahesh Mandya

Department of Environmental Science, Kuvempu University, Shivamogga, Karnataka- 577451.

Received: 02 June 2015; Revised:23 July 2015; Accepted:29 July 2015

Abstract: The density of Fejervarya limnochariswas studied in12 locations (including 2 control sites) of agro-ecosystems where the spent wash was sprayed and the influence of spent wash was assessed. Soil samples of each study site were collected and analysed for twenty variables. Total five species of anurans were identified in the study sites: F. limnocharis, Z. rufescence, F.caperata, H. tigerinus and E. cyanophlyctis. We observed considerable difference in the pattern of amphibian population structure at different locations in spent wash sprayed areas. Meanwhile, as a model species we concentrated on the density and abundance of F. limnocharis in both contaminated and uncontaminated sites, and the result showed the significant decrease in the population of F. limnocharis in contaminated sites compared to uncontaminated sites. The study is the first to find evidence for an association between effluent contamination and anuran distribution in southern Karnataka, adding to a small but growing body of evidence that distillery effluent pollution has contributed to amphibian population decline. Key words: Distillery effluent (spent wash), Fejervarya limnocharis, Density, Diversity and Distribution.

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Diversity … Mahesh Mandya.

INTRODUCTION Amphibian population are dwindling at shocking rates worldwide1-2. The reason for the decline and extinction of amphibian population are diverse and complex, which may involve multiple stressors3. Many researchers discourse that most past and existing amphibian decline are due to habitat destruction or modification4-13. Habitat destruction, emergent diseases, introduction of exotic species, and the pollution of both terrestrial and aquatic habitats have all been described as important causes for such decline2,14. The habitat quality depletion due to chemical contaminations, land uses managerial pattern are the important causes that forced status of many frog populations into stake. Anthropogenic activities have also altered the environment by damaging the habitat, introduction of predators, direct application of pesticides, herbicides and industrial chemicals and are generally accepted as immediate causes for some amphibian decline15. In the present investigation attempt has been made to assess the impact of distillery spentwash on habitat destruction, diversity and distribution pattern of amphibians in agro-ecosystems in Karnataka. A variety of pollutants occur in natural habitats including agrochemical runoff, fertilizers, pesticides, heavy metals, and effluents. The distilleries are recognized as the polluting units generating high volume of foul smelling and colored waste water known as spentwash16. These pollutants are increasingly introduced into the environment by direct application, runoff from crop field, urban and industrial sewage and by atmospheric deposition17-19. Now a days it has become a common practice in southern Karnataka to use a distillery spent wash to irrigate rice paddy, sugarcane and other croplands. However, it is also being used to enrich soil fertility as it contains many essential elements required for plant growth. The spent wash is also known to contain high concentrations of heavy metals, sulphates and phosphates20. Continued and indiscriminate disposal of spent wash in agro-ecosystem is known to induce heavy metal toxicity in biota21. In the southern part of India the agro-ecosystem of rice paddy and sugar cane provide good water resources for the occurrence of common frog Fejervarya limnocharis (Anura: Ranidae). The preliminary survey on the diversity and distribution of frogs in these areas have revealed considerable differences in diversity of frogs and particularly the F. limnocharis between those croplands treated with spent wash, and those croplands where such supplementary products are not used. In most of cases, toxic substances present in the effluent cause negative impact on the biota living in the place of its discharge. As these effluents are regularly applied in the agro-ecosystem and they are rich in organic content it is believed that the frog population has responded to these contaminations. This needs to be handled carefully to avoid any adverse effect on amphibian population. Many previous studies have elaborated the influence of these toxic chemicals present in the distillery effluent on amphibian population22. As the spent wash is rich in many substances such as sulphates, phosphates, magnesium chloride, cadmium, nickel, lead, zinc, copper, etc., it may cause serious damage to habitats where amphibians and other organisms inhabit. This could ultimately influence the diversity and distribution of amphibian population. Fejervarya limnocharis is a common inhabitant in wet soil condition of the rice paddy field and it gets directly exposed to spent wash. Hence, in the present investigation, we tried to examine the possible influence of distillery effluent (spent wash) on the density & distribution of the frog F. limnocharis which generally lives and breeds in rice paddy fields.

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Diversity … Mahesh Mandya.

MATERIAL AND METHODS Study Site: Study site is situated around NSL Sugars Ltd. Koppa, Mandya District. It is located at 25km North–East (Location: 77° 58' 41. 03'' E and 12° 42' 00. 51'' N; alt.: 641-704 m). Mandya District is one of the most agriculturally prosperous district in Karnataka, due to irrigation from K.R. Sagar reservoir leading to cropping pattern, composition of crops, better yield level, resulting in better economic conditions of the people. The total study area covers around 50 Km with the distillery spent wash being used (contaminated) and the control (uncontaminated) crop lands. Sampling sites were selected from the range of terrestrial/semi-aquatic habitats comprising twelve sites (Figure 1). Of these, Besagarahalli (Site 1) and Keregodu (Site 2) were the uncontaminated sites. Bidarakote (Site 3), Koppa (Site 4), Kesthur.a (Site 5), Huthagere.a (Site 6), Maraliga (Site 7), Dhundanahalli.a (Site 8), Athagur (Site 9), Huthagere.b (Site 10), Dhundanahalli.b (Site 11) and Kestur.b (Site 12) were the contaminated sites. The type of crop being cultivated in these sites is shown in Table 2. We grouped these twelve study sites into six based on on the type of cropsbeing cultivated.

Figure 1: Map showing the study sites (numbered S1 to S12) Spent wash and Soil Sample Analysis: The spent wash and soil assay was carried out using AAS analyzer (Atomic absorption spectroscopy) according to the standard method for the examination of water and waste water23.

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Diversity … Mahesh Mandya.

Sampling Methods: Sampling of amphibians was carried out at intervals of 30 days in three seasons for two successive years (2012-2014) to record the diversity and distribution of amphibian population in contaminated and uncontaminated sites. Sampling of amphibians was done using torchlight in the evening and during the night. Three man-hours was spent in each of the sampling sites (1Acre) and an equal time was spent in each sampling throughout the study. The diversity was calculated using the Simpson diversity index and Shannon-wiener diversity index for reference and contaminated sites. Density and mean abundance of frog population were also investigated. Similarly, jaccard similarity index was used to analyse the distribution of frogs. Shannon- Wiener diversity index, H = -∑ Pi ln Pi Simpson's diversity index, D = 1/∑ Pi2

Statistical Analysis: All the data obtained during the study were processed using Excel sheets. The data collected from sites 1 & 2 were taken as control, and data from all the other sites (3 to 12) represent sites with direct influence of spent wash. We calculated species diversity and distribution between the sites. Two way ANOVA was used to test the significance of differences in density between sites. We used SPSS 20 for all statistical analysis. RESULT The distillery effluent used in the study sites was light brown colored odorous waste released from distillery industry. The result of analysis of various physico-chemical characteristics carried out on the soil samples are summarized in Table 1. Out of 20 parameters analyzed, pH shows the insignificant difference between the soil samples of all sites. Temperature was found to be gradually increasing in the contaminated sites compared to uncontaminated (control) sites (S1 & S2). Concentrations of other parameters like sulphates, phosphates, sodium, potassium, magnesium, calcium chloride and heavy metals like iron, zinc, nickel, chromium & lead were found to be high in contaminated sites compared to uncontaminated (control) sites and the values were found to be significantly different at p < 0.01. Considering the availability of water, the parameters showed a high degree of concentration. The barren and Jowar land showed greater concentrations of all parameters, and it is statistically significant (F 17,2= 1370.300, p < 0.01). Species density: The most common species observed were F. limnocharis, Z. rufescence, F. caperata, H. tigerinus and E. cyanophlyctis in uncontaminated (control) and contaminated sites. While H tigerinus and E cyanophlyctis were absent from jowar and barren lands respectively, all other species were present in all the crop fields. We recorded a total of 529 frogs/ acre in control fields, 402 frogs/ acre in paddy fields, 365 frogs/ acre in ragi fields, 199 frogs/ acre in sugarcane fields, 129 frogs/ acre in jowar fields and 78 frogs/ acre in barren fields (Figure 2).

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Diversity … Mahesh Mandya.

Table 1: Physico-chemical parameters of soil exposed to distillery effluent

Sites (Crop field) Sl. Parameters No. Control Paddy Ragi Sugarcane Jowar Barren 1 pH 7.3 ± 0.09 7.9 ± 0.15 7.9 ± 0.08 8.2 ± 0.05 8.0 ± 0.09 8.0 ± 0.05 2 Temperature (º 25.2 ± 0.16 26.6±0.11 26.9 ± 0.12 27.5 ± 0.09 28.1 ± 0.25 29.2 ± 0.15 C) 3 Organic Carbon 2.4 ± 0.28 6.1 ± 1.29 6.1 ± 0.51 4.7 ± 0.06 6.3 ± 2.43 3.7 ± 0.02 (mgC/ Kg Soil ) 4 Sulphate 47.1 ± 0.26 124.3 ± 5.58 139.8 ± 6.38 128.1 ± 4.06 139.1 ± 3.78 175.1±13.87 (mg/Kg Soil) 5 Iron 248.6±11.37 281.8±49.39 598.5 ± 99.58 471.2±57.54 647.1±23.33 514.9±91.31 (mg/Kg Soil) 6 Zinc 3.4 ± 0.90 3.4 ± 1.01 3.1 ± 0.34 2.8 ± 0.07 3.7 ± 0.50 4.2 ± 1.00 (mg/Kg Soil) 7 Nickel 1.9 ± 0.62 8.6 ± 0.15 8.6 ± 1.44 10.1 ± 1.01 11.0 ± 1.53 11.1 ± 0.58 (mg/Kg Soil) 8 Chromium 0.7 ± 0.5 2.0 ± 0.17 3.2 ± 0.27 2.8 ± 0.41 3.4 ± 0.64 3.1 ± 0.47 (mg/Kg Soil) 9 Manganese 10.2 ± 0.44 11.1 ± 0.63 11.6 ± 4.31 9.3 ± 0.45 7.9 ± 1.02 9.2 ± 0.52 (mg/Kg Soil) 10 Aluminium 3.2 ± 0.50 8.7 ± 0.88 9.3 ± 1.05 10.7 ± 0.87 9.1 ± 0.26 11.3 ± 0.47 (mg/Kg Soil) 11 Copper 2.6 ± 0.37 2.3 ± 0.05 3.2 ± 0.98 2.2 ± 0.09 3.4 ± 0.82 2.7 ± 0.57 (mg/Kg Soil) 12 Lead 0.8 ± 0.04 1.3 ± 0.20 1.9 ± 0.42 2.2 ± 0.37 1.6 ± 0.10 1.8 ± 0.08 (mg/Kg Soil) 13 Cadmium 0.02 ± 0.00 0.1 ± 0.00 0.1 ± 0.01 0.1 ± 0.00 0.1 ± 0.00 0.1 ± 0.00 (mg/Kg Soil) 14 Chloride 90.7 ± 4.16 165.9 ± 5.83 182.0 ± 6.24 169.8±14.62 1.55 ± 7.58 171.3 ± 1.45 (mg/Kg Soil) 15 Sodium 26.8 ± 1.97 77.5 ± 0.87 91.8 ± 1.16 91.0 ± 2.41 77.4 ± 3.15 78.7 ± 6.38 (mg/Kg Soil) 16 Potassium 57.5 ± 4.56 148.4±14.55 188.5 ± 16.02 199.3±14.28 207.1±13.25 220.4 ± 3.08 (mg/Kg Soil) 17 Calcium 7 14.9 ± 3.06 77. 8.1 ± 6.53 77.6 ± 3.41 88.8 ± 3.12 80.4 ± 1.28 87.6 ± 1.54 (mg/Kg Soil) 18 Magnesium 9.8 ± 0.61 20.4 ± 1.45 18.1 ± 1.24 23.3 ± 1.05 21.2 ± 2.50 23.2 ± 1.19 (mg/Kg Soil) 19 Electrical 2.8 ± 0.05 3.9 ± 0.26 5.1 ± 1.09 3.7 ± 0.49 4.3 ± 0.12 5.7 ± 0.42 conductance (μmho/cm) 20 Phosphate 14.9 ± 0.79 20.4 ± 0.95 18.0 ± 0.39 22.4 ± 1.11 19.3 ± 1.07 23.1 ± 0.64 (mg/Kg Soil)

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Diversity … Mahesh Mandya.

Table 2: Description of the study sites

Sampling Sites Location Altitude Direction of sites Type of terrain and details of (Latitude - (m asl) from Distillery habitat Longitude) industry Besagarahalli 12º 38' 58.31" N 658-662 N 8300 Paddy field without distillery (Site 1) 76º 59' 54.34" E effluent application (control). Keregodu 12º 38' 50.68" N 698-704 N-E 9300 Undisturbed paddy fields (Site 2) 76º 54' 16.19" E located in the dry deciduous forest area (control). Bidarakote 12º 39' 59.12" N 677-685 E 5550 Rice paddy field with (Site 3) 76º 56' 66.12" E continuous agricultural activities & spent wash application Koppa 12º 42' 29.56" N 652-655 W 1650 Paddy field near to industry (Site 4) 76º 58' 10.20" E Kesthur.a 12º 41' 42.02" N 661-665 W 9500 Ragi crop with regular (Site 5) 77º 03' 52.32" E spentwash application Huthagere.a 12º 43' 40.02" N 650-663 S-W 6700 Disturbed ragi field with Semi- (Site 6) 77º 01' 01.86" E evergreen forest. Maraliga 12º 40' 25.37" N 641-644 N 5800 Sugarcane field having dry (Site 7) 76º 59' 24.11" E deciduous area with regular application of spentwash Dhundanahalli.a 12º 43' 35.99" N 662-665 S-W 8900 Moist deciduous area without (Site 8) 77º 02' 20.86" E regular water supply. Athagur 12º 39'44.52" N 641-642 N-W 13100 Jowar field disturbed regularly (Site 9) 77º 03' 25.46" E with the application of spent wash Huthagere.b 12º 43' 16.63" N 648-654 S-W 6400 Dry deciduous area with Jowar (Site 10) 77º 00' 39.81" E as main crop. Dhundanahalli.b 12º 43' 16.16" N 649-651 S-W 9750 Highly disturbed with (Site 11) 77º 02' 52.75" E regularly application of spentwash. Kestur.b 12º 42' 08.10" N 654-656 N-W 11700 Dry land with no surrounding (Site 12) 77º 04' 00.81" E vegetation

In the present investigation, we have sampled a total of 956 frogs belonging to F.limnocharis species. The density of F. limnocharis in these sites was found to be 302 frogs/ acre in control sites, 231 frogs/acre in paddy field, 207 frogs/acre in ragi field, 110 frogs/acre in sugarcane field, 76 frogs/acre in jowar field and 53 frogs/acre in a barren field. This clearly indicates that spent wash contaminated site shows less species density compared to uncontaminated sites (Figure2).

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FL ZR FC HT EC 600

400

200

individuals Number of Number

0

Study sites

Figure 2: Number of individuals in each study sites.

Species Diversity and richness: Highest Shannon diversity was found in Sugarcane fields followed by ragi, control, paddy, jowar and barren fields (Table 4). According to Simpson diversity index sugarcane fields showed highest diversity followed by ragi, control, paddy fields compared to jowar and barren fields (Table 3). The evenness of each field is listed in the Table 3.The values stated in table 4 indicates the diversity of frogs is highest in Sugarcane fields followed by ragi, control and paddy fields, compared to jowar and barren fields. Occurrence of amphibians in different location: Amphibian occurrence in all study sites were presented in Table 4. Three species of amphibians namely, F. limnocharis,F. caperata and Z. rufescens, were recorded in all the sites. Two species of anurans H. tigerinus and E. cyanophlyctis were absent in jowar and barren fields respectively but recorded in all the other sites (Table 4). Mean abundance of Fejervarya limnocharis: Species abundance was also recorded to verify the number of F. limnocharis speciesin uncontaminated and contaminated sites (Table 5). Highest abundance of F. limnocharis was seen in control-paddy fields (5.31 ± 0.02), followed by rice paddy (4.28 ± 0.01), sugarcane (4.17 ± 0.007), ragi (4.11 ± 0.00) and least in jowar and barren fields (3.08 ± 0.00 & 2.06 ± 0.007 respectively). Table 3: Species richness, diversity and evenness of Amphibians recorded from different study sites

Species richness Diversity index Locations Species recovered Simpson (D) Shannon (H) Evenness (J) Control-Paddy fields ( 1 & 2) 5 0.626 1.271 0.712 Paddy fields (3 & 4) 5 0.623 1.267 0.709 Ragi fields (5 & 6) 5 0.630 1.279 0.718 Sugarcane fields (7 & 8) 5 0.640 1.285 0.722 Jowar fields (9 & 10) 4 0.583 1.079 0.735 Barren fields (11 & 12) 4 0.501 0.964 0.655

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Table 4: Distribution of amphibian in different locations of study sites. Values in the parenthesisindicate range of frogs collected in each survey/acre.

Sites Species Fl Zr Fc Ht Ec Mean abundance Control- Paddy fields (1 & 2) + + + + + 25.2 ± 5.67 Rice paddy fields (3 & 4) + + + + + 19.3 ± 4.88 Ragi fields (5 & 6) + + + + + 15.3 ± 3.98 Sugarcane fields (7 & 8) + + + + + 9.2 ± 2.52 Jowar fields (9 & 10) + + + - + 6.3 ± 1.07 Barren fields (11 & 12) + + + + - 4.4 ± 1.68 Fl =Fejervarya limnocharis, Zr= Zakerana rufescens, Fc =Fejervarya caperata, Ht= Hoplobatrachu tigerinus, Ec= Euphlyctis cyanophlyctis

Table 5: Mean abundance (No/100 m2/ Yr) of F. limnocharis. Values in the parenthesis indicate range of frogs collected in each survey/acre.

Sites Mean abundance & range F. limnocharis Control-Paddy fields (1 & 2) 5.31 ± 0.02 (8 – 19)

Rice paddy fields (3 & 4) 4.28 ± 0.01 (4 – 13) Ragi fields (5 & 6) 4.11 ± 0.00 (3 – 10) Sugarcane fields (7 & 8) 4.17 ± 0.007 (4 – 12) Jowar fields (9 & 10) 3.08 ± 0.00 (2 – 4) Barren fields (11 & 12) 2.06 ± 0.007 (0 – 3)

Species Distribution: Figure 3 depicts, Jaccard similarity index of five different frog species assemblages in different crop fields. According to cluster analysis F. limnocharis, Z. rufescens and F. caperata showed more similarity (> 97.5 %) in their distribution in all crop fields. It is followed by H. tigerinus (83 %) and E. cyanophlytis (78 %) distribution. This clearly indicates that F. limnocharis, Z. rufescens and F.caperata are distributed in all crop.

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0.775

0.825

0.875 0.925

Similarity 0.975

0.85

0.95

0.8

0.9 0.6

1.2 EC 1.8

HT 2.4

3 FC 3.6

4.2 ZR 4.8

FL

5.4 6

Fl =Fejervarya limnocharis, Zr= Zakerana rufescens, Fc =Fejervarya caperata, Ht= Hoplobatrachu tigerinus, Ec= Euphlyctis cyanophlyctis

Figure 3: Cluster analysis based on transect data analysed by using the Jaccard similaity index representing F. limnocharis assemblage structure similarity between different study areas.

DISCUSSION

Amphibians possess biphasic life and permeable skin and are considered to be good indicators of environmental health and can provide an early warning of environmental degradation which may not be perceived by humans24. The present study suggests that the application of spentwash in an agro-ecosystem seriously affects the habitat there by decreasing the amphibian population. Meanwhile, this spentwash can also combine with pesticides which are regularly applied in the agro-ecosystem and can lead to detrimental effects on amphibian population. Though the impact of the use of industrial effluents on soil and soil dwelling organisms has been evaluated, database is not adequate to elucidate the influence of long term use of these effluent to agro-ecosystem as well as environmental as a whole. Although, previously there is little work carried out on the diversity and distribution of amphibians, in the present study area, attempt has been made to collect the information about F. limnocharis species in spent wash sprayed agro-ecosystem. In the present investigation, control sites showed comparable species richness, whereas density decreased towards the spent wash sprayed (disturbed) sites. Amphibian population decline in response to habitat degradation has been discussed for a variety of locations elsewhere25,26,9,27,28. In our study we noticed that species richness is less in jowar and barren lands compared to other sites. The possible reason behind this is the scarcity of water in soil (wet soil). Another problem associated with species richness is the immense application of spentwash to dry lands compared to wetlands; this may be the other reason to see good number of amphibian population in 3190 J. Chem. Bio. Phy. Sci. Sec. D, May 2015 – July 2015; Vol.5, No.3; 3182-3192.

Diversity … Mahesh Mandya. control, paddy and ragi fields. Further, the best strategy for utilization of industrial effluents for irrigation in agro-ecosystem should emerge from realization by all the concerned agencies like pollution control, industries, farmers and research organizations.

CONCLUSION

When frogs were exposed to spentwash, they bear irreparable damage to both their habitat and life cycle making them less fit for survival. These changes can alter the activities of the frog like jumping performance and growth. Thus, from this study it may be inferred that spentwash can be responsible for habitat destruction which may seriously affect the anuran F. limnocharis and this alteration may lead to loss of resistance of the frog, ultimately leading to a decline in its population in the agro-ecosystem. The present study also suggests that the spraying of spentwash has damaged the habitats, resulting in the decline of amphibian populations in and around the agro-ecosystem.

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* Correspondingauthor: MaheshMandya Department of Environmental Science, KuvempuUniversity, Shivamogga, Karnataka- 577451.

3192 J. Chem. Bio. Phy. Sci. Sec. D, May 2015 – July 2015; Vol.5, No.3; 3182-3192.