Ecological and laboratory studies on the role of luminous bacteria and their luminescence in coastal pollution surveillance

Marine Pollution Bulletin Uurme I'ol/utiou Bulletin, Volume 26. No. 4, pp. 191) 201. 1993. Printed in Great Britain.

1~o25 3 2 6 X / 9 3 S6,(Io+I).(}{~ © 1993 Pergamon Press [ ttl

Ecological and Laboratory Studies on the Role of Luminous Bacteria and their Luminescence in Coastal Pollution Surveillance N. R A M A I A H and I). C H A N D R A M O H A N National Institute o f Oceanography, Dona Paula, Goa 403 004, India

This study was aimed at finding out how bacterial bioluminescence, a trait very sensitive to toxicants, is affected in coastal environs which receive various types of effluents. For this, observations on occurrence and distribution of visibly luminous bacteria from both polluted and nonpolluted environments were documented during a 7 yr period. While luminous colonies were abundant, often contributing over 10% of the total colony-forming units in the pollutant free areas, none of over 200 water, sediment, fish, shellfish and plankton samples from the polluted localities yielded any visibly luminous colony. On analysing several bacterial strains originating from the latter environment, it was evident that there were many dark (nonvisibly luminous) strains of the usually luminescent Photobacterium leiognathL Laboratory studies on the effect of various chemicals on light emission by different luminous species suggested a strong depression (and/or irreversible loss) of luminescence in them. Results of this study suggest that in addition to several advantages known with these bacteria, they are useful as biomarkers in the assessment of environmental health.

Marine luminous bacteria have continued to interest microbial ecologists for more than one reason. They are ecologically versatile, utilize several nutrients and occupy many niches in the marine environment (Hastings & Nealson, 1981). Their bioluminescence, being extremely sensitive to the toxicants, has been employed in bioassays for detecting nano or picomolar concentrations of impurities in pharmaceuticals (Hastings, 1976) and in the food industry (Barak & Ulitzur, 1980; Ulitzur, 1986). Bioluminescent bacteria have also been employed in water quality testing (Bulich et al., 1981; Bulich, 1982; DeZwart & Sloof, 1983; Herschke, 1983; Slawinska & Slawinski, 1985). Many investigators have adopted Microtox+ bioassay technique that utilizes live luminous bacteria in the quick testing of ecotoxicity (McFerts et al., 1983; Ribo & Kaiser, 1983; Plotkin & Ram, 1984; Ribo et al., 1985). Application of luminous bacteria in ecotoxicity 190

testing was critically evaluated by Vasseuer et al. (1984). They suggested that a bacterial bioluminescence test is important in marine toxicity screening for its simplicity, high reproducibility, extreme sensitivity and rapidity. Although there are investigations employing Microtox® or other bacterial bioluminescence tests for detecting environmentally toxic agents (Schiewe et al., 1985; Buelow & Klein, 1987), from the available literature it appears that there are no attempts to enumerate these bacteria from coastal environments receiving industrial and domestic wastes. Such data are of practical importance for microbial ecologists to get a quick insight into the biological watermass characterization (Yetinson & Shilo, 1979; Ramaiah & Chandramohan, 1992). This study is a part of long term investigations on pollution microbiology, in which we monitored for luminescent bacteria and noticed a complete absence of luminous colonies in over 200 samples collected in the coastal regions receiving large amounts of industrial and/or domestic wastes. This paper reports results of a study of over 7 yr with the main objective of quantifying the abundance and detection of luminous bacterial species from the polluted and relatively pollution-free coastal environments. In view of the extreme sensitivity of their luminescence, this study also intended to compare the in vitro response of light emission by different species of luminous bacteria to toxic as well as nontoxic chemicals. This was done to see if the observed absence of visibly luminous bacterial colonies in all the samples from the polluted environments was because of the prolonged exposure to the toxicants. Materials and Methods

Water and sediment samples were collected from different locations (Fig. 1). Locations off Bombay', Madras, Haldia and Veraval are reported to be polluted (Anon., 1986, 1990; Zingde el al., 1989; Lodh, 1990). Off Bombay, sampling was carried out from a stretch of over 13(1 km in the coastal, estuarine and backwaters. These backwaters are essentially part of both coastal

Volume 2 6 / N u m b e r 4,'April 1993

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2 18°:35' - 19°00' N 72°45 ' -- 75°00 ' E 73°17 '

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4 16°05 ' -- 16°07 ' N

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A I 20°48 ~- 20°52 ' N 70°27 ' -- 70°29 ' E

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9 13°10~ -15°12 ' 80"18' -- 80°21 ' I0 21°45 ' -- 22°55' 88°05 ' - 88°10 '

N E N E

185"

-io"

5° 910 °

Fig. I Various sampling locations. Coordinates indicate area within which the samples were collected. Data from 2() different sampling sites off Bombay were included in this study. At the time of sampling for this study, there were no industries within distances of 2 5 - 3 0 km of the sampling sites off Sutrapada, Jaitapur, Karwar and Tutieorin.

and estuarine environs with salinities ranging from 936X 10-3 S (Lodh, 1990). Most parts of this stretch receive effluents of various types from the industrial estates, shipping and harbour activities. Coastal environs of Madras receive substantial loads of tannery and automobile wastes. Besides effluents from numerous industries, the River Hoogly also carries substantial amounts of domestic wastes, and salinities at sampling sites off Haldia range between 12 and 25 × 10 -~ during a tidal cycle (Anon., 1992). Sampling sites were spaced at distances of 4-5 km at all 10 locations shown. During most of these collections, seaward sites were at least 15 km from the shore. Several sessile, benthic, planktonic and nektonic samples collected from many of these locations were also examined for the presence of luminous bacteria. In most cases, Seawater Complete Agar (SWC) of Rheinheimer (1977) was used as the medium for enumeration of both viable and luminous heterotrophic bacteria. Glycerol (3% vol/wt) was added to this medium to facilitate the enumeration of luminous colonies (Nealson, 1978). For enumerating bacterial populations from the estuarine and backwater samples, this medium was prepared with half strength (50°/,,) aged seawater. Spread plating, pour plating and membrane filtration techniques were followed in these analyses. Many subsamples (6-10 replicates), particularly from off Bombay, Madras and Haldia were plated for confirming the absence of luminous colonies. All the plates were incubated aerobically at 26(_+3)°C for 2-3 days and checked periodically in a darkened room for luminous colonies.

After enumerating all the colony forming units, many isolations were made, particularly from samples off Bombay, mainly to check whether there were any strains of luminescent bacteria occurring in the polluted zones. Over 400 bacterial strains so collected were identified to their generic level by following standard microbiological methods (Oliver, 1982). This procedure assigns the strains to their genera unambiguously and many isolates assigned to Photobacterium were later identified to their species level along with many luminous ones, included for reference, by following the definitive identification scheme of Reichelt & Baumann (1973). Effects of different chemicals on the luminescence of Vibrio harvevi, V fischeri and Photobacterium leiognathi were examined as follows. Initial levels of light in these strains grown for 18-24 h at 26(+3)°C in the SWC broth were measured in a Turner 20e luminometer (Turner Designs, USA). Integration intervals and volumes of aliquots to be taken for measuring luminescence were standardized. Light output from the aliquots of 100 ~1 of broth cultures taken in the minivials was monitored after stabilizing these vials for a minute in the luminometer chamber. Known amounts of various chemical solutions were added separately to each vial to attain the final concentrations mentioned in Table 1 and the luminescence output was recorded with the integration duration of 20 s for 5 rain. All these analyses were done by taking replicates of 2-3 aliquots for each measurement. For reasons yet unknown, addition of arginine into the growing medium is reported to enhance bioluminescence in the dark 191

Marine Pollution Bulletin

variants (Hastings et al., 1987). We added arginine (0.2% wt/vol) to SWC to see if revival of bioluminescence was possible in some 80 strains which had turned dark following many transfers for subculturing, and added it also to a few nonvisibly luminous strains of t'hotobacterium isolated from the polluted waters. All these dark, strains formed a part of an extensive collection of over 1500 luminous isolates from the Arabian Sea (Ramaiah & Chandramohan, 1992). In addition, we examined whether this amino acid was TABLE 1

Various chemicals tested for their effect on the luminescence of different species of light emitting bacteria. ('heroical I)ecanol Ethyl alcohol Methyl alcohol Vormaldehyde Naphthalamine l)iaminonaphthalamine Alanine Arginim: Cysline Phen5 lalanine Prnline Tvrosinc (']flor'tmphenicol Nalidixic acid Penicillin Streptomycin Ammonium pyrrolidine dithiocarbomate ( A P I ) ( ) l'itriplex Bcnzenc hexachloride DI)T DDI) Lindanc PPDDE Calcium chloride Magnesium chloride Potassium chloride Sodium chloride Phenol Dichlorophenol l)ichlorophenoxy acetic acid Acetone Hexane Glucose lnositol Rihosc Al-scnous oxide Pot. hydroxide Sodium arsenite Sodium hydroxide 1)% qlydrochloride Cadmium chloride Cobaltus chloride Cupric sulphate l.ead acetate Mercuric chloride Mercurious nitrate 8il,,er nitrate Zinc chh)ride t')a rium chloride ("rude oil (aqueous extract 2-.24 h old) Aged seawater (salinity) Artificial seawater l)istilled water

Conc.* 50% 0.5% 0.5% 1.67% 25 3.16 5 5 5 5 5 5 5 1{I 5 5 3.28 7.45 1(I 0.5 5; 10 25 5 22.19 148.06 149.10 116.90 25 8 1I (}.(1058 50% 5 5 5 10 150 O. 1% 200 60.55 18.33:30.6 I: 45.83; 54.9(#; 61.04 38.75 20.71: 34.59:51.79; 62.13 32.53; 54.32: 81.32:97.58 27.15; 45.34; 67.88:81.4(~ 32.46; 54.22; 81.16; 97.40 16.98; 28.37; 42.47; 50.97 13.63; 22.75: 34.07; 40.88 14.14 50% 70 X 1()-~ S 711 X 10 -~ S

18 X 10 ~S

*Final concentration in the assay vial. All these concentrations unless mentioned are in ~tg (-=ppb). Many toxic chemicals were checked at various higher concentrations, which resulted in sharp falls in the luminescence. Therefore, such concentrations are not mentioned here. To note the concentrations of different chemicals mentioned in Figs 2 to 9, please refer to this table.

192

helpful in maintaining luminescence for prolonged periods during the assays, and also checked whether bacterial luminescence can detect the toxicity of trace metals in the presence of a nontoxic agent (e.g. aminoacids). For this, 15 additions of 5(1 gl arginine (10 [~g ml-~), one each at the end of every 30(1 s, to 50 gl broth culture of V harveyi in a luminometcr vial were made and the light levels monitored as described above. Influence of different trace metals such as cadmium, copper, mercury, nickel and lead at various ion concentrations on the growth of 1~ hart'o,L I/.. fischerL I'. leiognathi and F. phost)horettm was checked by monitoring the optical density at 6(10 nm in a DU-6 spectrophotometer (Beckman, USA). Salts of these heavy metals were added to a complex liquid medium of Baumann & Baumann (1981) with a composition (g l-l): 7?is-HCI (pH 7.5), 6.055; NHaCI, 1.02: K=HPO 4, 0.058; FeSO a, 0.0(128: NaCI, 11.69: MgSOa, 12.33: KCI, 0.65: CaCI~, 1.46: yeast extract, 5.0 and tryptone, 5.0 to get 0.1, 1.0, 2.5, 5.0, 10 and 25 gg metal ion ml -~ of this medium. All the tubes were inoculated uniformly with 18-24 h old cultures, incubated for 24 h and their growth monitored by measuring the optical density. Just prior to checking thc growth, luminescence in all these tubes was examined in the luminometer.

Results In Table 2, ranges of viable bacterial numbers with their geometric means and the percentages of luminous bacteria in water and sediment samples positive for luminous colonies are presented. Although the total viable and luminous bacterial numbers for each individual sample are available, in order to emphasize the presence of luminous colonies in different samples, only the ranges of their percentages (with their geometric means) are presented here. Luminescent colonies were absent around Bombay, off' Madras and in the Hoogly estuary (off Haldia). Luminescent colonies were abundant at most other coastal places (often accounting to 15% or more of the total viable counts) excepting a few sampling sites (closer to waste discharge points) off Sutrapada and Mangalore, where domestic or industrial effluents seem to exert stress. V. harveyi, V fischeri and P. leiognathi were the luminous species recorded from these collections. These numbers and species composition of luminescent bacteria are similar to those found in the pollution free coastal areas (Yetinson & Shilo, 1978: Ramaiah & Chandramohan, 1992). From the Bombay Harbour 'Fhana creek and Bassein area, 16 animal species were examined for the luminous bacteria. While the gut contents of all species of fish caught off Bassein (an area north of Bombay, relatively free of pollution) were positive for luminous bacteria, none of the fish or other benthic organisms collected from the Thana creek was positive for these bacterial colonies (Table 3). In contrast, all the nektonic, benthic, planktonic and sessile organisms examined from the coastal environs of Goa were positive for luminous colonies which contributed quite substantially to the total colony forming units of viable bacteria.

Volume 26/Number 4/April 1993 TABLE 2 Quantitative abtmdances of viable bacteria and the percent of luminous bacteria in water (W), expressed as No. ml -~ and sediment (S), No. g dry wt. (Multiply numbers from sediment samples by 10~.) Figures in parentheses are the number of samples positive for luminous colonies. Location/ Sampling month

No. of samples

Sutrapada, near Vera~al August 1984 W S November 1984 W S Bombay Augusl 1984

'~ S W S W S W S

Range

18 9 18 9

(8) (2) 112) (fl) ((I) (0) (0) (11) (0) (0) (0) (0) (0) (0) (0) (0)

Viable bacteria Geometric mean

Luminous bacteria* % Range % Geometric mean

1100.00-20 000.00 2.80-230.00 94.00-2720.00 8.80-19.40

3435.41 50.89 788.33 9.56

1.22-2.80 1.48-2.41 8.18-10.69 3.17-5.88

208.00 760.00 4.98-17.54 620.00-750.0(I 20.21 38.88 15.00-880.00 3.41-80.30 90.00 5600.00 13.51)-285.10 266.00-852.00 20.75 56.58 110.00-8400.00 3.36-582.62

436.38 8.79 682.17 28.99 345.13 33.82 846.57 83.82 453,53 30.37 14116.89 111.28

0 0 I) 0 11 0 (I 0 0 0 0 0

2.16 1.88 9.3~'~ 4.3t~

I)ecembcr 1986

~' S

March 1987

~' S

6 3 ~ 3 211 11 10 (~ 20 I1 10 6

aaitapur, near Ratnagiri December 19911 W S

11 4

(1 1) (3)

21,33-160./10 4,96-17.72

102.53 9.42

6.59-17.24 5.45-8.48

8.61 6.67

W S W S

4 2 9 3

(4) (2) (9) (3)

118,00 262.00 5,76-9.87 166,50-652.511 7,18 18.13

168.66 7.23 296.33 12.36

4.90-13.35 4.43-7.56 5.68-9.40 2.62-2.96

7.88 5.79 7.08 2.77

W S W S W S W S

28 14 18 9 18 9 I 4

(28) (12) (18) (7) (18) (8) (11) (4)

203,00-945.50 18,53-38.69 336,33-936.67 11,62 79.89 161,00-404.011 32,31-82.36 120.00-721.011 153,46 174.72

484.89 26.24 543.64 29.39 298.43 43.11 432.67 161.84

9.85-19.80 2.8/)-7.6/) 11.29-21.36 2.43-5.64 12.66-28.33 2.76-6.98 12.33-22.67 4.99-7.80

13.S6 4.67 16.63 4.26 19.18 4.39 16.76 4.59

W S W S

4 2 1 4

(4) (2) (11) (3)

118.00-262.110 57.60 98.70 144.66 1834.33 317.77 1386.88

210.07 72.33 642.12 552.86

4.90-13.35 3.44-6.57 6.42-8.87 1.73-2.38

8.78 4.47 7.74 2.08

W 8 W S W S

8 9 4 6 1I 4

(14) (5) (101 (3) (8) (2)

138.00-824.110 23.33-83.11 235.00-760.00 4.30-83.40 272.50-592.00 58.88 224.42

316.88 38.811 464.33 39.36 481.28 128,42

2.46-6.95 1.91-4.N2 2.86-5.46 1.48-3.46 2.26-3.94 1.71-2.29

5.06 2.97 4.72 2.11 3.52 1.98

W S W S

18 9 8 9

(18) (9) (18) (8)

112.0(/ 931.50 11.33 49.118 61.33-757.33 19.02-340.02

428.43 29.08 289.33 2119.88

3.19-9.14 1.56-2.92 4.67-8.46 2.82-3.16

8.06 2.52 7.63 1.89

W S W S W 8

8 9 2 9 22 6

(18) (9) (12) (9) (22) (6)

I 10.50 700.50 10.53-73.88 115.50-228.50 18.31 - 132./) 1 213.50 810.00 26.68 69.33

406.54 37.38 166.77 49.35 496.35 46.93

4.36-17.42 2.94-4.22 6.81-18.82 2.62-2.91 7.60-12.94 1.38-4.31

12.33 3.69 14.91 2.75 10.91 3.04

W 8

16 9

(0) (0)

558.50-3601L011 70.03-1240.12

1I124.67 32.44

0 0

W S W S

22 9 18 9

(11) (0) (0) (0)

37 500.00 87 400.00 642.67-1876.31 41 200.110-77 100.00 728.26-1179.62

46 762.68 1008.77 59 718.21 992.63

I) I) I) 0

November 1984 May 1986 September 1986

Malvan May 1988 December 199[} Goa September 1985 October 1c~86 March 1988 May 1988 December 199(I Kar,~ar .lune 1988 January 1991 Mangalnr March 1985 December 1987 January 1991 Totieorin May 1991 October 1991 Nagapattinam October 1990 February 1991 August 1991 Madras February 1991 Haldia June 1988 April 1991

*In the samples positive for luminescing colonies. 193

Marine Pollution Bulletin TIME ( S e c )

I00

o

ALCOHOLS

ALDEHYDE AMINES ANTIBIOTICS

DECANOL ETHANOL METHANOL FORMALIN ef.-NAPHTHALAMINE

200

I

I

lm =,

HEAVY METAL SALTS

Vib rio hcrveyi

PESTICIDES

PHENOLS

MISCELLANEOUS

ARSENOUS OXIDE SODIUM ARSENITE ACETONE KOH NoOH TRIS HE XANE LEAD ACETATE NALIDIXIC ACID DETERGENT

m

194

1

I00

200

I

Photoboeterium leio.~.gnath i

1 l l

l 1 1 1 l

m m m m

m

m

i

m iN

l I m

m l l

m Ill m

I I l m m m i N m m

II l l m

m

Thana Creek

Locations Bassein Bay

Off Goa

------NS NS

+ (8.78) +(8.33) + (6.47) + (9.34) +(9.01) + (3.71 ) + (2.61) NS NS NS

+ (9.16) + (7.17) NS + (8.76) +(11.81) NS NS NS + (12.99) + (8.45)

-NS

+ (4.86) + (2.88) + (4.68) NS

+ (12.23) + (9,36) NS + (21.24)

NS NS NS

NS NS NS NS NS

NS + (18.72) NS + ( 11.25) + (6.68)

---

+ (3.85) NS

NS NS

-

+ (4.89)

+ (9.63)

Zooplankton

Whole sample

V. fischeri

m

Polychaete

Tellina sp. ( "apitella sp.

m m

mm

m m m m

Molluscs

Modiolus sp. Perna viridis Pholas sp. ('n~ssostrea sp. l)onax sp.

:500

OR LESS

I

m mm

Crustaceans

Mempenaeus sp. Scvllaserram Squilla sp. Penaeus sp.

WAS 5 0 %

Ill

Fish

Atmdontosmma sp. Ariussp. Cynoglossus sp. Johnius sp. Mugilsp. Coilia dussumieri Harpodon nehereus Saurida Sardinella sp. Nemipterusfllponicus

LEVEL 200

l

TABLE 3 Different fish and other organisms collected and examined from different locations for the presence (+) or absence (-) of luminous colonies; NS, not sampled. Figures in the parentheses are percent of luminous bacteria of the total colony forming units in the gut contents of fishes or the whole animal homogenates of other organisms.

Organism

moo

l

STREPTOMYCIN APDC m TRITRIPLEX-'m" m

Cu s o 4 HgCI 2 HgNO 3 ZnCl 2 CdQ2 AgNO3 DDT PPDDE DDD ( 2 0 p p b ) DDD ( l O p p b ) PHENOL DCP DCPAA

,500

LIGHT

u

m m D IAMINONA~THALAMINE 1 CHLORAMPHENIC~ 1 PENICILLIN

CHELATORS

A T WHICH

Fig. 2 Reduction of luminescence to 50% or less by various chemicals.

Concentrations of chemicals other than trace metal salts are as given in Table 1. Final concentrations of these trace metal salts in the assay vials were 51.79 gg CuSO4; 67.88 ttg HgCI_~;81.16 ~g HgNO~; 34.(17 gg ZnCI;; 45.83 ttg; 45.83 CdCI_< 42.47 ug AgNO~ and 81.32 ~tg of lead acetate. Concentration of a commercial detergent was 250 p.g. A l t o g e t h e r , 53 c h e m i c a l s w e r e t e s t e d f o r t h e i r effect o n the light o u t p u t by d i f f e r e n t s p e c i e s o f l u m i n o u s b a c t e r i a . A s m a n y as 21 o f t h e m r e d u c e d t h e light to b e l o w 5 0 % in V. harveyi w i t h i n 2 0 s (Fig. 2). A l c o h o l s , formaldehyde, naphthalamine, diaminonaphthalamine, c h l o r a m p h e n i c o l , s t r e p t o m y c i n , c h e l a t o r s , all h e a v y m e t a l salts ( e x c e p t zinc a n d c a d m i u m ) , a c e t o n e , p o t a s s i u m h y d r o x i d e , s o d i u m h y d r o x i d e , p h e n o l , dichlorophenol (DCP), dichlorophenolacetic acid ( D C P A A ) a n d Tris-hydrochloride r e d u c e d the light to b e l o w 5 0 % in t h e first 2 0 s. Z n C I 2, CdC12 a n d P P D D E d e c r e a s e d the l u m i n e s c e n c e to b e l o w 5 0 % in 4 0 s. H e x a n e r e d u c e d it to 4 8 . 3 8 % in 180 s. H o w e v e r , 2 0 0 o r m o r e s e c o n d s w e r e r e q u i r e d by d e c a n o l , penicillin, s t r e p t o m y c i n a n d D D D (10 a n d 2 0 p p b ) to r e d u c e the light to b e l o w 5 0 % level. All the c h e m i c a l s t e s t e d f o r V. harveyi w e r e n o t i n c l u d e d f o r V. fischeri o r P. leiognathi. B u t t h o s e c h e m i c a l s t e s t e d a g a i n s t all t h r e e s p e c i e s s h o w e d a s i m i l a r t r e n d in t h e r e d u c t i o n o f light levels. O n l y s t r e p t o m y c i n did n o t b r i n g d o w n the light to b e l o w 5 0 % w i t h i n 5 m i n in b o t h o f t h e s e species. G e n e r a l l y , all h e a v y m e t a l s d e c r e a s e d t h e light to b e l o w 1 0 % w i t h i n t h e first 4 0 - 6 0 s. C o m p a r e d to o t h e r c h e m i c a l s t h e y are m o r e toxic e v e n at v e r y l o w c o n c e n t r a t i o n s . It is a l s o c l e a r that t h e r e d u c t i o n o f light by

Volume 2 6 / N u m b e r 4/April 1993

Ag

Zn CI 2

NO 3

I00

50

Hg NO 3

Hg CI2

o--o All strains H V. harveyi

I00

V. fischeri _P. leiognothi LLI

u

50

Z L~ CO i,i

z_

o Lead acetate

Cu S04

_J

[DO

50

L____.__ Co CI 2 I00

50

0

I00

200

300 0 T IM E (Sec.)

100

200

3(30

Fig. 3 Pattern of light reduction among different luminous species due to the effect of heavy metals. Tested concentrations of these sails are 51.79 gg CuSO4; 38.75 ~tg COCI2; 67.88 gg HgCI2; 81.16 ~tg HgNO3; 34.07 ~tg ZnCI2; 45.83 ~g; 45.83 CdCI2; 42.47 ~tg AgNO 3 and 81.32 gg of lead acetate. Similar or same reduction pattern of light in these species by man~ metals, are included as 'all strains'.

the heavy metal salts was similar in all the luminous bacterial species (Fig. 3). HgC12, HgNO 3, AgNO 3 and lead acetate had the same effect on all three species. But, CoCL increased the light output in V. harveyi during the first 140 s. After this, a sharp decline to about 12.5% was seen at the end of 5 min. Even the lowest concentration of all the six heavy metals (tested to see whether there are any variation in the rate of reduction of luminescence in 1/. harveyi), decreased the light to less than 10% within 300 s (Fig. 4) and it dropped to 50% levels within the first 40 s, except for CdC12 which enhanced it in the first 60 s and, reduced sharply to 10% or less at the end of the experimental period (Fig. 5). Amino acids (Fig. 6), sugars (Fig. 7) and salts of essential inorganic ions (Fig. 8) increased the lumin-

escence by over 1-3 fold in all three species and this increase was rapid in the first 150 s. After this, a steady state or gradual decline of light was seen for many amino acids and sugars. Cystine showed a slightly different effect on the luminescence of V fischeri and V. harveyi. In the former species the light output went up by over 300% and in the latter it fell drastically to about 32% but recovered to over 100% within 200 s. Very low concentrations of DDD (2 ppb), and ethyl alcohol (0.1%) had an enhancing effect. As can be seen from Fig. 9, water soluble fractions of the crude oil (extracted for 2 h or 24 h) had slightly stimulated the light. Addition of aged or sterile seawater as well as sterile distilled water had neither enhancing nor deleterious effect. By adding arginine it was possible both to increase 195

Marine Pollution Bulletin 1o0 Lead

Ag

acetate

NO3

2o

15

I.~ ~.) Z W (..) (") W Z

I0

J

0

z

I00

•

= 3 2 ' 5 3 J.lg

o

= 54'32

,,

A

= 81" 3 2

,,

nM

• = 16"9@ Jag

EQUIVALENT

CONCENTRATIONS

0 = 2@'37 " -

-

IOOnM

o---o 1 6 7 n M 250nM o---.o 3 0 0 n M

5

y.

Cu SO4 t,d

• l=

n-' n

Hg CI 2

15

Hg N 0 3

20"71~.g

•

=27"15hg

O = 34" 59 "

0

=45'34

A = 51" 7 9 "

A =67.88

,

0 =54'22

O =62"

0

"

A

:

•

14 ,,

=81"46

IO

I00

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and maintain the light at 100% or above for over 75 rain. Interestingly, during this ensuing period it was not adversely affected though the repeated additions of arginine had decreased the salinity to as low as 2.18 ppt. Addition of 50 gl each of 10 ~tM NaC1, CaC12 and MgSO 4 also enhanced and maintained higher levels (109.66%) of light. However, addition of 50 gl of 100 nM (=27.15 ~tg) solution of HgCI 2 to the same vial 196

drastically r e d u c e d the light to less than 4 0 % within 40 s. B i o l u m i n e s c e n c e was revived in m a n y strains ( 3 0 % of 24 m o n t h s old; 8 5 % o f 12 m o n t h old a n d 9 4 % of 6 m o n t h old from their initial isolation) when arginine was a d d e d to the S W C broth. But. the isolates identified as P h o t o b a c t e r i u m sp. from the p o l l u t e d areas, and all the strains which were grown in the m e d i u m c o n t a i n i n g

Volume 26,. Number 4/April 1993

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Discussion Rapid industrial developments result in the disposal

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Marine Pollution Bulletin

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fore, pertinent and essential on a continuous basis for ecological surveillance. On a global scale, much of the research in estuarine and coastal biology has currently been on the description and observations of biota, biotope and biome (Watermann & Kranz, 1992). 198

Total absence of luminous colonies in all the water and sediment samples collected from the nearshore, creek or estuarine environments of Bombay, Madras and Haldia reflects higher levels of toxicants. Many studies in Bombay harbour-Thana creek (Zingde & Desai, 1981; Patel et al., 1985; Zingde et al., 1989) have indicated higher concentrations of heavy metals, sometimes 2-3 orders of magnitude higher than those observed in the coastal or offshore waters (Sengupta et al., 1978: Ouseph, 1992). Several biological studies have also noted the deterioration and nonsuitability of these environments for many planktonic organisms (Lodh, 1990; Nair et al., 1991; 1992). Analysis of over 200 samples collected from the regions reported to be under pollution stress, i.e. off Bombay, Madras and Haldia (Zingde et al., 1989; Anon., 1986, 1990, 1992) has confirmed the sensitivity of luminescent bacteria to the toxicants. Currently, many researchers employ phyto and zooplankton species in the pollution assessment studies (Krishnakumari & Nair, 1992). Owing to the adaptive responses of many of these organisms, it is often difficult to pinpoint the pollution stress (Jensen et al., 1974; Simkiss & Mason, 1984). It is clear from this study that luminescent bacteria loose their ability to emit light in the presence of a toxicant even at very low concentrations. Monitoring for bioluminescent bacteria can curtail the laborious biological studies for assessing the status of environmental health. Five to six replicates of a sample analysed either by the spread plating or membrane filtration method certainly confirm their presence or absence (Orndorff & Colwell, 1980). Since the presence of luminescent cokmies in the coastal samples ascertains the environment to be healthy. combining the ecological approach of this study with that of detection of both visibly and nonvisibly luminous bacterial colonies by the gone probe techniques as was very recently done by Lee & Ruby

Volume 2 6 / N u m b c r 4,'April 1993

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(1992), would greatly reduce the dependancy on many laborious, time consuming yet uncertain methods. It was possible through the biochemical characterization of several bacterial strains in this study to detect the occurrence of nonvisibily luminescent strains (dark variants) of P. leiognathi in these collections. However, such elaborate procedures can be circumvented, and not always needed in the environmental assessment programmes, if one looks for light emitting colonies. Although there are other luminous species, mainly in the genus Vibrio which contains numerous nonluminous species, looking for Photobacterium is preferable since it has just two species, and P. leiognathi is easy to characterize. Experiments with arginine, besides signifying its use in prolonged maintenance of light output, highlighted as well the usefulness of bacterial luminescence for detecting any toxic substance even at picomolar concentrations. Failure of this amino acid to revive luminescence in the dark variants of P. leiognathi isolated from the polluted regions as well as all other strains exposed to heavy metal ions, signifies that luminescence would be irreversibly lost when these bacteria are under constant exposure to the toxicants. No study seems to have examined this aspect of heavy metals (and many other toxicants) on the loss of bioluminescence. From this study, it is highlighted that this system is lost irreversibly in these bacteria grown in the presence of these ions and those occurring in polluted waters. It is well known that bacterial bioluminescence is very sensitive to toxic materials and several pure

compounds examined in this study have already been tested by various earlier studies (Bulich et al., 1981; Schieve et al., 1985; Buelow & Klein, 1987). This study however, has compared the sensitivity of luminescence in different species, and highlighted that any one species of these bioluminescent bacteria can be employed in laboratory assays. There are many advantages in employing these mesophilic species in tropical countries. It is easier to isolate, maintain and handle these tropical species than the psychotrophic P. phosphoreurn (the species currently in use for Microtox ® assays). Even the sublethal levels of varieties of waste materials reaching the marine environment might affect bioluminescence by acting synergistically or antagonistically. It appears that such sublethal effects on marine biotopes can be sensed or evaluated by looking for bioluminescent colonies. This may not be possible by including higher planktonic organisms for bioassays in view of their adaptive responses. Bacteria are known to concentrate and excrete varieties of metal ions (Doelle, 1975). There are several studies on the effect of heavy metals on the growth of different species of bacteria (Kushner, 1978). But there are no such studies on bioluminescent bacteria (Hastings et al., 1985). Results of this study indicated that 2.5 to 5 mg 1-~ can drastically reduce their growth. Concentrations as low as 0.1 mg 1-~ of Hg, Cu, Cd or 2.5 mg 1-~ of Ni and Pb were found to completely inhibit luminescence. Since subsequent transfers of many loopfuls of bacterial cultures from the tubes containing these metal ions at these concentrations did 199

Marine Pollution Bulletin

not yield luminescence, it is probable that it was lost irreversibly. Ecological studies on bioluminescent bacteria are thus useful in a quick and sure way of pollution assessmerit in coastal v,aters. Now that the gene probe techniques are increasingly used in microbial ecology, their right application is further helpful. Concentrations of many toxicants tested in this study are around the maximum allowable concentrations as per the guidelines of the USEPA (1980) and U N E P 119861. Nonetheless, bacterial luminescence is far more sensitive to most toxic agents (e.g. pesticides, phenolic compounds, trace metals, A P D C and titriplex) examined in this study when compared to zooplankton or other animal assays t U N E R 1990 and ref. therein). Although we had not aimed at arriving at the guidelines for water quality classification, we are of the opinion that there is a need for reassessing the current maximum acceptable toxicant concentrations (MATC) through bacterial bioluminescence assays. In any case, all these observations suggest the strong possibility of employing ecological data on bioluminescent bacteria in coastal surveillance programmes. We thank l)r. A. H. F'arulekar, Head, Biological Oceanography Division and l)r. B. N. l)esai, Director, for encouragements and facilities. Rcferees" suggestions were helpful in re,:ising this manuscript.

Anon. (1986). Lnvironmental Quality' and Dispersion Studies off Unto rib,rib(G;. Tec. Rcp. National Institute of Oceanography. Goa. Anon. ( 19901. Em'ironmenlu[ SurvQ',[or Murine ['~luent Disposal fi'om Reverse Osnlo,~is Plunt of Madrus Refineries. Tec. Rep. National Institute of Oceanography, Goa. Anon. (19921. Environntental hnpact Assessmetu attd Management I'lun with Regurd to l)redging Operutions owr Yiggerkhali Hut in the RiEvr qf'ttoog@ Tech, Rep. National Institute of Oceanography, Goa. Barak. M. & Ulitzur, S. (1980). Bacterial biotuminescence as an early indication of marine fish spoilage, k, to: J. AppL Microhiol. Biolechnol. 10, 155-165, Baumann. P. & Baumann. L. 11981). Marine gram negative eubacteria: genera Photobaeterinnr Beneckea, Alteromonas, Pseudon~onas and Alealigens. In 7"he Prokutyotes (M. P. Starr. H. Stolp, H. G. Truper, A. Bak)ws & H. G. Schlegel, eds), pp. 13112-1331. Springer-Verlag 1no., New York. Buclov< W. & Klein, B. ( It~871. Preliminary investigations on the utility of biolumincscent bacteria for ecotoxicological evaluation of marine and limnic sediments. [n Etnironmental t'recuution, pp. 339-355. Nieder Umwelt. Hanno~er. Bulich. A. A. (I982). Practical and reliable method for monitoring toxicity of aquatic samples. Process Bioehenr 17, 45-47. Bulich. A. A., Greene, M, W. & lsenberg, D. L. (1981). Reliability of the bacterial luminescence assay for determination of the toxicity ol pure compounds and complex effluents. ASTM Spee. 7t'dr PuhL 737. 338 356. l)eZ'aart, D. & Sloof. W. (1983). The microtox as an alternative assa} in the acute toxicity assessment of water pollutants. AquuL 7izvicoL 4. 12t)-138. Docile. H. W. (1975). Bacteriul Metaholism. 2nd edn. Academic Press, Ne,a York. 1 lastings, J. W. ( 19761. Biohnninescence. Oeeumrs 19, 17 27. Hastings, J. W., Makemson, .I. & Dunlap, P. V. (1987). How are growth and luminescence regulated independently in light organ symbionts? ,Slvmbio.sis 4, 3 24. Hastings, J. W. & Nealson, K. H. (19811. q'he svnflfiotic luminous bacteria. In "lhe 15wearvoles" (M. P. Slam H. Stolp, H. G. Truper, A. Balows & It. G. Schlegel, eds), pp. 1332 1345. Springer-Vcrlag Inc.. New York. Hastings, J. W., Potrikus, ( . J., Gupta, S. C., Kurfust, M. & Makemson. ,1. (19851. Biochemistry and physiology of bioluminescent bacteria. Adv. Microbial. physiol. 26. 236-291. l lerschke, B. (1983). Use of marine bacteria to determine the toxicily of products of effluents. I:'au. Ind. Nuisance,s 75, 68-72.

200

,Icnscn, A., Rystad, t3. & Melson, S. (1974). Heavv metal tolerance of marine phytophmkton..I, eap. mar. Biol. k, eoL! 5, 145 - 157. Krishnakumari, L. & Nair, V. R. (1992). Uptake and hioaccumulation of copper and zinc by the mollusc ,~'uecoslreu eHcullulu (Born) and ('erithium rubto (Desh) from the coastal waters of Bombay. Ind..I. Mar Sci. 2 1 . 7 4 - 7 6 . Kushner, D. ,I. (ed.) (1978). 31i(rohiul li[~" in exlreme em'ironmenlv Academic Press, London. Lee, K. H. & Ruby, E. O. ( 19921. l)etection of the light organ symbionl, l')'hrio fiLSCheri, in Hawaiian seawater by using luxgenc probes. Api d. era: 3licrobiol. 5 8 , 9 4 2 947, t.odh, N. M. (1990). Ecologicul ,stttdie,s on plattkton /)om neur shore waters of Bomhay. Ph.D. Thesis, LIniv. Bombay. McFerls, G. A.. Bond, P. J., Olson, S. B. & "Ichan. Y. T. 119831. A comparison of microbial assays lor the detection of aquatic toxicants. Water Res. 17, 1757-1762, Nair, V. R., (iajbhiye, S. N. & l)esai, B. N. 11991 ). Effect of pollution on the distribution of chaetognaths in Ihc nearshore v;atcrs of Bombay. hM. ,I. Mar 5ci. 20, 43 48. Nair, V. R., Nagasawa, S., Lodh, N. M. & Ncmoto, T. (19921. Unusual thickening of collcrette m Sugitm bedoti from the creek environments of Bombay. ImL J. Mur ,Sci. (in press). Ncalson, K. H. 11978). Isolation, identification and manipulation of luminous bacteria. In ,'llethod~ m En',.ymology, Vol. 57 (S. P. ('olov, ick & N. O. Kaphm, eds), pp. 153-165. Academic Press, New York. Oliver. J, I). (1992). Taxonomic scheme for the idcntificatioi1 of marinc bacteria. Deepsea Res. 2 8 , 7 9 5 798. Orndorf, S. A. & Colwell, R. R. 1198(t). Distribution and identification of luminous bacteria from the Sargasso Sea. AppL env Mierohiol. 39,983-987. Ouseph, P. P. (19921. Dissolved and particulate trace metals in the Cochin estuary. Mar. Poller. BulL 24, 186-192. Patel, B., Bhargava, V. S., Patti, S. & Balani, M. ('. (1985). Hcaxv mctals in the Bombay harbour area. liar I'olhtt. Bull. 16, 22-28. Plotkin, S. & Ram, N. M. (1q84). Muhiplc bioassays to assess the toxicity of a sanitary landfill Ic'achalc, ,lretr l:m'iron. ¢'omam. li,xicoL 13, lu7 206. Ramaiah, N. & Chandramohan, 11. t1992). Ecolog', and biology of luminous bacteria in the Arabian Sca. In OeeuEtographv of the Indian (h'ean (B. N. Desai. cd.), pp. I I 23. Oxford & [BH, New l)¢lhi. Reichclt, J. 1,. & Baunmml, P. 11973). I'axonomy of the marine, Inminous bacteria. Areqr ,llilo'ohiol. 94,283-3311. Rheinheimer, G, ted.) (1977)..Uicrohiul ecology of u hrackish wuter em'iromnem. Springe>Verlag, Heidelberg. P,ibo, J. M. & K a i s c r . K. L. E. (1~,J83). ('orrelation of acute and sublethal effects of selected chemicals to rainbow trot.t (,Sahno gairdneri) with acute toxic effects on I~hotohacleritmr l¥oc. 26It* ('re(f2 Greutluke Re~. 7 15. R i b o . . I . M . , Zaruk, B. M., tiuntcl. It. & Kaiser, K. L. E. (1985). Microtox toxicity test rcsuhs fi~r ~atcr samples form the Detroit River..I. (heut&ke Res. 11,297- 3114, Schiewe, M. H., Hawk. E. G.. Actor, D, 1. & Krahn. M. M. 119851. Use of a bacterial bioluminescencc assay to assess toxicity of contaminated marine sediments. ('un. J. kZqr Aquat. Sci. 42, 1244-1248. Sengupta, R., Singbal, S. Y. S. & Sanzgiri, S. (1978). Atomic absorption analysis of a few trace metals in Arabian Sea. Ind. ,I. Mar. &'i. 7, 295 299. Simkiss, K. & Mason, A. Z. (1984). Cellular responses of molluscan tissues to environmental metals. In Responses o(marine organis'ms to pollutants' (,I. ,I. Steigman & (i. W. Hcalth, cds). pp. 103 l lg. Elscvier, London. Slav, inska, 1). & Sla;~inski, J. (I 985). Applications o f bioluminesccnce and Iov, level luminescence lrom biological obiects. In ('hemi- uml I~iohmmmsceme (J. G. Burr, cd.), p p 533- 50U. Marccl l)ekker, New York. Ulitzur, S. (1986). Bioluminesccncc test for gcnotoxic agents. In Uetlzod.s in l:ncvmologv Vol. 13.t (S. P. ('olov, ick & N. O, Kaplan, cdsj, pp. 264 274. Academic Press, Nee 5ork. UNEP (1986). (;I']SAMP: En,Aummcntal capacity: an approach to marine pollution prevention, pp. 22 46. tlNf!P Regional Seas Reports and Studies, No. 80. UNEP (1990 I. (iESAMP: The state ol marinc enx,iromnent, pp. I26 213. UNEP Rcgilmal Seas Rcporls and Sttldics, No. I15. USEPA (19g0). l(n'dronmental Protection Agency water quality document, pp. 79318-79371). Federal Register, part 5. Vasseucr, P. ,I., Fcrard, F.. Rasl, ('. & Larbaigt. G. (1984). Lummcscent marine bacteria it] acute loxicit} Icsting. In I:cozmi<'ul wsliHg lor the environment. Vol. 2 ((i. Persoonc, F. ,laspcrs & ('. ('laus, cds), pp. 281 396, Slatc k:nivcrsitv Ghent. Bclgiunl. Watcrmaun, B. & Kranz, H. (IC192). Pollution and fish diseases in the North Sea: some historical aspects, llu*: I'ollut. Ihdl. 24, 131-138.

Volume 2 6 / N u m b c r 4/April 19~)3 Yetinson, Y. & Shilo, M. (1979). Seasonal and geographic distribution of luminous bacteria in the eastern Mediterranean Sea and the Gulf of Elat. Appl. era: Microhiol. 37, 1231) 1238. Zingde, M. D.. Bhonsle, N. B., Narvekar, P. V. & Desai, B. N. (1989).

Hydrography and water quality of Bombay harbour, l'.m'iron. Strut. Biosci. 14, 37-58. Zingde, M. D. & Desai (1981). Mercury in Thana creek and Bombay Harbour. Mar Pollut. Bull. 12,237-241.

IHI2> 326X 93 S¢~.0{)+(b.00 'cj 1t)¢~ Pergamon Press l l d

Marlin' Pollution Bulletin. V,~lumc 21~,Nt~ 4, pp. 20] 20~J. 1993, Printed ill (}real t~ril;lin

Geographical Distribution of Chlorinated Biphenyls (CBs) and Polycyelic Aromatic Hydrocarbons (PAHs) in Surface Sediments from the Humber Plume, North Sea HANS J. C. K L A M E R and L I S B E T H F O M S G A A R D Tidal Waters Division, Rijkswaterstaat, Ministry of Transport and Public" Works, P.O. Boa 20Z 9750 A E Haren, The Netherhmds E-mail address: klamer'~dgw.rws.nl

Concentration distributions of chlorinated biphenyls (CBs) and polycyclic aromatic hydrocarbons (PAHs) were determined in the < 6 3 pm grain size fraction of surface sediments of the Humber plume, North Sea. Concentration ranges of XCB and 12PAH were 2 . 9 2 19.07 pg kg -l and 0 . 7 0 - 2 . 7 0 mg kg -t, respectively. The general distribution pattern of CBs and PAHs followed the general path of the Humber plume, with little influence from the Wash. The pattern of lower chlorinated CBs suggested a possible source of these compounds north of the region studied; PAH patterns and PAH-compound ratios demonstrated the influence of urban pollution on offshore sediments. On average, Humber plume sediments had lower CB concentrations than sediments in other areas of the North Sea, while PAH levels were similar or, for some compounds, higher. Concentrations of CBs and PAHs in the Humber mouth, however, showed increased levels.

It has been well established, that organic micropollutants (e.g. CBs, PAHs) have pronounced adverse effects on the health of marine biota (Reijnders, 1986; Malins el al., 1988; Tanabe, 1988). At the Third International Conference on the Protection of the North Sea it was decided to reduce the loads of primary pollutants to the North Sea by 5 0 - 7 0 % (Anon., 1990a). To monitor the reduction measures adequately, detailed information on sources (emission), input into the system and actual concentrations is essential. A literature survey on CB

concentrations in the North Sea showed that data on CB levels in English waters are very scarce (Klamer et al., 1990a). However, there are reasons for believing that sediment quality in the area has been adversely effected by industrial activities at e.g. Teesside and Humberside (Grogan, 1984). The present study aims to fill part of this gap in knowledge, by an investigation of CB and PAH levels in sediments from the Humber plume area. It focuses on determining distribution patterns of individual CB congeners and PAH compounds, and compares concentrations in the area with those found in sediment samples from the rivers Rhine, Scheldt and Ems/ Dollard and from sedimentation areas like the Wadden Sea and the Oyster Grounds. Materials and Methods The sediment samples were collected during cruise C H 69/90 of the RRS Challenger (26 July-7 August 1990), affiliated to UK-NERC's North Sea Project. The location of the sampling stations is shown in Fig. 1. Surface sediment samples were collected using a Day grab. If a relatively undisturbed and muddy top layer existed (0-2 cm), it was subsampled using a P T F E spatula; otherwise, the top 5 to 10 cm were sampled. The subsamples were transferred to pentane-washed polyethylene bottles. All samples were sieved on board, using a specially designed sieving apparatus with 63 btm nylon mesh (Klamer et al., 1990b). The collected fine fractions were kept frozen until further treatment. 201