Toxicity of three oils to bivalve gametes and larvae

Volume 6/Number 8/August 1975 TABLE 1 Attained size and long diameter increments of soft clam transplants (diameter in mm)

Shefl no.

Jan. 1973

June 1973

Total Increase

Monthly Increase

Annual Rate

April 1974

Total Increase

Monthly Increase

Annual Rate

Avg. % decline in annual growth rate

1 2 3 4 Mean

44.5 44.3 46.2 42.2 44.3

50.1 50.9 64.5 48.8 53.6

5.6 6.6 18.3 6.6 9.3

.9 1.1 3.1 1.1 1.6

10.8 13.2 37.2 13.2 18.6

53.3 55.8 73.6 51.2 58.5

3.2 4.9 9.1 2.8 5.0

0.3 0.5 1.0 0.3 0.5

3.6 6.0 12.1 0.3 6.4

67 55 67 72 65

survive. In four monthly-samples o f transplanted clams, all but 586 were recovered or accounted for, indicating that many may have surfaced from their burrows and died as had occurred at Searsport, Maine, in March 1971 when a spill of about 14 metric tons of No. 2 fuel oil mixed with JP5 killed approximately 4.5 million commercial size clams in less than 2 weeks (Air Force contract report). During continued 1974 studies, four previously unrecovered clams from the June 1973 experimental transplant were found to have survived the winter of 1 9 7 3 - 7 4 . Total survival of transplanted clams, including 227 collected at monthly intervals after June 1973, was 12.8% as compared with 78.1% survival of clams in the control area. The average 1973 growth of the four clams prior to transplanting was 9.3 m m in length, or a monthly growth rate o f 1.6 mm. The average linear growth of these same clams from 20 June, 1973, to 9 April, 1974, was 5.0 mm, or a monthly rate of 0.5 mm, a 65% reduction in growth after transplanting to the oiled area. Annual growth rate of the experimental clams before transplanting was 18.6 mm,

as contrasted with 6.4 m m after transplanting. The decline of 65% in the annual growth rate of the surviving transplanted clams appears to have been caused by the residual oil (Table 1).

ROBERT L. DOW Department o f Marine Resources, Augusta, M E 04333, U.S.A.

Dow, R. L. (1972). Fluctuations in Gulf of Maine sea temperature and specific molluscan abundance. 3". Cons. int. Explor. Met., 34(3), 532-534. Mayo, D. W., Donovan, D. J., Jiang, L., Dow, R. L. & Hurst, J. W. (1974). Long-term weathering characteristics of Iranian crude oil: the wreck of the Northern Gulf. NBS Spec. Publ. 409, Marine Pollution Monitoring (Petxoleum), Proc. Syrup. and Workshop held at NBS, Gaithersburg, Md., 13-17 May, 1974, pp. 201-208. Final Report, Sears-port Oil Spill Survey (1972). U.S. Air Force Contract No. F33600-72-C-0540, Dept. of Sea & Shore Fish., Augusta, ME.

Toxicity of Three Oils to Bivalve Gametes and Larvae Water soluble extracts of three crude oils, Kuwait, Nigerian and Prudhoe Bay have been tested using the sperm and eggs of two marine bivalves. Fertilization is depressed and developmental abnormalities sometimes appear after exposure to these toxins. The spermatozoa, in particular, are very sensitive to the water-soluble fractions of these oils. Nigerian crude is particularly toxic. Pollution of oceans, seas, rivers and lakes has been increasing at a very high speed in recent years. Among the most c o m m o n pollutants are crude oil and oil derivatives, deriving from well spillages, refinery effluents, washing of tankers and from collisions, accidents and wreckages of tankers. Degradation, especially by microbial oxidation and decomposition, is the normal fate of an oil or an oil derivative once it is at sea; but this process can be retarded or nullified if the water basin is already highly polluted and its various ecosystems partially or totally disrupted. Small fractions of crude oils as well as of oil derivatives are water-soluble and their toxicity to marine and fresh-water

organisms has been proved repeatedly. In this note, continuing some observations previously reported, we refer some data on the influence of the water-soluble fractions of three crude oils on gametes, embryos and larvae of two marine bivalves. Material and Methods The experiments were undertaken with Mulinia lateralis Say and Crassostrea virginica Gmelin, two marine bivalves of extreme interest because of the short life cycle of the former (Calabrese~ 1969) and the commercial value as a food of the latter. After being laboratory-conditioned for several weeks~ the animals were forced to spawn by increasing the water temperature to 2 9 - 3 0 ° C for a few hours. The eggs, f'iltered after spawning, were poured into previously filtered and u.v. sterilized sea-water; the sperm were collected in filtered and u.v. sterilized sea-water. The crude oils examined were from three very distant areas: Kuwait, Nigeria (Bonny) and Alaska (Prudhoe Bay); the first is one of the most common Arabian oils, the second 125

Marine Pollution Bulletin

Results

TABLE 1

Crassostrea virginica: p e r c e n t a g e s o f f e r t i l i z a t i o n , of embryo development a n d l a r v a l s u r v i v a l a t v a r i o u s o i l - i n - w a t e r c o n c e n t r a t i o n s . Crude oil origin

Alaska (Prudhoe)

Nigeria (Bonny)

Kuwait

Concn of the oil (ml 1-1)

% of fertilization

% development of embryos

% survival o f larvae

0.001 0.01 0.1 1

89.9 88.2 83.7 71.4

91.5 86.5 81.9 68.5

83.0 80.2 75.2 55.0

0.001 0.01 0.1 1

90.1 80.3 75.2 63.3

89.0 77.9 66.4 50.4

77.1 72.5 68.7 43.3

0.001 0.01 0.1 1

89.9 88.7 85.2 73.2

92.8 88.9 85.9 69.3

88.3 81.8 77.7 53.8

All values a r e a v e r a g e s f o r d u p l i c a t e c u l t u r e s a n d a r e e x p r e s s e d as percentage of the values obtained from control cultures.

proved to be of high toxicity in preliminary (unpublished) investigations, and the last one will shortly be the most common crude oil in the North-American west coast areas. The water soluble fractions (wsO were obtained as follows: the oil and the sea water were vigorously shaken in a separating funnel for 30 min; the 'solution' was left standing for 3 h and most of the oil reformed as a film on top. The underlying water with the water-soluble fractions was drawn from the bottom of the separating funnel and the chosen dilutions were prepared in the test jars. The wsf of the three oils were tested as follows: (a) by adding them immediately after mixing the gametes in unpolluted water (Mulinia lateralis and Crassostrea

virgin ica ) ; (b) by adding them to the eggs, before fertilization with untreated sperm (Mulinia lateralis); (c) by adding them to the sperm, and fertilizing untreated eggs with the treated sperm (Mulinia lateralis). Gametes of (b) and (c), after being treated with the wsf for an hour, were repeatedly washed before the fertilization. In both bivalves, fertilization, development of embryos and survival of larvae (48 h ) w e r e examined; in Mulinia lateralis, the increase in mean length (14 days) of larvae was also determined. In all cases data are expressed as percentage of the results obtained with simultaneously run control experiments. The counting was done under a compound microscope; in some cases immediately, and, in others, later after preservation in 5% neutral formalin. TABLE 2

Mulinia lateralis: p e r c e n t a g e s o f f e r t i l i z a t i o n , o f embryo development, o f l a r v a l s u r v i v a l a n d of the i n c r e a s e in m e a n l e n g t h at various oil-in-water concentrations. Crude off origin

Alaska (Prudhoe) Nigeria (Bonny)

Kuwait

Cohen of the off (ml F 1 )

%of fertilization

%

0.001 0.01 0.1 1 0.001 0.01 0.1 l 0.001 0.01 0.1 1

93.8 91.9 85.1 68.8 89.4 88.5 83.7 67.8 96.8 94.2 89.5 73.8

development of embryos 91.9 89.0 86.9 77.1 89.0 87.0 77.8 62.8 95.8 94.1 91.1 72.9

% surviv~ oflarvae 84,8 85.3 80.6 71.0 86.4 82.6 71.3 59.2 88.0 83.8 80.9 62.0

% increase in mean length oflarvae 82.8 82.5 74.5 55.6 81.7 72.6 63.3 40.3 81.3 82.5 74.5 50.1

All v a l u e s a r e a v e r a g e s f o r d u p l i c a t e c u l t u r e s a n d a r e e x p r e s s e d as percentage of the values obtained from control cultures.

126

(1) In both species the wsf of the three crude oils show a degree of toxicity to fertilization processes, to the development of embryos and to survival of larvae (Tables 1 and 2) that is strictly correlated to their concentration. (2) The influence of the wsf on the rate of growth of the Mulinia larvae kept in the polluted water for 2 weeks can be clearly seen in Table 2; it should be noted that new wsf were added every 48 h when the water was changed and typical algal food (Isochrysis, Monochrysis and Chlorella) was added. (3) In Mulinia, the structure that was the most affected by the toxicant is the sperm, as is evident from the results reported in Table 3. (4) Besides the influence of the wsf upon the reproductive success of both species and on the larval growth of Mulinia, the presence of the toxicants in the culture water determines the appearance of abnormal embryos at the various concentrations considered, and especially at the highest (Fig. 1 a,b). Among the various abnormalities, one occurred frequently in our experiment. It consisted of larger embryos, with variable shape and with some highly vacuolated areas alternating with others containing cells with turbid (apparently degenerating) cytoplasm. Another abnormality, though considerably less frequent, is found in embryos that, though they reach the straight-hinge stage and present the typical behaviour of normal larvae of this age (especially as far as swimming activity is concerned), are smaller and lack the shell (Fig. 1 c *). Another abnormal aspect, especially evident in the lower concentrations of the wsf in the culture medium is the appearance, among the majority of larvae swimming normally, of some larvae unable to swim and with the velum constantly protruding from the shell and bearing cilia that alternate periods of movement with periods of inactivity (Fig. 1 c). (5) In all experiments performed for this research the toxicity of the wsf of the Nigerian oil was higher than that of other oils (from Kuwait and from Alaska), statistically significantly higher (P < 0.01) in experiment c. Research is underway to study the hydrocarbon content of the wsf for all three oils in greater detail.

Discussion Marine and especially estuarine waters present increasing levels of petroleum contamination from various sources, therefore estuarine benthic species come into continuous contact with the water solube fractions of oil (as well as of other oil residues). For this reason we are continuing laboratory experiments with typically estuarine species and particularly with those of commercial interest for human consumption, such as Crassostrea virginica (Engle, 1966), and those of a great potential usefulness for laboratory research (Calabrese, 1969). Whereas data from field studies conducted after large oil spills are numerous and, quite frequently, concordant, laboratory studies to evaluate the toxicity of crude off and its derivatives are not many, and quite often the results obtained are discrepant. Many of the differences in the results are evidently to be correlated to the different chemical composition of the crude oil, to the age of the petroleum at the time of laboratory extraction, to the length of oil-water mixing in the laboratory and to

Volume 6/Number 8/August 1975

the different species tested (Mironov, 1972; Anderson e t al., 1974; Evans & Rice, 1974; Rice, 1974). Our extraction method was chosen after many trials and after a comparative evaluation of the total dissolved hydrocarbon concentration; various authors have been following the same method in the recent past (Rice, 1973; Evans & Rice, 1974). Our data demonstrate once more the high toxicity of the wsf of all three oils to gametes, embryos and larvae of two estuarine species and the values obtained closely resemble those previously reported for taxonomically related bivalves (Renzoni, 1973; Le Gore, 1973). Another finding, which by confirming previous results of Renzoni seems to indicate a common trend in bivalves, is the greater sensitivity to the wsf of the male gametes than of the eggs. That sensitivity is so great that, at some concentrations, the sperm fertility is drastically reduced. The occurrence of abnormal embryos as well as that of live larvae~ unable to swim and with the velum protruding from the shell, is the new finding of the present research. Findings of abnormal embryos, though in taxonomically distant species (such as herring embryos), have been reported by Ktihnhold (1969); pictures similar to our larvae, with the velum protruding from the shell have been reported by Galtsoff (1964) for larvae of Crassostrea virginica under experimental laboratory narcosis. Normally hatched larvae of herring showed phenomena of narcotization when passed from clean to crude oil-contaminated water (K'tihnhold). In both cases some of the separated crude oil fractions are responsible for the occurrence of such deformed larvae.

U.@.

& ql

S

Neither in the former research of Renzoni when using the crude oil-dispersant mixture, nor in the present when testing the wsf only, did we. have the opportunity of observing signs of irritation of the swimming embryos or larvae of both species, comparable to those reported by Smith (1968) for larvae of Sabellaria. Notwithstanding the great difficulty of obtaining data reproducibility in experiments with hydrocarbon extracts (because of the many reasons previously mentioned and from many possible others still unknown), with our extraction method we obtained consistent results with both species of bivalves. For both of them and for all parameters considered so-far, the water soluble fractions of the Nigerian (Bonny) crude oil are clearly more toxic to marine representatives than those derived from the Alaskan and Kuwait oils. It is well known that "among separated fractions the lower and more aromatic fractions, are by far the most toxic" (Nelson-Smith, 1972).

TABLE 3 Mulinia lateralis: percentages of fertilization at 1 ml/l oil-in-water

concentration. Crude oil origin Alaska (Prudhoe) Nigeria (Bonny) Kuwait

Eggs and sperms mixed in water with oil

Eggs treated and inseminated with untreated sperms

Eggs untreated inseminated with treated sperms

88.58+3.28 83.68~-:4.00 89.56+1.84

90.72+1.46 87.84!2.04 92.32+1.08

75.76:!:8.11 61.84-+7.59 77.92+-6.16

All values are averagesof five experiments and are expressed percentage of the values obtained from control cultures, + standard deviation.

Fig. 1 Mulinia lateralis: (A) abnormal embryos (arrow), with

degenerating ceils (two arrows), scattered among normal embryos and unfertilized eggs (u.e.) in cultures kept in Nigerian oil-in-water at 1 ml/1 concentration; (B) the same as (A) at 0.1 ml/l concentration; (C) straight-hinge stage larvae kept in water with Nigerian oil (0.01 ml/l) showing the protruding velum. An abnormal small larva without shell is visible (*). 127

All the experiments were conducted at the National Fisheries Laboratory, Milford, Connecticut. The courtesy of Dr. Hank, the Director, and Dr. Calabrese, Chief of the Pollution Branch, has been appreciated very much. Research supported by the Consiglio Nazionale Ricerche grant No. 73/00653.

ARISTEO RENZONI

I s t i t u t o di A n a t o m i a Comparata, Universit~ di Siena, 5 3 1 0 0 Siena, Italy.

Anderson, J. W., Neff, J. M., Cox, B. A., Tatem, H. E., & Hightower, G. M. (1974). Characteristic of dispersions and water-soluble extracts of crude and refined oils and their toxicity to estuarine crustaceans and fish. Mar. Biol., 27, 75. Calabrese, A., (1969). Mulinia lateralis: molluscan fruit fly. Proc. Nat. Shell. Ass., 59, 65. Engle, J. B., (1966). The molluscan shellfish industry; current status and trends. Proc. Nat. Shell. Ass., 56, 13. Evans, D. R. & Rice, S. D. (1974). Effects of oil on marine ecosystems: a review for administrators and policy makers. Fishery Bull., 72, 3 625. Galtsoff, P. S. (1964). The American oyster. Fishery Bull., 64, 480. Kuhnhold, W. W. (1969). Der Einfluss wasserliSslicher Bestandteile yon RohiSlen und Rohblfraktionen auf die Entwicklung yon Heringsbrut. Bet. deutsch, wiss. Kommiss. Meeresforsch., 20, 165. Le Gore, R. (1973). A preliminary assessment of the effects of Alaskan North Slope crude oil on developing larvae of the Pacific oyster, Crassostrea gigas. Tech. paper at the 1973 N.S.A. Convent. Proc. Nat. Shell. Assoc. 64, 4. Mironov, O. G. (1972). The effect of oil pollution on the flora and fauna of the Black Sea. (FAO tech. Conf. mar. Poll.). In: M. Ruivo Ed.: Marine Pollution and Sea Life, ed. Ruivo, M., p. 222. London: Fish. News (Books). Nelson-Smith A. (1972). Oil Pollution and Marine Ecology. Paul Elek (Scientific Books). Renzoni, A. (1973). Influence of crude oil, derivatives and dispersants on larvae. Mar. Pollut. Bull., 4, 9. Rice, S. D. (1973). Toxicity and avoidance tests with Prudhoe Bay oil and pink salmon fry. Proc. Joint Conf. Prey. Contr. Oil Spills. Amer. Petr. Inst., E.P.A. and U.S. Coast Guard, Wash. D.C.p. 667. Rice, S. D. (1974). Personal Communication. Smith, J. E. (1968). 'Torrey Canyon' pollution and marine life. Cambridge University Press.

N e w B o o k s you should s e e Aeration of A c t i v a t e d

Di harge of Sewage

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from Sea Outfalls

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Proceedings of an International Symposium held in London, August 1974

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Pergamon 128

180pp 133 illus

Editor: A. LH. GAMESON, Water Research Centre, Stevenage, Hefts., England Published as a S u p p l e m e n t to Progress in Water Technology The Symposium examined three main topics: microbial standards for bathing beaches, treatment of sewage before discharge into the sea, and the various mechanisms to which microbial mortality in the marine environment has been attributed. CONTENTS: Introduction. Opening Address. Regional Problems. Indicators of Sewage Pollution. Microbial Standards. Effects of Non-microbial Pollutants. Sewage Treatment. Microbial Mortality. initial Dispersion. Subsequent Dispersion. Predictive Modelling. Predictive Modelling and Conclusions. Participant List. Name Index, Subiect Index. OF INTEREST TO: Microbiologists, Sewage treatment technologists. Environmental Scientist~. ISBN 0 08 018302 6 June 1975 464pp $35.00 £14.60 Price to non-suhscribers Special price to subscribers £11.60 $28.00 Headington Hill Hall, O x f o r d O X 3 0 B W , England Fairview Park, Elmsford, New Y o r k 10523, USA

400