The abundance, heterotrophic activity and taxonomy of bacteria in a stream subject to pollution by chlorophenols, nitrophenols and phenoxyalkanoic acids

War. Res. Vol. 20, No. I, pp. 85-90, 1986 Printed in Great Britain.All fights reserved

0043-1354/86 $3.00+0.00 Copyright © 1986PergamonPress Ltd

THE A B U N D A N C E , HETEROTROPHIC ACTIVITY A N D T A X O N O M Y OF BACTERIA IN A STREAM SUBJECT TO POLLUTION BY CHLOROPHENOLS, NITROPHENOLS A N D PHENOXYALKANOIC ACIDS C. R. MILNER and R. GOULDER Department of Plant Biology and Genetics, University of Hull, Hull HU6 7RX, England (Received June 1985) Abstract--Analysis by HPLC identified 14 individual chlorophenols, nitrophenols and phenoxyalkanoic acids in a small stream in West Yorkshire; concentrations fluctuated markedly over 12 months. Abundance of bacteria (as direct counts and colony-forming units) and heterotrophic activity (as fraction of glucose substrate mineralized) were measured in water and artificial gravel substratum at two sites in this stream. Mean bacterial abundance, and at one site activity, were at least similar to those found in an adjacent unpolluted stream. Sometimes, however, colony-forming units and activity were depressed. Multiple-regressionanalysis demonstrated relationships between bacteria and pollution-related variables; low pH and nitrophenols (particularly picric acid), were identified as possible causes of bacterial inhibition. Taxonomic investigation of isolates from sediment demonstrated that bacteria in the polluted stream were dominated by Pseudomonas, whereas a wide range of mainly Gram-negative bacteria was present in the unpolluted stream. Key words--acridine-orange direct-counts, chlorophenols, colony-forming units, freshwater bacteria, heterotrophic activity, nitrophenols, phenoxyalkanoic acids, river pollution INTRODUCTION

Aquatic bacteria exhibit a variety of response to pollutants. Organic wastes frequently provoke increase in bacterial abundance; e.g. in river waters (Rheinheimer, 1965; Deufel, 1972; Coleman et al., 1974; Kachan, 1976; Starzecka, 1979) and sediments (Ossowska-Cypryk, 1981). Heterotrophic activity of bacteria may also be positively related to organic pollution (Albright and Wentworth, 1973; Carney and Colwell, 1976; Goulder et al., 1980). The bacteria presumably thrive in response to enhanced availability of organic substrate for heterotrophic utilization. Toxic pollutants, in contrast, may inhibit aquatic bacteria; e.g. metal-bearing effluents may locally reduce heterotrophic activity in estuarine and river waters (Goulder et al., 1979, 1980; Milner and Goulder, 1984). Pollution may also cause change in the taxonomic composition of aquatic bacterial communities; e.g. in mercury-contaminated estuarine sediments (Nelson and Colwell, 1975) and in seawater contaminated by pharmaceutical wastes (Peele et al., 1981; Grimes et al., 1984). With some pollutants, the likely response of aquatic bacteria is problematic. For example, phenolic compounds and phenol derivatives might potentially act either as enhancing biodegradable energy sources, or as biotoxins. Freshwater microbial communities can degrade, for example, chlorophenols (Aly and Faust, 1964; Baker et al., 1980) and 2,4-dichlorophenoxyacetic acid (Aly and Faust, 1964; Watson, 1977; Nesbitt and Watson, 1980). In addition, laboratory-adapted microbial communities 85

from freshwater sediments can degrade a wide range of phenolic compounds (Tabak et al., 1964; Spain and Van Veld, 1983). Phenolic compounds in natural waters are, however, potential biological inhibitors (Buikema et al., 1979). In laboratory studies, interactions between pH and toxicity of 2,4-dichlorophenoxyacetic acid to Salmonella typhimurium are described by Zetterberg et al. (1977), and the effect of molecular structure on toxicity of halogenated phenols to a Bacillus sp., isolated from activated sludge, is described by Liu et al. (1982). The aim of the work described herein was to investigate the effects of pollution of a small stream by a mixture of phenolic compounds and phenol derivatives on (1) the abundance and heterotrophic activity of bacteria in water and artificial gravel substratum, and (2) on the taxonomic composition of the sediment bacterial community. This study was facilitated by comparison of the polluted stream with an adjacent non-polluted stream. MATERIALS AND METHODS

Site description Sugden Beck rises from the Carboniferous coal measures of industrial West Yorkshire and flows, partly in culvert, for about 2500 m before joining Hunsworth Beck, a tributary of the River Calder, at Grid Ref. SE 185 268. The stream receives leachate from old mine workings which are historically contaminated by phenolic wastes. The water frequently has a distinct yellowcoloration and phenolic odour. Stubbs Beck, a similar sized but unpolluted stream, merges with Sugden Beck, in culvert, about 400 m upstream of its junction with Hunsworth Beck. The combined discharge of both streams, estimated from velocity measurements in a channel of regular section, ranged from 0.01 to 0.14 m3s-:.

86

C . R . MILNER and R. GOULDER

Routh~e sampling and field measurements Sites for routine sampling were; (1) Sugden Beck (upstream)--about 1300m above the confluence with Stubbs Beck, (2) Sugden Beck (downstream)---about 300 m below the confluence with Stubbs Beck, (3) Stubbs Beck--about 700 m above the confluence with Sugden Beck. At monthly intervals, from April 1981 to April 1982, surface-water samples were taken from mid-stream in acetone-washed glass bottles for analysis by HPLC and in sterile glass bottles for bacteriological analysis. Also, preexposed mesh bags of artificial gravel substratum were collected into sterile polythene bags. These mesh bags ( 10 × 15 cm) were of non-biodegradable polyester mesh (4.4 strands per cm, aperture 1.8 mm, open area 61~) sewn with polyester thread. The bags were each filled with 55 g of acid-washed, rinsed and dry-sterilized, crushed, calcined flint (>99°(i silica), as supplied commercially for aquarium gravel, and were tethered, submerged, on the stream bed for one month before collection. Water temperature, pH, dissolved oxygen concentration and conductivity were measured in the field using portable instruments. The Stubbs Beck site was dry on two sampling occasions (July and September 1981).

Determination o/phenolic compounds and phenol derivatives Concentration of these compounds in stream water was determined by HPLC. The apparatus consisted of paired high-pressure pumps (model 750/03) controlled by a (model 750/36) deci-linear gradient programmer (Applied Chromatography Systems) connected to a 10 cm Hypersil ODS5 (Shandon) column. Detection of separated compounds, by u.v. absorbance at 280 nm, was with a LC-UV detector (Pye-Unicam). Sensitivity ranged from 0.04 to 0.16 absorbance units for full-scale deflection with output recorded on a Linseis recorder (model LS5) at chart speed 200 mm h-~. Prior to analysis, samples were preserved by acidification (1 ml concentrated HC1 per 300 ml sample) and stored at 4' C. Solids were removed by membrane filtration (0.22/am) and 250 ml of filtrate were concentrated to 2-3 ml in a rotary evaporator at 75C. Next, 2.5 ml of a saponification mixture (0.2 M KOH made up in a mixture of equal volumes of isopropanol and twice-distilled water) were added to the concentrate, to convert ester or amine forms of phenoxyalkanoic acids to the acid form (Skelly et al., 1977). The saponification mixture also contained 500mg l4-bromophenol as internal standard. A volume (20#1) of saponified concentrate was injected onto the column. Solvents used were acetonitrile and 0.1 M, pH 3.0, citrate buffer. The following programme, at flow rate 3 ml min -~, gave adequate separation; 10~o acetonitrile for 10min, increase of acetonitrile concentration at 1~o rain -~ for 10min, constant acetonitrile concentration for 10min, increase of acetonitrile at 1~o min -~ for 20rain, constant acetonitrile concentration at 40~o to end of run at 60 min. Compounds in the sample were identified by comparing their retention times with those of known standards run under identical operating conditions. The concentration of each compound was calculated from peak height and the instrument's response factor to that compound, relative to 4-bromophenol. The response factors were obtained from calibration runs in which 20 t~l of 100 mg 1-~ test compound and 100rag 1-~ 4-bromophenol (in the saponification mixture) were injected onto the column.

Abundance and heterotrophic activity of bacteria Concentration of bacteria in water samples was determined by the acridine-orange direct-count (AODC) method Oones and Simon, 1975). Bacteria in sub-samples, preserved for up to 24 h with 2°~ formaldehyde (Daley and Hobbie, 1975). were stained with 10rag 1-~ acridine orange for 10 rain and concentrated onto black 0.22/.tm (Millipore) membrane filters. Bacteria were then counted in 30 fields using a Zeiss epifluorescence microscope (Goulder, 1976).

Concentration of colony-forming units (CFUs) in water samples was determined by spreading, within 3-5 h of sampling, a precise volume (0.1-0.25ml) of appropriately diluted (sterile 25~o Ringer solution) sample onto each of ten CPS plates (Jones, 1970; Staples and Fry, 1973). Colonies were counted after 7 days at 20°C. Pigmented colonies were also recorded separately, and chromogenic colony-forming units (CCFUs) were expressed as a percentage of total CFUs. The abundance of bacteria associated with artificial gravel substratum was expressed in terms of release per unit weight of gravel. 1.0 g of gravel, plus 1.0 g of sterile sand, in 10 ml of sterile 25~o Ringer solution were treated for 5 min with a Mickle disintegrator (Mickle Laboratory Engineering Co). The bacteria suspended in the solution were then counted using the AODC method and CFUs and '!;, CCFUs were determined as above. The measure of bacterial heterotrophic activity, in water samples, was the fraction of added glucose substrate mineralized to CO 2 in unit time. For artificial gravel substratum, the measure was the fraction mineralized per unit weight of gravel in unit time. The procedure with water samples was as described by Milner and Goulder (1984). Briefly, four 20 ml sub-samples, and an acidified control, were incubated with [~4C]glucose at about 100/~g I ~(radioactivity about 3.7 kBq), a concentration which should swamp the natural glucose present (Wright and Burnison, 1979). Incubations (1 h at IO'C in darkness on a rotary incubator at 100 oscillations min ~) were stopped by acidification. J4CO2 released was trapped on 2-phenylethylamine-impregnatedpaper wicks and radioactivity determined by liquid scintillation counting. The fraction of substrate mineralized = (mean radioactivity of 14CO~ released minus release from the control)/radioactivity ~4 of initial [ C]glucose. Incubations to measure heterotrophic activity of bacteria associated with artificial gravel substratum were performed as above except that 10 g of gravel (dispensed using an aluminium scoop of appropriate volume), plus 20ml of sterile 25~ Ringer solution, were substituted for the 20ml sub-samples. Incubations were begun within 2-3 h of sampling.

Bacterial taxonomy Natural stream-bed sediment was collected, into sterile containers, from the Sugden Beck (upstream) site and from the Stubbs Beck site, on 21 July and 16 August 1982. The sediment was diluted 105 times, with sterile 25~o Ringer solution, and aliquots were spread onto CPS agar plates and incubated for 7 days at 22°C. Fifty colonies from each site were selected at random, on each occasion, and transferred to nutrient agar (Oxoid CM3). Of the 200 isolates, 13 were later lost or discarded because of contamination. Tentative identification of the isolates to genus level followed Harrigan and McCance (1976) with reference to Buchanan and Gibbons (1974), the following tests being employed; Gram's stain, colony pigmentation and morphology, Hugh and Leifson's glucose oxidationfermentation test, catalase production, oxidase production, penicillin sensitivity (Oxoid disks, penicillin G 1.5 units), sensitivity to vibriostatic compound 0/129 (2,4-diamino-6,7-diisopropylpteridine), production of extracellular fluorescent pigments, liquefaction of gelatin, starch hydrolysis, arginine hydrolysis and motility by hanging drop. Position of flagella was determined by silver staining (Rhodes, 1958), or in difficult cases by transmission electron microscopy following negative staining with uranyl acetate (Gregory and Pirie, 1973). RESULTS Fourteen phenolic c o m p o u n d s and phenol derivatives were identified in Sugden Beck (Table 1) although other, unidentified, c o m p o u n d s were also

Bacteria in a stream

87

Table I. List of phenolic compounds and phenol derivatives identified in Sugden Beck and summary of concentrations, April 1981-April 1982 Upstream site Downstream site Mean

(Range)

CV

Mean

0.67 0.57 3.5 2.9 7.6

(0-2.9) (0-1.9) (0-19.0) (0-14.8) (0-34.6)

133 97 146 153 123

0.41 0.30 1.6 1.5 3.9

0.07 2.0 0.03 0.01 2.1

(0-0.24) (0.36-4.4) (0-0.09) (0-0.05) (0.394.7)

109 61 101 207 61

0.05 0.83 0.02 <0.01 0.90

(Range)

CV

(0-2.2) (0-2.0) (0-8.6) (0-10.6) (0-21.5)

179 182 142 193 150

(0-0.29) (0.16-1.9) (0-0.09) (0-0.02) (0.16-2.2)

159 72 148 244 75

Chlorophenols (mg 1-I) 3-Chlorophenol 4-Chlorophenol 2,4-Dichlorophenol (2,4-DCP) 4-Chloro-2-methylphenol (PCOC) Total chlorophenols

Nitrophenols (mg I-I) 2,4-Dinitrophenol 2,4,6-Trinitrophenol (picric acid) 2-Methyl-4,6-dinitrophenol 2-sec-butyl-4,6-Dinitrophenol Total nitrophenols

Phenoxyalkanoic acids (mg l -L) 2,4-Dichlorophenoxyacetic acid (2,4-D) 2.1 (0-6.4) 2-Methyl-4-chloropbenoxyacetic acid (MCPA) 2.1 (0-9.0) 2-(2,4-Dichlorophenoxy)proprionic acid (2,4-DP) 7.4 (0.65-17.3) 2-(2-Methyl-4-chlorophenoxy)-proprionic acid (CMPP) 8.1 (0-21.6) 4-(2,4-Dichlorophenoxy)-butyric acid 0.47 (0-1.3) 4-(2-Methyl-4-chlorophenoxy)-butyric acid 0.16 (0-1.2) Total phenoxyalkanoic acids 20.3 (4.4-53.4) Total phenolic compounds and phenol derivatives (rag I ~) 30.0 (4.8~9.7) Values are means (n = 13), ranges and coefficients of variation (%); abbreviations used in the present. N o phenolic compounds or phenol derivatives were detected in Stubbs Beck. Concentrations o f phenoxyalkanoic acids in Sugden Beck (principally 2,4-D, M C P A , 2,4-DP and C M P P ) were generally higher than concentrations of chlorophenols (principally 2,4-DCP and PCOC) and nitrophenols (principally picric acid)--Table 1. The concentrations were less at the downstream site, presumably because of dilution by Stubbs Beck. All compounds showed much irregular variation in concentration over the sampling period, note the wide ranges and high coefficients of variation (Table 1). Other physico-chemical differences were found between the two streams (Table 2). Mean p H was lower in Sugden Beck and the range was wider. The p H in Sugden Beck was negatively correlated with concentration of total phenolic compounds and phenol derivatives (r = - 0 . 4 8 , n = 2 6 , P <0.05). Conductivity in Sugden Beck was greater and more variable than in Stubbs Beck, but there was no significant correlation with total phenolic compounds (r = 0.37, n = 26, P > 0.05). Temperature and oxygen concentration were similar in the two streams, the lower mean temperature in Stubbs Beck is due to there being no data when the stream was dry in July and September 1981. Substantial mean concentrations of directlycounted bacteria and C F U s were found at all three

91 0.94 (0-3.7) 135 0.91 (0-3.7) 77 3.6 (0-7.6) 66 4.6 (0-17.0) 108 0.18 (0-1.1) 227 0.11 (0-0.60) 69 10.3 (0.7%25.7) 60 15.1 (0.94-48.3) text are given in parentheses.

121 132 65 90 207 199 65 78

sites in both water and artificial gravel substratum (Table 3). Mean concentration of directly-counted bacteria in water, and of C F U s in water and gravel, was notably highest at the Sugden Beck (downstream) site. Mean concentrations in water and gravel were similar at the other two sites. Mean beterotrophic activity (Table 3) was markedly lower at the Sugden Beck (upstream) site than at the other two sites. O f particular interest is the variability of bacterial concentration and activity (ranges and coefficients of variation are included in Table 3). C F U s and activity, in both water and gravel, showed much greater variability at the two Sugden Beck sites than in Stubbs Beck, and on some occasions extremely low values were recorded. The presence of inhibitory relationships between bacteria and pollution-related variables in Sugden Beck was confirmed by multiple-regression analysis (SPSS New Regression--Hull and Nie, 1981) using combined data from both sites. Directly-counted bacteria, C F U s and activity, in water and gravel, were used separately as dependent variables; the independent variables were pH, conductivity and concentration of total identified phenolic compounds and phenol derivatives. Each bacterial variable, apart from directly-counted bacteria in gravel, had a significant relationship (P < 0.05) with pollutionrelated variables (Table 4). The maximum multiple

Table 2. Summary of physico-chemicalconditions in Sugden Beck and Stubbs Beck, April 1981-April 1982 Sugden Beck (upstream site) Stubbs Beck Sugden Beck (downstream site) Mean

(Range)

CV

Mean

(Range)

CV

Mean

Temperature (°C) 9.9 0.0-19.0) 49 9.2 (2.0-19.0) 50 7.6 pH 5.0 (2.7-7.4) 38 6.4 (3.3-7.6) 26 7.5 Oxygen (mg I-~) 10.3 (8.2-12.1) 12 10.4 (8.5-12.6) 12 10.0 Conductivity (pS cm ~) 2 3 7 2 (529~i292) 77 1506 (429-4576) 88 512 Values are means (n = 13 for Sugden Beck sites, 11 for Stubbs Beck), ranges and coefficients of variation (%).

(Range)

(I.0-17.0) (7.0-8.0) (7.7-12.0) (329-1001)

CV

60 5 14 39

88

C. R. MILNER a n d R. GOULDER

Table 3. Summary of bacteriological data from Sugden Beck and Stubbs Beck, April 1981-April 1982 Sugden Beck (upstream site)

Sugden Beck (downstream site)

Stubbs Beck

Mean

(Range)

n

CV

Mean

(Range)

n

CV

Mean

(Range)

n

CV

16.5 7.7 2.7 t 10

(2.4-49.2) (0.003~,6.7) (0.3-18.5) (2.4-25.0)

13 13 13 13

85 176 184 62

25.4 25.0 16.8 14.3

(3.1 73.7) (0.002 79.4) (0.2 74.8) (0.5-40.0)

13 12 12 11

77 110 129 80

17.3 6.0 22.6 26.7

(5.4~48.1) (1.2-14.8) (4.2-74.2) (19.0-36.2)

II 11 It 11

84 66 98 22

3.0 9.7 2.8 14.0

(0.211.0) (0.0003-54.2) (0.006-8.2) (0.9 75.0)

I1 I1 11 11

113 166 123 151

4.5 33.3 15.2 25.9

(1.0 13.2) (0.004-90.9) (0,0004-25.1) (12.3--50.0)

10 9 10 7

80 88 59 50

2.5 9.0 14.9 33.0

(1.6-2.9) (1.3-17.7) (9.8 21.7) (19.4-43.6)

6 6 6 6

20 63 27 28

For water samples

10 5 × Direct counts (ml ~) 10 -4 x C F U s (ml -~) 104 x Activity (h L) % CCFUs

For artificial gravel substratum 10-7× Direct counts (g ~) 10 6 x C F U s (g i) 103 x Activity (g ~ h ~) !~ C C F U s

Values are means, ranges, number of samples and coefficients of variation (%)

"Fable 4. Results of multiple-regression analysis between bacterial variables and pollution-related variables; Sugden Beck, April 198 l-April 1982

Bacterial variable

n

R "~

F

P

26 25 25

0.35 0.52 0.34

4.0 7.6 3.6

0.02 0.001 0.03

0.23 0.48 0.59

1.7 5.0 8.0

022 0.0l 0.002

For water samples

Direct counts CFUs Activity

For artificial gravel substratum Direct counts CFUs Activity

21 20 21

The three pollution-related independent variables (pH, conductivity, total phenolic compounds and phenol derivatives) were forced into the equation. Values are number of samples (n), multiple coefficient of determination (R2), overall F ratio (F) and the significance of regression (P). Data from the upstream and downstream sites were combined.

coefficient of determination (R 2), which is the proportion of variation in the dependent variable which might be explained by variation within the set of independent variables, was 0.59 (for activity in gravel). Especially striking was the relationship between bacteria and pH in Sugden Beck. This may be demonstrated by comparison of results obtained when the stream was acid with those from nearneutral conditions (Table 5). Bacterial abundance (especially CFUs) and activity were much lower under acid conditions. The relationships between bacteria and phenolic compounds were further investigated by multiple regression analysis with the following as independent

variables; total chlorophenols, total, nitrophenols, total phenoxyalkanoic acids. A forward-entry stepwise procedure was used with P-to-enter set at 0.05. With directly-counted bacteria, in water and gravel, as dependent variables, no regression steps were performed (i.e. P > 0.05 for all independent variables). With CFUs and activity, in both water and gravel, one independent variable (total nitrophenols) entered the equation. Further analysis was performed with CFUs and activity, in water and gravel, as dependent variables and the 14 individual, identified, phenolic compounds as independent variables. In all four analyses, picric acid was the sole independent variable to enter the equation. Mean values of Yo CCFUs (Table 3), in water and gravel, were lower at the Sugden Beck sites than in unpolluted Stubbs Beck. The taxonomic study showed marked differences between the two streams; 95 out of 97 isolates from Sugden Beck were Gram-negative compared to 68 out of 90 from Stubbs Beck. The isolates from Sugden Beck were dominated by Pseudomonas (Table 6). Those from Stubbs Beck were much more varied, most frequent were Gram-negative genera (Pseudomonas, Flavobacterium, Aeromonas, and Acinetobacter) but a moderate number of Grampositive rod-shaped bacteria was also present. DISCUSSION

Bacteria in the water samples may have been transient whereas those in the artificial gravel sub-

Table 5. Abundance and activity of bacteria in Sugden Beck under acid and near-neutral conditions Acid conditions _

Mean

(Range)

10.2 0.2 0.6

1.9 0.9 1.9

Near-neutral conditions n

CV

Mean

(Range)

(2.4-32.3) (0.002-2.2) (0.2--1.2)

11 95 I1 305 11 62

28.8 28.4 16.4

(12.6-73.7) (2.3-79.4) (1.2 74.8)

(0.2--7.6) (0.0003-6.0) (0.0004-14.7)

8 130 7 258 8 280

4.9 30.8 12.9

(1.4 13.2) (3.3-90.9) (0.3-25.1)

n

CV

For water samples

10 -~ x Direct counts (ml ~) 10 -4 × C F U s (ml -I) 104×Activity (h -I)

15 61 14 85 14 122

For artificial gravel substratum

10 -7 × Direct counts (g-~) 10 ~ x C F U s (g- ~) 103 x Activity (g ~ h ~)

13 13 13

75 84 65

Under acid conditions mean pH (and range) = 3.7 (2.7-5.7), n = 11; under near-neutral conditions mean pH (and range) = 7.1 (6.3-7,6), n = 15. Values in the table are means, ranges, number of samples and coefficients of variation (%). Results from the upstream and downstream sites were combined.

Bacteria in a stream Table 6. Results of identificationof randomly-selectedbacterial isolates from the naturalsedimentof SugdenBeckand StubbsBeck, July-August 1982 Number of isolates Sugden Beck Identification (upstream site) StubbsBeck Pseudomonas 91 25 Flavobacteriura Aerornonas Acinetobacter

0

16

0

14

0

6

UnidentifiedGram-negativerods

4

3

Chromobacterium Escherichia Yersinia

0 0 0

1 1 1

0 2 0 0 97

1 17 2 3 90

UnidentifiedEnterobacteriaceae UnidentifiedGram-positiverods UnidentifiedGram-positivecocci Streptomyces group Total

stratum represented a more permanent bacterial flora which developed in response to relatively long-term conditions in the stream. The presence of substantial mean concentrations of directly-counted bacteria and CFUs, in both water and gravel, at both Sugden Beck sites, and mean activities at the downstream site similar to Stubbs Beck (Table 3), suggest that the polluted Sugden Beck was not, overall, a particularly hostile environment for bacteria. The extreme variability, and at times very low values, shown by CFUs and activity at the Sugden Beck sites suggest, however, that there were instances of severe inhibition of bacteria, presumably in response to pollution. Regression analysis (Table 4) confirmed the existence of relationships between bacteria and pollution-related variables (pH, conductivity, total phenolic compounds), but partitioning the overall R 2 between the independent variables was not feasible because of interrelationships between the independent variables (e.g. between pH and sum of phenolic compounds). Of these independent variables, however, it is probably unlikely that elevated conductivity inhibited bacteria through osmotic shock, since the highest values (Table 2) were no more than that of the 25% Ringer solution used as diluent, and incubation medium for gravel-activity determinations. The relationship between pH and bacteria was, however, very obvious (Table 5) and it is probable that low pH values (down to 2.7) adversely affected bacteria. The optimum pH range for most aquatic bacteria is about 6.5-8.5 (Rheinheimer, 1980) and Rao et al. (1984) observed a critical pH of 5.5, for lake bacteria, below which concentration of CFUs and actively-respiring bacteria decreased by about two orders of magnitude. The phenolic compounds may have been toxic of themselves, also their toxicity may have been enhanced by low pH (Zetterberg et al., 1977 found that 2,4-D toxicity to Salmonella typhimurium increased as dissociation decreased with reduction in pH). Further regression analysis implicated nitrophenols, particularly picric acid, in the inhibition of bacteria. Some phenolic compounds which were inhibitory may, however, have been excluded from the regression

89

equations because of interrelationships between concentrations of the different phenolic compounds. These results form a basis for ecologically-meaningful comparative toxicity testing of phenolic compounds against isolates of stream bacteria. The reduction in ~o CCFUs at the Sugden Beck sites, relative to Stubbs Beck (Table 3), points towards change in the composition of the bacterial community in response to pollution. Other studies have shown change in ~ CCFUs related to pollution (Guthrie et al., 1974; Cherry et al., 1977). The bacterial taxonomy (Table 6) showed a genuscomposition in unpolluted Stubbs Beck which was broadly similar to that found in other river waters (Baker and Farr, 1977; Bell et al., 1980; Nuttall, 1982) and in lake sediments (Inniss and Mayfield, 1979). In Sugden Beck, however, diversity had decreased. Pseudomonas dominated and it is interesting that Tabak et al. (1964) found that Pseudomonas predominated in mixed cultures, from a range of soils and aquatic sediments, which had been enriched with chlorophenols and nitrophenols. Pseudomonas was presumably partly tolerant of conditions in Sugden Beck and may have been capable of metabolizing the phenolic substances and phenol derivatives present in the stream. thank H. Fennell and Dr C. Urquhart for their support and advice. One of us (CRM) held a Science and Engineering Research Council CASE studentship which was in co-operation with the Yorkshire Water Authority. Acknowledgements--We

REFERENCES

Albright L. J. and Wentworth J. W. (1973) Use of the heterotrophic activity technique as a measure of eutrophication. Envir. Pollut. 5, 59-72. Aly O. M. and Faust S. D. (1964) Studies on the fate of 2,4-D and ester derivatives in natural surface waters. J. agric. Fd Chem. 12, 541-546. Baker J. H. and Farr I. S. (1977) Origins, characterization and dynamics of suspended bacteria in two chalk streams. Arch. Hydrobiol. 80, 308-326. Baker M. D., Mayfield C. I. and Inniss W. E. 0980) Degradation of chlorophenols in soil, sediment and water at low temperature. Water Res. 14, 1765-1771. Bell C. R., Holder-Franklin M. A. and Franklin M. 0980) Heterotrophic bacteria in two Canadian rivers--l. Seasonal variations in the predominant bacterial populations. Water Res. 14, 449-460. Buchanan R. E. and Gibbons N. E. (1974) Bergey's Manual o f Determinative Bacteriology, 8th edition. Williams & Wilkins, Baltimore. Buikema A. L., McGinniss M. J. and Cairns J. (1979) Phenolics in aquatic ecosystems: a selected review of recent literature. Mar. envir. Res. 2, 87-181. Carney J. F. and Colwell R. R. (1976) Heterotrophic utilization of glucose and glutamate in an estuary: effect of season and nutrient load..4ppl, envir. Microbiol. 31, 227-233. Cherry D. S., Guthrie R. K., Singleton F. L. and Harvey R. S. (1977) Recovery of aquatic bacterial populations in a stream after cessation of chemical pollution. Wat. Air Soil Pollut. 7, 95-101. Coleman R. N., Campbell J. N., Cook F. D. and Westlake

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