The effect of physicochemical, phytoplankton and seasonal factors on faecal indicator bacteria in northern brackish water

Water Resear('h Vo[. 14. pp. 279 to 285 Pergamon Press Ltd 1980. Printed in Great Britain

THE EFFECT OF PHYSICOCHEMICAL, PHYTOPLANKTON AND SEASONAL FACTORS ON FAECAL INDICATOR BACTERIA IN NORTHERN BRACKISH WATER JORMA HIRN*. HILKKA VILIAMAA'["and MARKKU RAEVUORI:~ *College of Veterinary Medicine, H~meentie 57, 00550 Helsinki 55 tCity of Heisinki Building Office, Water Conservation Laboratory, Kylisaarentie 10, 00550 Helsinki 55. Finland ~,State Veterinary Medical Institute, Hiimeentie 57. 00550 Helsinki, 55, Finland

(Received for publication 20 Auoust 1979) AbslraetnThe relationships existing between the numbers of coliforms, faecal coliforms` faecal streptococci, C. perfrinoens, certain physicochemical parameters, phytoplankton, and seasonal factors in an eutrophic northern brackish water were investigated during a period of I year. Seasonal fluctuation of the faecal indicator bacteria was noted. A highly significant correlation was found between the non spore-forming faecal indicator bacteria examined. Of the physicochemical parameters examined the pH-value and temperature were found to have the most effect on the ntlmber of faecal indicator bacteria but nutrients, especially total nitrogen and nitrate nitrogen were also significant. The lack of correlation between C. perfringens and the other indicator bacteria was found to be related, in part, to the variation in pH. Thus C. perfrinaens can be considered as a useful indicator in ecosystems having stress factors, The results in this study show that many stress factors affect the number of faecal indicator bacteria. Therefore it is necessary to estimate the quality of water using a combination of several parameters.

INTRODUCTION

Coliforms, faecal coliforms, faecal streptococci and Clostridium perfrin¢ens are accepted bacterial indicators of faecal pollution of water. Different kinds of physicochemical and plankton analyses have also been used to indicate this pollution. Carnery et al. (1977) did not find any correlation between total and faecal cofiforms and dissolved oxygen, temperature, turbidity and conductivity in a fiver basin. Jonas et al. (1977) investigated the relationship between the bacterial flora and water chemistry in a salt marshestuarine ecosystem. They found that the number of bacteria determined by plate counts corrrelated well with the nitrogen and phosphorus concentrations of the water and the salinity. Pagliardini et al. (1976) studied the correlations between total coliforms, Escherichia coli and Streptococcus faecalis along a seashore. Their study indicated that the number of faecal coil correlated well with that of total coliforms, while the temperature did not. Ambraziene (1976) found a linear relationship between microbial population and organic pollution concentration in 33 rivers. Seasonal variation has been demonstrated in the bacterial flora of water. Faecal indicator bacteria survive better in water during the winter than the summer (Gordon, 1972; Cohen & Shuval, 1973). Bacterial counts are generally found to be higher in the autumn and winter than in the spring or early summer (Jonas et al., 1977). The purpose of this study was to investigate the relationships existing between the numbers of the fac279

cal indicator bacterial groups, certain physicochemical parameters, phytoplankton, and seasonal factors in an eutrophied northern brackish water. MATERIALS AND

METHODS

Study area and sampling stations The study area and sampling stations are shown in Fig. 1. In this area of the Gulf of Finland the air temperature generally remains above 0oc from April to November and the inshore waters are normally ice-covered for about 100 days from January to April. In the shallow inner bays the temperature during the summer may rise above 20°C throughout the water column. In these waters the pH values range normally from 7.0 to 10,0 and the salinity from 0.4 to 0.6?/0. The sampling stations were located west of Helsinki. Station I was in an eutrophic inner archipelago with an average depth of 7 m and station 2 was in a very eutrophic bay area with an average depth of 3 m. This area receives a heavy load of sewage from the treatment plants of Helsinki. The study was conducted during the 1 year period of November 1976--October 1977. Samples were taken at intervals of 14 days into glass bottles using a Ruttner sampler. The samples were collected in the morning, taken to the laboratories within 3 h, and analysed immediately.

Bacterial analysi.~ The MF technique (Standard Methods, 1975) was used throughout the study for the determination of coliforms, faecal coliforms and faecal streptococci. Millipore HC illters (porosity 0.70 pro) were used for faecal coliforms and Gelman GN-6 (porosity0.45/am) for the two other groups, The growth media used were LES Endo agar (Orion Diagnostica, Finland) for coliforms,m F C agar (Difco)for faecal coliforms and Slanetz and Bartley agar (Orion Diagnos tic& Finland) for faecal streptococci. For the detection c

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C. perfringen~ the tryptosc-sulfite-cycloserine-eggyolk agar mgmbrane filter method was used (Hirn & Raevuori, 1978). No confirmation tests were done for the faecal indicator bacteria. Both Salmonella and Yersinia enterocolitica detections were made after the filtration (Geiman GN-6) cd" I 1. of the sample. Y. enterocotitica was isolated using a modified cold enrichment method (Highsmith er al., 1977). Aliquots from the enrichment broth were taken after 2, 4 and 6 wvgks for the isolation of Y. enterocolitica to SS agar (Difco) and desoxycholate-citrate agar (BBL). Tetrathiohate broth (BBL) was used as an enrichment medium for

Salmonella. The broth was incubated with the filter for 1 and 2 days at 41.5"C. The isolation of the organism was made from brilliant greta agat (Orion Diagnostica, Finland), which was incubated at 35°C for 24 h. Typical colonies were tran~'erred to TSI and urea agars (Orion Diagnostica, Finland) and further identified by serological tests. Phytoplunkton analysis The phytoplankton was preserved with Kede's solution (Keefe, 1926) and analysed by Utermt~hl's technique (Uterm~hl, 1958). The biormm~ were delermined by the

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Physicochemical analysis Temperature was measured immediately at the sampling stations with a thermometer fastened inside the Rutmer sampler. Salinity was detected electrically with a conductimetric salinometer (Autolab Industries P/L, m o d e l 601 MK Ill) and pH potentiometrically (Findip 555 A pH meter). Oxygen was measured using a modified Winkler's method (Stand, rd Methods, 1975~ Orthophosphate phosphorus was measured using the ammonium molybdate acid method and total phosphorus by the persulfate digestion method (Standard Methods, 1975). Concentrations were measured with a photome~-r (Vitatron DCP). Nitrogen components were detected by the following methods: ammonia by the indophenol method (Verlag Chemie, 1975), nitrite through the formation of an azo dye (Standard Methods, 1975), nitrate by reduction to nitrite with a Cd-Cu column, and total nitrogen was determined by oxidation to nitrate followed by the reduction to nitrite (Standard Methods, 1975). Statistical analysis A logarithmic transformation, y ' = log (y+ 1[ was made for the bacterial counts before the statistical analysis using the computer programs (Computing Centre, University of Helsinki, 1978) for correlation and regression anal),sis. RESULTS The indicator bacteria data is shown in Figs. 2-5. The results of the analyses of coliforms, faecal coilforms, faecal streptococci and C. perfrinoens of the samples taken at station 1 are shown in Fig. 2. The corresponding results of station 2 are given in Fig. 3. Figures 4 and 5 show the moving averages of three consecutive indicator bacteria counts, Salmonella was detected only once during this survey. $. haelsinbor0

was found in a sample from station 2 in late December. Y. enterocolitica was not detected in any of the samples, whereas Y. pseudotuberculosis was found four times during this survey. The results of the physicochemical and phytoplankton analyses from sampling stations 1 and 2 are shown in Table 1. These results are expressed as the mean values of the analyses of the 20-25 samples taken and their standard deviations. The maximum and minimum values are also shown for each analysis. The correlation coefficient table for all the parameters studied is shown in Table 2. Partial correlation coefficients after removal of the effect of pH are shown in Table 3. The combined data from the both stations were used to construct Tables 2 and 3. DISCUSSION

Seasonal fluctuation of the indicator bacteria counts is clearly seen at sampling stations 1 and 2. The bacterial numbers are higher in the autumn and winter especially when the sea is ice-covered. The numbers of indicator bacteria decrease very soon after the stratification in the spring and stay relatively low during the summer. In the autumn the counts of bacteria begin to increase again. V~ifiinen (1976) also observed that the bacterial numbers are generally higher in the autumn and winter than in the spring and summer. The pattern of C. perfrinoens counts differed from that of the other indicator bacteria analysed. At both stations (Figs. 4 and 5) the numbers of C. perfrinoens did not fluctuate as remarkably as the numbers of the other indicator bacteria used. The seasonal fluctuation is partly dependent on the tempera-

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with the die-off index of E. coll. Their data also show that between 40 and 50% of the variation of the dieoff index is either directly or indirectly governed by the insolation. Highly significant correlations (p <0.01) were found between the counts of cotiforms and faecal coliforms, coliforms and faecal s ~ as well as faecal coBforms and faecal smsptoco~ (Table 2). These correlations were also hilhly significant (p < 0.01).

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The effect of physicochemicaL phytoplankton and seasonal factors on faecal indicator bacteria Table !. The results of the physicochemical and phytoplankton analyses of the 20-25 samples taken from each of the two sampling stations Variable 6

11

Phytoplankton biomass (rag 1-~) Proportion of Cyanophyta in total biomass (To) Proportion of Chlorophyta in total biomass (%) Proportion of Diatoma¢ in total biomass (%) Proportion of other groups in total biomass (%) Temperature (°C)

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Total phosphorus (rag m - a) Orthophosphate phosphorus (rag m-3) Total nitrogen (mg m-3) Nitrate nitrogen (mg m - 3) Nitrite nitrogen (rag m -3) Ammonium nitrogen (mg m - ~) Salinity (°,/o)

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4.0 26.3 8.2 23.6

4.8 24.2 8.9 26.8

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15.6 78.0 31.8 69.9

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21.0 32.8

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6.7 6.9 7.8 7.9 52.2 271 35.2 231 775 3910 184 603 7.4 47.2 164 2260 0.56 0.44 11.9 I0.5

7.3 7.3 0.4 0.8 21.4 414 29.1 484 537 4430 283 722 6.4 68.7 215 3680 0.09 0.13 1.5 2.4

- 0.4 -0.3 7.2 7.0 18 17 0 2 220 880 0 0 1 0 3 3 0.25 0.06 9.0. 5.8

22.0 18.7 8.8 9.3 96 1900 87 2000 2600 16000 1200 2600 19 270 870 13000 0.67 0.58 14.8 14.6

Table 2. The correlation coefficient table (combined data from both stations) for all of the parameters studied. The variables 6 to 20 are explained in Table ! Variable 2 2 3 4

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Coliforms Faecal coliforms Faecal streptococci

C. perfrirugens

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1.000 0.861

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0.865 0.423 -0.133 -0.125 - 0.007 -0.247 0.270 -0.679 -0.672 0.412 0.438 0.614 0.639 0.597 0.548 --0.361 --0.518

0.839 0.489 -0.099 -0.151 0.082 -0.168 0.202 -0.635 -0.618 0.387 0.391 0.561 0.641 0.497 0.511 --0.420 --0.420

1.000 0.482 -0.104 -0.099 - 0.020 -0.086 0.140 -0.613 -0.594 0.478 0.504 0.634 0.671 0.638 0.558 --0.522 --0.398

1.000 0.320 0.336 0.230 0.031 -0.338 0.017 0.089 0.243 0.178 0.315 0.362 0.216 0.254 --0.510 --0.116

283

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284

Table 3. The partial correlation coefficient table (combined data from both stations) after the removal of the effect of pH. The variables 6 to 20 explained in Table 1 Variable 2 2 3 4

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Coliforms Faecal coliforms Faecal streptococci

C. perfringens

Correlation coefficient variable 3 4

5

1.000 0.765

1.000

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0.747 0.695 0.262 0.154 0.227 -0.043 -0.166 -0.302

1.000 0.667 0.233 0.208 0.088 0.055 -0.235 -0.289

1.000 0.316 0.331 0.220 0.012 -0.341 -0.087

0.299 0.265 0.458 0.427 0.441 0,372 -0,330 -0.307

0.269 0.213 0,392 0,452 0,309 0,332 -0,399 -0,185

0.390 0.373 0.501 0.508 0.513 0,406 -0.524 -0.165

0.283 0.231 0,406 0.499 0.289 0.330 -0.537 -0,180

when the material was divided according to the sample station. The correlation coefficients were slightly higher, however, in the very eutrophic area (station 2), rather than in the eutrophic area (station 1). C. perfrinoens also correlated highly significantly (p < 0.01) with coliforms~ faecal colfforms and faecal streptococci (Table 2). In the material from the separate sites (divided material) no significant correlation between C, perfrinoens and the other indicator bacteria could be shown in the very eutrophic area. In the eutrophic area C, l~rfrinoens had a significant correlation (p < 0.05) with coliforms and faecal streptococci and a non-significant correlation with faecal coliforms. Thus there are evidently certain factors which affect the other faecal indicator bacteria more in the very eutrophic area than in the other sampling area. That is why it should be necessary to evaluate the quality of water using more than one indicator bacteria or bacterial group. Salmonella and Y. enterocolitica were the only pathogenic enterobacteria that the water samples were analysed for. We isolated S. haelsinobory from one sample, This serotype is not a common causative agent of human salmonellosis in Finland. The indicator bacteria counts were not elevated in the Salmonella positive sample. Y. enterocolitica was not found in any of the samples. This may be partly due to the fact that the selective agars used for the isolation are relatively inhibitory for the environmental strains of Y. enterocolitica. The results, however, indicate that these pathogens are not common in the water area studied. When examining the effect of the phytoplankton biomass and the percentile proportion of different

algae groups no significant correlations were found with coliforms, faecal coliforms and faecal streptococci. A significant correlation (p < 0.05) was found in the case of C. perfrln@ens but the correlation coefficient was relatively low (Table 2). On the basis of our results it is impossible to show that some algal groups have any direct effect on the counts of the faecal indicator bacteria. Indirectly phytoplankton could influence the numbers of bacteria by rising the pHvalue. When comparing the faecal indicator bacteria data to the concentrations of phosphorus and nitrogen compounds it is notable that coliforms, faecal coilforms and faecal streptococci correlated highly significantly (p < 0.01) (Table 2) with phosphorus (total P; PO4--P) and nitrogen (total N: NO3--N; N O 2 - - N ; NH4--N). This is evidently due to the sewage input to the area. The correlation coefficients of the nitrogen compounds were higher than those of phosphorus, because phosphorus sediments rapidly. The correlations were again considerably stronger in the eutrophic sample station than in the very eutrophic area. Jonas et al. (1977) have reported an association between the number of bacteria determined by viable counts and the nitrogen and phosphorus concentrations of a salt marsh-estuarine water. Correlations (p < 0.05) between C. perfrinoens and phosphorus and nitrogen compounds were found only in the case of total nitrogen and nitrate nitrogen (Table 2). In the divided material there was no correlation in the very eutrophic area. In the eutrophic area highly significant correlations (p < 0.01) were found between C. gerfringens ,and total nitrogen and nitrate nitrogen and significant correlations (p < 0.05) between C. perfrin~ens and nitrite nitrogen and ammonium nitro-

The effect of physicochemicaL phytoplankton and seasonal factors on faecal indicator bacteria gen. The highest correlation coefficient between nitrate nitrogen and C. perfringens can be explained by the stability of the nitrate nitrogen. Negative correlations were found between coliforms, faecal coliforms and faecal streptococci, and temperature, pH value, salinity and oxygen when the whole data was used (Table 2~ Similar results were also found for the divided material. No significant correlations between temperature and coliforms, E. coil, S. faecalis (Pagliardini et al., 1976), temperature and coliforms, faecal coliforms (Goyal et al., 1977) have been found in field studies. The effect of water temperature on the indicators have, however, been reported in laboratory studies (McFeters & Stuart, 1972; McFeters et al., 1974). The negative correlation between the temperature and the non-sporeforrning faecal indicator bacteria could be explained by the increased die-off of the bacteria. An association was found between oxygen concentration and the indicator bacteria except C. perfringens. These correlations were not found in the divided material from the eutrophic area, whereas in the very eutrophic area a highly significant negative correlation Lo < 0.01) in the case of coliforms and a significant negative correlation (p < 0.05) in the case of faecal coliforms and faecal streptococci were found. It is noteworthy that in the very eutrophic area the amount of dissolved oxygen was low especially when the area was ice-covered. Jonas et al. (1977) found a correlation between the viable count of bacteria and salinity. In our study there were highly significant negative correlations (p < 0.01) between all the studied indicators and salinity in the whole data. This negative correlation indicates a diluting effect of sewage. No correlations were found, however, between the indicators except C. perfringens and salinity in the divided material. In one of our partial correlation .studies the effect of salinity was excluded from the material. There were no great differences in the correlation coefficients compared to the results in Table 2. This indicates that the low salinity in brackish water ( < 1%) has little if any effect on the survival of the indicator bacteria. pH had also a highly significant correlation with the indicator bacteria other than C. perfringens. Goyal et al. (1977) found no significant correlation between pH and coliforms and faecal coliforms i.n coastal canal communities. The results after the elimination of the effect of pH (Table 3) show that C. perfringens also had a highly significant correlation (p < 0.01) with coliforrns` faecal coliforms and faecal streptococci. The correlation coefficients after the elimination of pH indicate that this parameter had a great effect on the commonly used faecal indicator bacteria in eutrophic areas where pH is elevated. For this reason C. perfringens and especially its spores, which are much more tolerant to various physicoebemical effects than the other faecal indicator bacteria, could serve as a useful indicator in ecosystems having stress factors.

285

REFERENCES

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