New procedures for the toxicity testing of oil slick dispersants in the United Kingdom

Marine Pollution Bulletin J Black, R. (1973). Growth rates of intertidal molluscs as indicators of unexpected incidents of pollution. J. Fish. Res. Board Can., 30, 1385-1388. 2Hood, M. A., Bishop, W. S., Jr., Bishop, F. W., Meyers, S. P. & Whelan, T., III (1975). Microbial indicators of oil-rich salt marsh sediments. Appl. Microbiok, 30, 982-987. 3Jangaard, P. M. (1972). Effects of elemental phosphorus on marine life: Collected papers resulting from the 1969 pollution crisis, Placentia Bay, Newfoundland. Circular No. 2, Atlantic Regional

Office, Fisheries Research Board of Canada, Halifax, N.S. 313 pp. 4Oppenheimer, C. H., Gunkel, W. & Gassman, G. (1977). Microorganisms and hydrocarbons in the North Sea during July-August 1975. Proc. Oil Spill Conference (Prevention, Behavior, Control, Cleanup), New Orleans, March, 1977. pp. 593-609. s Payne, J. F. (1976). Field evaluation of aryl hydroxylase induction as

a monitor for marine petroleum pollution. Science, N.Y., 191, 945 -946. 6Payne, J. F. (1977). Mixed function oxidases in marine organisms in relation to petroleum hydrocarbon metabolism and detection. Mar. Poll. Bull., 8, 112-116. 7Payne, J. F. & Penrose, W. R. (1975). Induction of aryI hydrocarbon (benzo[a]pyrene) hydroxylase in fish by petroleum. Bull. Environ. Contain. Toxicol., 14, 112-116. 8Thomas, W. A. (1972). Indicators of E n vironmental Quality, Plenum Press, New York. 9Vandermeulen, J. H., Keizer, P. D. & Penrose, W. R. (1977). Persistence of non-alkane components of Bunker C oil in beach sediments of Chedabucto Bay, and lack of their metabolism by molluscs, Proc. Oil Spill Conference (Prevention, Behavior, Control, Cleanup).

New Orleans, March, 1977. pp. 469-474.

Marine Pollution Bulletin, Vol. 9, pp. 234-238 .~ Pergamon Press Ltd. 1978. Printed in Great Britain

0025 -326X/78/0901-0234.$02.00,0

New Procedures for the Toxicity Testing of Oil Slick Dispersants in the United Kingdom R. A . A. B L A C K M A N , F. L. F R A N K L I N , M. G. N O R T O N a n d K. W. W I L S O N * Fisheries L a b o r a t o r y , B u r n h a m - o n - C r o u c h , Essex, U . K . *Present address: North West Water Authority, P.O. Box 12, New Town House, Buttermarket Street, Warrington, U.K.

Under the Dumping at Sea Act 1974 the use of oil stick dispersants requires a iicence from the Ministry of Agriculture, Fisheries and Food in England and Wales. These licences are issued or refused on the basis of tests to assess the toxicity of the dispersant when used at sea or on beaches. This paper describes the rationale behind the development of the two toxicity tests used, together with the test methods adopted and the results of the tests. T h e toxicity o f oil d i s p e r s a n t s to m a r i n e o r g a n i s m s has been e v a l u a t e d for a n u m b e r o f years at the Fisheries L a b o r a t o r y , B u r n h a m - o n - C r o u c h , using a s t a n d a r d static b i o a s s a y e m p l o y i n g the d i s p e r s a n t a l o n e ( P o r t m a n n & C o n n o r , 1968). This test a l l o w e d the 48 h LCs0 o f the d i s p e r s a n t to the b r o w n s h r i m p ( C r a n g o n c r a n g o n L.) to be d e t e r m i n e d , a n d the p r o d u c t s r a n k e d a c c o r d i n g to their toxicity. P r o d u c t s with a s a t i s f a c t o r y p e r f o r m a n c e in efficiency tests a n d a low toxicity in the static test were a p p r o v e d for use in the UK; lists o f a p p r o v e d p r o d u c t s were p u b l i s h e d by the W a r r e n Spring l a b o r a tory o f the D e p a r t m e n t o f T r a d e a n d I n d u s t r y ( D e p a r t m e n t o f T r a d e a n d I n d u s t r y , 1975). A l t h o u g h this test m e t h o d w o r k e d well with the original types o f d i s p e r s a n t with 48 h LCsos in the r a n g e 1-1000 p p m , as dispersants o f lower toxicity b e c a m e available, the test c o n c e n t r a tions necessary to derive a 48 h LCs0 b e c a m e higher with the result that the d i s p e r s a n t n o longer m i x e d a d e q u a t e l y with seawater in the test tanks, b u t f o r m e d a surface layer or emulsion. Test a n i m a l s were t h e r e f o r e e x p o s e d 234

to c o n c e n t r a t i o n s o f d i s p e r s a n t much lower t h a n those theoretically present, giving unrealistic estimates o f the 48 h LC50. F u r t h e r m o r e , the different b e h a v i o u r o f dispersants i n v a l i d a t e d the previous r a n k i n g system a d o p t e d for toxicity (Wilson, 1974). New m e t h o d s of toxicity assessment were thus sought to o v e r c o m e these problems. A t a b o u t the same time the D u m p i n g at Sea A c t 1974 ( H M S O , 1974) c a m e into being and, as the licensing a u t h o r i t y for E n g l a n d a n d Wales, the M i n i s t r y of A g r i c u l t u r e , Fisheries a n d F o o d assumed responsibility to " p r o t e c t the m a r i n e e n v i r o n m e n t a n d the living resources which it s u p p o r t s f r o m any adverse consequence o f d u m p i n g . . . " . U n d e r the w o r d i n g o f the Act, the use o f oil dispersants was technically an act o f ' d u m p i n g ' a n d so f u r t h e r emphasis was placed on the requirements to i m p r o v e the testing procedures. In recognition o f the different conditions o f use of dispersants at sea a n d on beaches, two different testing p r o c e d u r e s have been developed. A m a j o r c o n s i d e r a t i o n in the d e v e l o p m e n t o f test m e t h o d o l o g y was the need, for licensing purposes, to evaluate a large n u m b e r o f p r o d u c t s routinely a n d r e p r o d u c i b l y whilst simulating, as far as possible, the a c t u a l c o n d i t i o n s e n c o u n t e r e d when a dispersant is a p p l i e d to a n oil slick at sea or to oil s t r a n d e d on beaches. This influenced the selection o f the c o n d i t i o n s o f e x p o s u r e o f the test o r g a n i s m s as well as the level o f effect to be m e a s u r e d .

Volume 9/Number 9/September 1978

The Sea Test Rationale and development Where dispersants are properly applied to an oil slick at sea marine organisms are exposed, not to a solution or suspension of the dispersant alone, but to an oil and dispersant suspension. Experience has shown that natural mixing processes (e.g. those due to waves) may cause some physical dispersion of the untreated oil but that addition of a dispersant increases markedly the extent of dispersion, even when additional mixing energy is not applied. A test to evaluate the additional environmental effect of using a given dispersant was therefore based on a comparison of the toxicity of physically and chemically dispersed oil. The chemical dispersion of small slicks of oil in open waters leads to initial concentrations of up to 50 p p m v / v of oil, reducing rapidly (in well mixed waters such as are found in the North Sea) to I ppm or less within a few hours (Cormack, 1977). Physical dispersion of a small test slick has been reported to give only a few p p m in the water column (Cormack, 1977; Nichols, 1973), but higher concentrations of up to 50 p p m have been reported following larger spills (Spooner, 1970). Since a continuous dilution system (as a simulation of natural dispersion processes) was impractical for routine application, in the test finally developed test organisms were exposed to a single concentration for a predetermined period. For routine testing, the mortality of the readily available species of brown shrimp (Crangon crangon L.) was selected as the effect to be measured. To overcome the deficiencies of the static test, it was necessary to develop a system which gave a homogenous suspension of the oil/dispersant mixture or the oil alone. Early work by Connor (1972) used stirring to maintain the suspension. Alternatives such as a circulating pump and jet were tried but the most efficient and reproducible system was found to be a cylindrical tank with the rotating propeller enclosed by a central Perspex cylinder having apertures at the top and bottom, covered by a plastic mesh screen to exclude test animals. With the propeller driven at between 1350 and 1450 rpm water was drawn through the upper apertures and expelled through the lower ones producing a uniform and reproducible dispersion without causing stress to the test organisms. A magnetic coupling between the propeller shaft and an induction motor allowed the tank to be closed by a Perspex lid. The apparatus is shown in Fig. 1. Developmental work using this apparatus showed that an initial exposure period of 100 min to a concentration of 1000 ppm produced a measurable toxic effect using the physically dispersed oil, against which the effect of the dispersant/oil mixture could be evaluated. The concentration of oil used in the test is high relative to concentrations observed in the field, and thus includes a safety factor for species more sensitive than shrimps to the acute toxic effects of oil.

Method Test animals are caught from wild populations and acclimated over 2-3 days to the test temperature of

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15 ° C. Only shrimps of total length between 50 and 70 m m are used. The test tanks are filled to the correct level with filtered ( < 1 Mm) seawater at 30-32%0 S and aerated for 1 h after which 20 test animals are added to each tank. After a further 2 h the aerators are removed, the motors put in place and the lids displaced to one side to allow the test substance to be added. Two methods of applying the oil and dispersant to the tanks have been used. In the first method the agitation is started and the appropriate amount of test substance (oil alone or pre-mixed oil and dispersant) is added from a disposable syringe near to the vortex to ensure immediate mixing, and the lids repositioned. In the second method, the oil is added to the surface before the agitation is started. Dispersant is then added to the surface layer of oil from a syringe and distributed as evenly as possible. After 1 min, the motors are started and the lids repositioned. The second method reflects more closely the application of dispersant to slicks at sea, and is now the standard method used for routine testing for licensing purposes. At the end of the 100 min exposure period, the motors are switched off, the surface oil and oily water siphoned out of the tanks, and the animals gently transferred to tanks of clean, gently flowing, aerated seawater at 15°C. Mortalities are recorded after 24 h. Routine tests employ 3 replicates of 1000 ppm of oil (fresh Kuwait crude) dispersed with 1000 p p m dispersant (concentrates are tested as a 10% mixture with seawater), and 3 replicates of 1000 p p m oil alone. The mortalities in the oil control and the oil and dispersant mixture are compared statistically (Student's t-test) to determine whether the oil dispersed with the aid of the dispersant is significantly (P = <0.05) more or less toxic than the oil dispersed by mechanical agitation alone.

Results The results of tests on a number of dispersants using the standard sea test conditions are presented in Table 1. The conventional dispersants tested have a generally neutral or inhibitory effect on the toxicity of the dispersed oil, while concentrates may increase the toxicity of the dispersed oil. (Concentrate dispersants have been tested using both methods of introducing the oil and dispersant into the test tanks.) 235

Marine Pollution Bulletin

TABLE 1 Sea test results for a range of dispersants (3 replicates per test). Mean Mortality Oil alone (control) Dispersant

%

Standard error

Oil and dispersant %

Standard error

(a) Oil and dispersant premixed before addition to the test tanks Conventional (hydrocarbon solvent based) dispersants A 40 4 20 B 65 2 50 C 80 5 90 D 60 8 65 E 35 8 30 F 60 8 40 G 60 8 50 H 60 8 25 1 65 2 35 J 65 2 70 K 60 8 60 L 35 12 35

7 15 6 3 3 9 2 6 6 4 4 16

Concentrates(tested as a 10%emulsionin seawater) M 50 3 70 N 50 12 80 O 55 2 100 P 80 5 100 Q 50 3 60 R 50 12 60 S 60 6 75 T 35 8 95 U 85 6 100 V 50 3 70

12 1 1 2 4 4 3 5 2 3

(b) Dispersant added to oil film in test tanks before agitation M N O P Q R S T U V

60 20 30 55 60 55 60 30 30 20

11 8 5 4 11 4 11 5 5 8

100 40 80 70 75 60 80 70 55 25

7 7 9 4 16 6 16 4 8 5

Variations in the toxicity of the physically dispersed oil and of the oil/dispersant mixtures can arise from seasonal and other changes in the sensitivity of the test species. It is thus essential to compare simultaneously the toxicity of a dispersant/oil mixture with that of a physically dispersed standard oil using the same stock of test animals. The values of the standard error which appear in Table 1, however, show that, within a set of replicate tests, variations in the toxicity of a given oil or oil/dispersant mixture may be kept low enough to allow detection of differences in toxic effect between different dispersants.

shores however, dispersants will be applied directly to the exposed foreshore, and direct spraying of intertidal organisms can occur. Although toxic effects of spraying are likely to have only limited impact on commercial fisheries the death of grazing organisms such as gastropods (winkles and limpets) on rocky shores can lead to significant and unacceptable ecological changes due to extensive algal growth, as happened following the use of toxic dispersants after the Torrey Canyon oil spill (Smith, 1968). Consequently, the toxicity test was developed using a typical intertidal grazing organism, the c o m m o n limpet, Patella vulgata L. When dispersants are used to clean oil from beaches, animals are exposed to very different conditions from those at sea. Both oiled and unoiled animals may be exposed to undiluted dispersant and left in air until either the incoming tide or the use of water hoses washes off the contaminants. The method of assessing the suitability of dispersants for beach use has therefore been based on these exposure conditions. Preliminary tests in the laboratory showed that the mortality of limpets exposed to oil for several hours is high and the measurement of toxic effect due to the dispersant over and above that of the oil would be difficult and less accurate than the determination of the effect of the dispersant alone. Additionally, dispersant is likely to be applied over wide areas of foreshore and the evaluation of a particular dispersant should take into account the effect of the dispersant on those parts which were unoiled as well as those which were oiled. Therefore the test finally adopted was designed to assess whether the effect of dispersant sprayed onto unoiled limpets was greater than the effect of the oil alone and followed the sequence (a) spraying with neat dispersant (concentrates as 10070 v / v emulsion in seawater) or with oil control, (b) exposure in air, (c) rinsing in tidal cycles of clean seawater. The amount of dispersant applied in the effective dispersal of oil on beaches ranges from 0.09 to 1.0 1. m -2 but is normally near 0.4 1. m -2 (Department of Trade and Industry, 1975). The rate of application used in the test is thus 0.4 1. m -2. The remaining variables are the time the test organism is exposed after spraying and until rinsing, and the time at which subsequent mortalities are recorded. The former was selected as 6 h to reflect the 'average' time before rinsing by tidal action if sprays were applied at mid tide level. Initial tests showed that mortalities resulted within the first 2-3 days of immersion in the seawater rinsing system, and that little additional mortality occurred over the next 2 days. Consequently the period over which mortality was measured was selected as 72 h.

T h e B e a c h Test Rationale and development

Method

The intertidal zone may be of value to commercial fisheries, e.g. for oysters, mussels, cockles, etc, or may be primarily of amenity interest, whether as bathing beaches or accessible rocky shores. In general, it is not recommended that dispersants should be used on oiled shellfish beds until the latter have been immersed by the tide, in which case the dispersants approved under the sea test may be used. Where oil is stranded on amenity

All experiments are carried out at a constant temperature of 15°C. Twenty test animals of 30-40 m m shell width are transferred to one face of a series of Perspex test plates of approximately 440 cm 2 area. The plates are left on racks in gently flowing, aerated seawater overnight for the limpets to attach and are then suspended vertically in stock tanks which are equipped with a simulated tidal seawater system. After 3 days the

236

Volume9/Number 9/September 1978 plates are removed from the stock tanks, drained and placed horizontally, attached animals uppermost, in the spraying tanks. Each limpet on each of 5 plates is then sprayed from a height of about 10 cm with 0.7 ml of dispersant (concentrates as a 10070 emulsion in seawater) from a hand-operated spray. Another 5 control plates are sprayed in a similar manner with fresh Kuwait crude oil. The plates of sprayed limpets are left in moist air for 6 h before being washed for 15 s with running seawater and suspended vertically in the full recovery tanks, one plate per tank, in which further rinsing takes place through a simulated tidal cycle. Limpets detaching from the plates are recorded as dead and removed from the tanks immediately after rinsing, 24 and 48 h after the start of the test. After 48 h any remaining limpets, which are not firmly attached to their plates, are gently detached and placed on the tank floor. If these have not re-attached within 24 h they are counted as dead, since this effect in the field would almost certainly result in their death by burial, wave damage, aerial exposure or predation. The mortalities after 72 h, for the dispersant treated plates and oil control, are compared statistically by means of Student's t-test to determine whether the dispersant is significantly (P = 0.05) more or less toxic than the oil.

Results

The beach test results for the dispersants used in the sea test are given in Table 2. Exposure of limpets to fresh Kuwait crude oil leads to mortalities of 67 to 93°70. Application of the dispersant alone leads to a wider range of mortalities varying from 22 to 96%. In most cases the variability of replicates within each test is low.

Licensing Standards in the UK Control in the UK via the licensing of dispersants under the Dumping at Sea Act has to be based on an assessment of whether the marine environment is likely to be harmed by the use of the dispersant. This is a difficult general judgement to make since dispersants are only used where harm may already be occurring due to the presence of a spill. Indeed, in the absence, at the present time, of any effective methods to recover or contain a slick under normal North Sea conditions, dispersion by chemical means may be the only way of protecting seabirds, inshore shellfish resources or beaches of high ecological, fishery or amenity value. Where the use of dispersants cannot be avoided, it is desirable to use only those dispersants causing no more damage (particularly to fisheries) than would result if the slick were left to disperse by natural means. Since the sea test is a direct comparison of the toxicity of oil dispersed by physical and chemical means under identical conditions of mechanical agitation, licences for sea use are restricted to those products which do not increase the toxicity of oil physically dispersed in the sea test by a statistically significant amount. The beach test results show that the majority of dispersants are less toxic for limpets than the control exposed to oil only. The licensing of dispersants for beach use is thus based on the philosophy that foreshore organisms sprayed by an approved dispersant should have a better chance of recovery than those already contaminated by oil or those which might be contaminated if oil was to spread further as a result of spraying being withheld. Products which pass the beach test are thus those which can be shown statistically to be no more toxic than the oil control. Since oil and dispersant are washed off by the incoming tide and dispersed into the water column, products are only licensed for beach use if they pass both the sea and beach tests.

TABLE 2

Beach test results for a range of dispersants (5 replicates per test).

Effects of the New Tests

Mean Mortality Oilalone(control)

Dispersant

%

Standard error

Oiland dispersant O7o

Standard error

Conventional (hydrocarbon solvent based) dispersants

A B C D E F G

80 70 80 70 80 70 70

2 4 3 4 3 4 4

60 40 40 25 75 95 85

5 4 7 7 4 2 2

I J K

80 80 70

4 2 4

50 55 85

4 6 5

L

80

4

70

6

Concentrates (tested as a 10% emulsion in seawater)

M N O P Q R S T U V

80 95 95 80 80 95 95 80 95 80

3 3 3 2 3 3 3 3 3 3

40 45 95 90 30 80 55 90 20 70

7 13 2 5 5 7 6 2 6 7

The majority of dispersants approved on the basis of the original static test have shown themselves to be of acceptably low toxicity under the new tests. A small number of concentrates have, however, been shown to generate toxic dispersions with oil and have not received licences under the Dumping at Sea Act for sea use. Additionally a number of dispersants (both concentrates and hydrocarbon based) have been shown to be highly toxic in the beach test. T h e n e w tests have thus identified effects in the laboratory which were not apparent from the simpler static tests and which are more likely to simulate effects of dispersant use. It is thus believed that these tests offer a better means of ensuring that only dispersants with low environmental impact are used. This paper describes the technical evaluation of oil slick dispersants under the Dumping at Sea Act. Details of the licensing procedures including the method of application may be obtained from Ministry of Agriculture, Fisheries and Food, Marine Pollution Branch, Great Westminster House, Horseferry Road, London SW1. A more detailed description of the apparatus and experimental procedures involved in the sea and beach test (Blackman et aL, 1977) is available from the Directorate of Fisheries Research, Pakefield Road, Lowestoft,

Suffolk. 237

Marine Pollution Bulletin Blackman, R. A. A., Franklin, F. L., Norton, M. G. & Wilson, K. W. (1977). New Procedures for the toxicity testing of oil slick dispersants. Fish Res. Tech. Rep., M A F F Direct. Fish. Res. Lowestoft, 39, 7 pp. Connor, P. M. (1972). Further investigations into the toxicity of oils and dispersants. ICES C.M. 1972/E: 14 (mimeo). Cormack, D. (1977). Oil Pollution. Chemy. Ind., 16, 605-608. Department of Trade & Industry (1975). Warren Spring Laboratory, Newsletter, No. 6 (August 1975). FIMSO (1974). Dumping at Sea Act 1974. Eliz. II, Chap. 20, ISBN 0 10 542074 3.

Nichols, J. A. (1973). Non-persistent oils in the marine environment. Warren Spring Laboratory CR 703 (OP). Portmann, J. E. & Connor, P. M. (1968). The toxicity of several oilspill removers to some species of fish and shellfish. Mar. BioL, 1, 322-329. Smith, J. E. (1968). Torrey Canyon Pollution and Marine Life. Cambridge University Press, London. Spooner, M. F. (1970). Oil spill in Tarut Bay, Saudi Arabia. ,War. Pollut. Bull., 1, 166-167. Wilson, K. W. (1974). Toxicity testing for ranking oils and oil dispersants. In Ecological Aspects o f Toxicity Testing o f Oils and Dispersants. L. R. Beynon & E. B. Cowell (eds). pp. 11-22. Allied Science Publishers, London.

0025-326X/78/0901-0238502.00 0

Marine Pollution Bulletin, Vol. 9. pp. 238-241 :~ Pergamon Press Ltd. 1978. Printed in Great Britain

Use of a Harpacticoid Copepod in Toxicity Tests B E N G T - E R I K BENGTSSON

The National Environment Protection Board, Brackish Water Toxicology Laboratory, Studsvik, S-611 O1 NykOping, Sweden The harpaedcoid copepod Nitocra spinipes has been tested for acute toxicity of 12 metal chlorides in brackish water. Their order of toxicity, expressed as 96 h LCs0, was in good agreement with other investigalions performed in freshwater and seawater. The 96 h LCs0-values were of intermediate levels compared to these two environments. The organochlorines p,p'-DDE and p,p'-DDE methyl suiphone were tested for effects on reproduction and mortality during two weeks, and it was found thatp, p'-DDE was the most toxic. It is concluded from the investigation that N. spinipes is a suitable toxicity test organism in brackish water. This study is the first step in an attempt to develop a simple standardized laboratory toxicity test for substances that occur or might be discharged into the brackish water of the Baltic Sea. The test was required to be fast, not space-consuming, cheap and easy to perform, even for personnel with very little biological training. It should also be applicable to the brackish water environment with a wide range of salinities. An advantage would be if the animal used also could serve as a potential fish-food organism in the laboratory. The usefulness and ecological significance of reproduction studies in aquatic toxicity is very well recognized. One shortcoming, however, is that they are usually very time-consuming, especially when fish are used and only tropical fish allow one-generation tests in much less than one year. Accordingly, in the search for a suitable test animal, much attention was paid to a comparatively short life-cycle. Small crustaceans like Artemia salina, Daphnia spp. and harpacticoids have been used for studies of toxic effects on reproduction (Grosch, 1973; Biesinger & Christensen, 1972; Winner & Farrell, 1976; Maki & Johnson, 1975; Canton et al., 1975; Beisinger et al., 1974; D'Agostino & Finney, 1974; Hoppenheit, 1975 etc.). After exploring the literature, the search for a test animal was concentrated on a hardy harpacticoid 238

species, which could tolerate a wide range of relevant external factors. This report describes results obtained in the search for this organism and the usefulness of this organism in some toxicological experiments with the chlorides of 12 metals and two DDE-compounds.

The Test Organism A number of harpacticoids were caught in the sediment from a static brackish water aquarium with prawns that had run for about one year at room temperature ( 2 0 - 2 2 ° C ) , and in which the prawns had been randomly fed with pelleted salmon food. Some different harpacticold forms were found, and 20 ovigerous females (females with egg-sac) were pipetted and isolated one by one in dishes with brackish water (70700 salinity) and small amounts of finely ground salmon food (Ewos 2, Astra-Ewos, S0dert~tlje, Sweden). To select the most tolerant laboratory animal, the offspring from the individual'females was cultivated to a number of about 3 0 - 1 0 0 animals which were exposed to a wide range of temperatures and salinities. After about 4 weeks, the offspring of 2 of the original 20 females had demonstrated a superior tolerance to the offered laboratory conditions. Ten adult males from each of the two progenies were sent to Dr. Wilfried Scheibel, Zoological Institute of the University of Kiel for taxonomic determination. It was established that the animals were all Nitocra spinipes Boeck. The species has been described by many authors and a short review on its biology and distribution is given below. The adult animal has a fusiform body with a length of 0 . 7 - 0 . 8 mm. According to Lang (1948) N. spinipes has an amphiatlantic and wide geographic distribution. It is often very numerous, especially on sand bottoms, interstitially or free, and in the phytal (Noodt, 1970). Dussart