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Toxicity of No. 2 fuel oil to coon stripe shrimp
Marine Pollution Bulletin Anon. (1971). Pollution and the Maritime Industry, 2, Alcan Shipping Services Limited. Brunnock, J. V., Duckworth, D. R. & Stevens, G. G. (1968). Analysis of beach pollutants. In Scientific Aspects of Pollution o f the Sea by Oil, edited by P. Hepple. pp. 12-27. London: Institute of Petroleum. Butler, J. N., Morris, B. F. & Sass, J. (1973). Pelagic Tar from Bermuda and the Sargasso Sea. Bermuda Biological Station Special Publication No. 10. Dodimead, A. J., Favorite, F. & Hirano, T. (1962). Review o f Oceanography of the Subarctic Pacific Region. International North Pacific Fisheries Commission Bull. 13. Heyerdahl, T. (1971). Atlantic ocean pollution and biota observed by the Raexpeditions. BioL Conserv., 3, 164-167. International Petroleum Encyclopedia. (1973). Petroleum Publishing Co., Tulsa, Oklahoma. International Petroleum Encyclopedia. (1974). Petroleum Publishing Co., Tulsa, Oklahoma.
Marumo, R. & Kamada, K. (1973). Oil globules and their attached organisms in the East China Sea and the Kuroshio area. J. Ocean. Soc. Japan, 29, 155-158 (in Japanese). Nasu, K., Ueyanagi, S. & Kitani, K. (1975). Oil contamination in the open ocean. Notes coastal Ocean Res., 12,100-105 (in Japanese). Ohya, M., Otsuki, T. & Saito, M. (1973). Oil pollution in the Izu Islands waters. J. ocean. Soc. Japan, 29,121-129(in Japanese). Petroleum in the Marine Environment. (1975). Workshop on inputs, fates, and the effects of petroleum in the marine environment, 21-25 May 1973. National Academy of Sciences, Washington, D.C. Ramsdale, S. J. & Wilkinson, R. E. (1968). Identification of petroleum sources of beach pollution by gas-liquid chromatography. In Scientific Aspects o f Pollution of the Sea by Oil, edited by P. Hepple. pp. 28-34. London: Institute of Petroleum. Wilson, R. D., Monaghan, P. H., Osanik, A., Price, L. C. & Rodgers, M. A. (1974). Natural marine oil seepage. Science, 184,857-865.
Toxicity of No. 2 Fuel Oil to Coon Stripe Shrimp Bioassay of a N o . 2 fuel oil dispersion with shrimp in a continuous flow system using measured waterborne oil as the indicator of oil concentrations reveals a treatment more definable than those previously described in terms of volume ratios and produces lower lethal concentrations. Shrimp 96-h LCs0 was 0.8 mg/I in this study as compared to values from 1.5 to 50 mg/I reported for other methods. Mean concentrations in tests do not give significant differences in concentration with respect to day of the test or spatial distribution in the exposure tanks.
Concern for the effects of crude and refined oil on marine biota is growing commensurately with the growth of marine transport and production of oil. The objective in this study was to measure short-term (96 h) lethal toxicity of a No. 2 fuel oil-in-seawater dispersion to coon stripe shrimp (Pandalus danae) under continuous flow conditions. A useful starting point in assessing the effects of contaminating materials is by determining short-term lethal concentrations. The bioassay of petroleum and refined oils has resulted in highly divergent and apparently contradictory findings with respect to short-term lethal effects. This is due, in part, to the method of expression of petroleum-in-seawater concentration. The early literature, and some recent literature (e.g. Jacobson & Boylan, 1973; Kittredge, 1971; Swedmark et al., 1973; Tagatz, 1961; Mironov, 1970; Eisler, 1975; McAuliffe et al., 1975; Morrow, 1974) expressed concentration in terms of oil:seawater volume ratios. Expression of oil concentration by such ratios has yielded data indicating lethally toxic concentrations from a few to several thousand mg/l, depending on the manner of oil-seawater contact and the duration of exposure. The most obvious reason for this wide variation is the complex manner in which oil reacts with seawater and the transient nature of toxic forms. Because oil-seawater volume ratios do not define waterborne oil, such data are difficult to relate to real-world situations where the concern is for oil that enters the water column and is available to biota. 106
Recent studies by Vaughan (1973), Anderson et al. (1974), Rice et al. (1975), Templeton et al. (1974) and Vanderhorst et al. (in press), have employed estimates of hydrocarbon concentration in bioassay. These studies have also considerably narrowed the range and lowered the LCs0 levels for several marine species. However, allbut the studies listed by Vaughan (1973) were conducted in 'static' systems receiving a one-time dose of hydrocarbons. Anderson et al. (1974) and Vanderhorst et aL (in press) show that the concentration of waterborne oil diminishes rapidly under these conditions; thus, refined estimates of lethal concentration require complex mathematical expressions. Sprague (1970) has indicated that the development of such expressions is quite feasible. However, a more efficient approach, especially with a view to development of future methods for long-term studies, is to use continuous flow systems for bioassay.
Materials and Methods The coon stripe shrimp (Pandalus danae) was selected for study because of its biological and economic importance and also because comparative data are available from 'static' bioassay. Shrimp were obtained by small otter trawl from Sequim Bay, Washington. During pretest holding the shrimp were fed freshly-cracked littleneck clams (Protothaca staminea). They were not fed during bioassay. There was no appreciable mortality in the shrimp stock during pretest laboratory holding and acclimatization. The test oil was a No. 2 fuel oil (API Reference Oil III, 38°70 aromatics) for which data are available concerning chemical composition of oil-in-water dispersions and water soluble phase (Anderson et al., 1974). The apparatus used to prepare the oil-water mixture for testing is described in detail in a manuscript by Vanderhorst et aL now being prepared for publication. Essentially, the process involves vigorous physical contact
Volume 7/Number 6/June 1976
of 2.4 ml/min No. 2 fuel oil and 15 000 m l / m i n seawater, followed by flotation and discard of insoluble oil. Waterborne oil is continuously withdrawn from the apparatus and diluted with raw seawater. Tests were conducted in fibreglass aquaria (93 × 44 x 30 cm) containing 751 o f test solution. Flow to the exposure tanks was 2 l/rain. Water quality measurements were made daily and varied little. Average values for dissolved oxygen, temperature and salinity were 8.6 mg/1, 10.5°C, and 30 ppt, respectively. Sampling for oil in the exposure tanks was at 0.5 h intervals for 6 h, 1.0 h intervals for the next 6 h, and in triplicate daily for the remainder of the tests. Sampling was accomplished through a glass siphon placed in each exposure tank prior to test initiation. Intake of the siphon was at the mid-point of each of the exposure tanks. Supplementary samples were collected by preplaced glass siphons at 12 vertically and horizontally distributed locations in the intermediate concentration exposure tank. All samples were collected directly into Teflon stopcocked 1 1. separatory funnels. Analysis o f C C l 4 (Burdick & Jackson, Analytical Grade) extracts followed the procedures of Simard (1951).
Results and Discussion Based on three replicate bioassays, the 96 h LCs0for coon stripe shrimp was 0.8 mg/1. The 72 h LCs0 was 1.3 mg/1. Insufficient mortality occurred at 24 and 48 h intervals to calculate LCs0 values. In this study, LCs0 concentration is based on mean measured concentration. Table 1 lists the mean concentration and standard deviation for the three replicate bioassay tests. These data are consistent and reproducible, offering an improved estimate of fuel oil toxicity. Anderson et al. (1974) and Vanderhorst et al. (in press) found in 'batch-treated' bioassay a reduction o f hydrocarbon content from 60 to 10 mg/1 in 6 h and 4.0 to 1.0 m g / l i n 24 h for the two studies, respectively. Since the constituents of oil a n d / o r refined products have diverse physical and chemical properties, the rates at which individual components leave a bioassay test system will differ. This implies that under conditions such as used by Anderson et al. (1974) and Vanderhorst et al. (in press), test organisms will be exposed to a medium that has a continually changing chemical composition. While we have not measured composition for this study, the stability of total waterborne oil increases the probability that composition will also be stable. We are currently gathering data on compound composition and its variation with time. In addition to the data presented in Table 1, tests of statistical significance were performed with respect to the day of bioassay and spatial distribution of total oil in the exposure tanks. There were no significant differences TABLE 1 Bioassay tank concentration of total oil (CC14 extracts, IR). Tank
(N)
Control Experimental (1) Experimental (2) Experimental (3)
45 45 45 45
Concentration (mg/l) Mean SD 0 0.4 1.0 1.7
0 0.03 0.14 0.21
( P = 0 . 0 5 ) in concentration with respect to the day of sampling either within a single replicate or for all the replicates. For the intermediate concentration, supplementary sampling at 12 vertically and horizontally distributed points in the bioassay tank did not reveal significant differences (P--0.05) in concentrations for depth or horizontal position. These data are relevant to findings by Vaughan (1973) and Eisler (1975) dealing with 'depth' effects. In order to adequately estimate the concentration of waterborne oil to which a test species is exposed in bioassay, Vaughan (1973) proposed the formula: EEC = D1 × 1 6 + / ) 2 × 16+D3 × 16+Dp ×48 96 w h e r e : D1 : analytically determined hydrocarbon con-
centration at V2 in below the water surface; DE = analytically determined hydrocarbon concentration at mid-depth; /)3--analytically determined hydrocarbon concentration 1 in from the tank bottom; and Dp= analytically determined hydrocarbon concentration at the normal vertical location of the test fish so that Dp, depending on the fish species, will be either Dj,/92, or/)3. One can readily see the difficulty in applying such a formula to systems in which the composition is rapidly changing. A major finding of Eisler (1975) indicated a 'depth-protective effect', i.e. the organism is protected from exposure by remaining near the bottom of the tank. Eisler recognized that this effect probably related to toxicant availability. Spatial distribution of toxicant, as produced in this study, greatly enhances control over this major variable. The reduction in LCs0 with increased time indicates that a threshold concentration has not been reached (Sprague, 1970). This is an important finding because from available data (Anderson et aL, 1974) concerning water soluble fraction and oil-in-water dispersions of the same No. 2 fuel oil, one may reach the opposite conclusion. From the data in the present study, we conclude that apparent lethal thresholds reported for static systems are indicative of hydrocarbon loss from those systems rather than threshold resistance levels for the species in question. Thisworkwas conductedby Battelle,PacificNorthwestLaboratories, for the US Atomic Energy Commission (now Energy Research and Development Administration) under contract AT(45-1)-1830. J. R. V A N D E R H O R S T C. I. GIBSON L. J. MOORE Batelle, Pacific Northwest Laboratories Marine Research Laboratory Sequim, Washington, 98382, USA Anderson, J. W., Neff, J. M., Cox, B. A., Tatum, H. E. & Hightower, G. M. (1974). Characteristicsof dispersions and water-soluble extracts of crude and refined oils and their toxicityto estuarine crustaceans and fish. Mar. Biol., 27, 75-88. Eisler, R. (1975). Toxic, sublethal, and latent effects of petroleum on Red Sea macrofauna. Proc. Joint Conf. on Prevention and Control of Oil Spills. API, EPA, USCG. pp. 535-540. Jacobson, S. M. & Boylan, D. B. (1975). Effect of soluble fraction of kerosene on chemotaxis in a marine snail, Nassaris obsoletus. Nature, 241,213-217. 107
Marine Pollution Bulletin Kittredge, J. S. (1971). Effects of the water soluble fraction of oil on chemoreception by crabs. US Dept. of Commerce, AD-73-8-505, 5 pp. McAuliffe, C. D., Smalley, A. E., Groover, R. D., Welsh, W. M., Pickle, W. J. & Jones, G. E. (1975). Chevron main pass block 41 oil spill: Chemical and biological investigations. Proc. Joint Conf. on Prevention and Control o f Oil Spills, San Francisco, Calif. pp. 555-566. Mironov, G. (1970). The effect of oil pollution on flora and fauna of the Black Sea. MarinePollution andSealife, Ed. M. Ruivo, London: Fishing News (Books) pp. 222-224. Morrow, J. E. (1974). Effects of crude oil and some of its components on young coho and sockeye salmon. Office of Research and Development. USEPA, Washington, DC. EPA-660/3-73-018. 37 pp. Rice, S. D., Moles, D. A. & Short, J. M. (1975). The effect of Prudhoe Bay crude oil on survival and growth of eggs alevins, and fry of pink salmon, Oncorhynchus gorbuscha. Proc. Joint Conf. on Prevention and Control o f Oil Spills, San Francisco, Calif. pp. 503-507. Simard, R. G., Hasegawa, I., Bandaruk, W. & Headington, C. (1951). Infrared spectrophotometric determination of oils and phenols in water. A nal. Chem., 23,1384-1387.
Sprague, J. B. (1970). Measurement of pollutant toxicity to fish. If. Utilizingandapplyingbioassayresults. WaterRes., 4,3-32. Swedmark, M., Granmo, A. & Kollberg, S. (1973). Effects of oil dispersants and oil emulsions on marine animals. Water Res., 7, 1649-1672. Tagatz, M. E. (1961). Reduced oxygen tolerance and toxicity of petroleum products to juvenile American shad. Ches. Sci., 2, 65-71. Templeton, W. L. (1974). Study of effects of oil discharges and domestic and industrial wastewaters on the fisheries of Lake Maracaibo, Venezuela. Fate and Effects of Oil, Vol. 2. Battelle, Pacific Northwest Labs, Richland, Wash. 97 pp. plus Appendix. Vanderhorst, J. R., Gibson, C. I. & Moore, L.J. (1975). The role of dispersion in fuel oil bioassay. Bull. Env. Contain. Toxicol., (In press). Vanderhorst, J. R., Gibson, C. I. & Moore, L. J. (1975). Continuous flow apparatus for use in petroleum bioassay. Battelle, Pacific Northwest Labs, Richland, Washington. Vaughan, B. E. (1973). Effects of oil and chemically dispersed oil on selected marinebiota--alaboratory study. API Report No. 4191. p. B3. American Petroleum Institute, Washington, DC.
The Effect of Detergents on Larval D e v e l o p m e n t of a Crab The effect of a mixture of anionic (ethoxylate) and non-ionic (alkylate) detergents on developmental stages of the crab Rhithropanopeus from final embryogenesis to the Megalopa larval stage has been studied. Larval resistance increases with age. Larvae still in the eggshells are only slightly sensitive because of the thick and impermeable chorion. Low concentrations of detergents exert a favourable effect, causing a decrease in larval mortality, but during moults there is an increasing mortality and a lengthening of the developmental period of the larvae. The mixture of detergents is more toxic than single detergents.
Since the 1950s many investigators have been engaged in the estimation of detergent toxicity (Corner et al., 1968; Lemke, 1963; Marchetti, 1965; Hidu, 1965). The aim of these studies was to analyse the effect of high concentrations of detergents over a short period of time (BellanetaL, 1972; Eisler, 1965; Kaim-Malka, 1972a,b,c) and the estimation of the long-term effect of low concentrations of detergents (Arthur, 1970; Wilson, 1968a). The results of these investigations showed that the activity of living organisms is decreased by exposure to detergents, and prolonged exposure usually leads to damage of biological functions and, in consequence, to the paralysis and death of animals. It has been observed that at very low concentrations of detergents organisms display'self-defence' (Sprague & Drury, 1969) manifested as a closing of the shell, for instance, in shellfish and snails. This reaction is gradually inhibited with increasing concentration of detergents because of a limited physiological activity of individuals under study. The effect of detergents on respiratory activity has also been stressed in the literature. There can be no doubt that these agents 108
unfavourably change the gas exchange and injure the respiratory epithelium. Depending on the structure of respiratory organs, they affect the intensity o f respiration. Generally speaking, the effect of detergents on both developmental stages and adult specimens manifests itself in reduced survival, but it should be remembered that these conclusions were drawn from laboratory studies. An important supplement to these conclusions is the discovery that a sublethal effect observed in the laboratory may be a lethal effect under natural conditions when the whole complexity of the ecosystem is taken into consideration.
Material a n d M e t h o d s
The present studies, conducted at the Biological Station of Gdafisk University in G6rki Wschodnie, were undertaken to determine the effect of non-ionic and anionic detergents on larval survival at different developmental stages and on their duration of these stages. We were also interested in changes in physiological function and morphology. The mixture of detergents used is marketed under the commercial name 'Solo' and consists of 19.4°70 Alfenol 8, 4.85 070Olbrotol 18 and 7.25 070Detepan T. The remainder is 67.8°7o water and 0.7°70 complementary additives including odorant oils. Alfenol 8 is a non-ionic surfaceactive agent, ethoxylate alkylophenol, which is soluble in water and organic solvents because of its oxyethylene chain. Olbrotol is also non-ionic and is ethoxylate deacetylate alcohol. It dissolves in water, benzene and fats. Detepan T is a surface-active anionic agent consisting of a solution of the triethanolamine salt of alkyl benzene-
Marumo, R. & Kamada, K. (1973). Oil globules and their attached organisms in the East China Sea and the Kuroshio area. J. Ocean. Soc. Japan, 29, 155-158 (in Japanese). Nasu, K., Ueyanagi, S. & Kitani, K. (1975). Oil contamination in the open ocean. Notes coastal Ocean Res., 12,100-105 (in Japanese). Ohya, M., Otsuki, T. & Saito, M. (1973). Oil pollution in the Izu Islands waters. J. ocean. Soc. Japan, 29,121-129(in Japanese). Petroleum in the Marine Environment. (1975). Workshop on inputs, fates, and the effects of petroleum in the marine environment, 21-25 May 1973. National Academy of Sciences, Washington, D.C. Ramsdale, S. J. & Wilkinson, R. E. (1968). Identification of petroleum sources of beach pollution by gas-liquid chromatography. In Scientific Aspects o f Pollution of the Sea by Oil, edited by P. Hepple. pp. 28-34. London: Institute of Petroleum. Wilson, R. D., Monaghan, P. H., Osanik, A., Price, L. C. & Rodgers, M. A. (1974). Natural marine oil seepage. Science, 184,857-865.
Toxicity of No. 2 Fuel Oil to Coon Stripe Shrimp Bioassay of a N o . 2 fuel oil dispersion with shrimp in a continuous flow system using measured waterborne oil as the indicator of oil concentrations reveals a treatment more definable than those previously described in terms of volume ratios and produces lower lethal concentrations. Shrimp 96-h LCs0 was 0.8 mg/I in this study as compared to values from 1.5 to 50 mg/I reported for other methods. Mean concentrations in tests do not give significant differences in concentration with respect to day of the test or spatial distribution in the exposure tanks.
Concern for the effects of crude and refined oil on marine biota is growing commensurately with the growth of marine transport and production of oil. The objective in this study was to measure short-term (96 h) lethal toxicity of a No. 2 fuel oil-in-seawater dispersion to coon stripe shrimp (Pandalus danae) under continuous flow conditions. A useful starting point in assessing the effects of contaminating materials is by determining short-term lethal concentrations. The bioassay of petroleum and refined oils has resulted in highly divergent and apparently contradictory findings with respect to short-term lethal effects. This is due, in part, to the method of expression of petroleum-in-seawater concentration. The early literature, and some recent literature (e.g. Jacobson & Boylan, 1973; Kittredge, 1971; Swedmark et al., 1973; Tagatz, 1961; Mironov, 1970; Eisler, 1975; McAuliffe et al., 1975; Morrow, 1974) expressed concentration in terms of oil:seawater volume ratios. Expression of oil concentration by such ratios has yielded data indicating lethally toxic concentrations from a few to several thousand mg/l, depending on the manner of oil-seawater contact and the duration of exposure. The most obvious reason for this wide variation is the complex manner in which oil reacts with seawater and the transient nature of toxic forms. Because oil-seawater volume ratios do not define waterborne oil, such data are difficult to relate to real-world situations where the concern is for oil that enters the water column and is available to biota. 106
Recent studies by Vaughan (1973), Anderson et al. (1974), Rice et al. (1975), Templeton et al. (1974) and Vanderhorst et al. (in press), have employed estimates of hydrocarbon concentration in bioassay. These studies have also considerably narrowed the range and lowered the LCs0 levels for several marine species. However, allbut the studies listed by Vaughan (1973) were conducted in 'static' systems receiving a one-time dose of hydrocarbons. Anderson et al. (1974) and Vanderhorst et aL (in press) show that the concentration of waterborne oil diminishes rapidly under these conditions; thus, refined estimates of lethal concentration require complex mathematical expressions. Sprague (1970) has indicated that the development of such expressions is quite feasible. However, a more efficient approach, especially with a view to development of future methods for long-term studies, is to use continuous flow systems for bioassay.
Materials and Methods The coon stripe shrimp (Pandalus danae) was selected for study because of its biological and economic importance and also because comparative data are available from 'static' bioassay. Shrimp were obtained by small otter trawl from Sequim Bay, Washington. During pretest holding the shrimp were fed freshly-cracked littleneck clams (Protothaca staminea). They were not fed during bioassay. There was no appreciable mortality in the shrimp stock during pretest laboratory holding and acclimatization. The test oil was a No. 2 fuel oil (API Reference Oil III, 38°70 aromatics) for which data are available concerning chemical composition of oil-in-water dispersions and water soluble phase (Anderson et al., 1974). The apparatus used to prepare the oil-water mixture for testing is described in detail in a manuscript by Vanderhorst et aL now being prepared for publication. Essentially, the process involves vigorous physical contact
Volume 7/Number 6/June 1976
of 2.4 ml/min No. 2 fuel oil and 15 000 m l / m i n seawater, followed by flotation and discard of insoluble oil. Waterborne oil is continuously withdrawn from the apparatus and diluted with raw seawater. Tests were conducted in fibreglass aquaria (93 × 44 x 30 cm) containing 751 o f test solution. Flow to the exposure tanks was 2 l/rain. Water quality measurements were made daily and varied little. Average values for dissolved oxygen, temperature and salinity were 8.6 mg/1, 10.5°C, and 30 ppt, respectively. Sampling for oil in the exposure tanks was at 0.5 h intervals for 6 h, 1.0 h intervals for the next 6 h, and in triplicate daily for the remainder of the tests. Sampling was accomplished through a glass siphon placed in each exposure tank prior to test initiation. Intake of the siphon was at the mid-point of each of the exposure tanks. Supplementary samples were collected by preplaced glass siphons at 12 vertically and horizontally distributed locations in the intermediate concentration exposure tank. All samples were collected directly into Teflon stopcocked 1 1. separatory funnels. Analysis o f C C l 4 (Burdick & Jackson, Analytical Grade) extracts followed the procedures of Simard (1951).
Results and Discussion Based on three replicate bioassays, the 96 h LCs0for coon stripe shrimp was 0.8 mg/1. The 72 h LCs0 was 1.3 mg/1. Insufficient mortality occurred at 24 and 48 h intervals to calculate LCs0 values. In this study, LCs0 concentration is based on mean measured concentration. Table 1 lists the mean concentration and standard deviation for the three replicate bioassay tests. These data are consistent and reproducible, offering an improved estimate of fuel oil toxicity. Anderson et al. (1974) and Vanderhorst et al. (in press) found in 'batch-treated' bioassay a reduction o f hydrocarbon content from 60 to 10 mg/1 in 6 h and 4.0 to 1.0 m g / l i n 24 h for the two studies, respectively. Since the constituents of oil a n d / o r refined products have diverse physical and chemical properties, the rates at which individual components leave a bioassay test system will differ. This implies that under conditions such as used by Anderson et al. (1974) and Vanderhorst et al. (in press), test organisms will be exposed to a medium that has a continually changing chemical composition. While we have not measured composition for this study, the stability of total waterborne oil increases the probability that composition will also be stable. We are currently gathering data on compound composition and its variation with time. In addition to the data presented in Table 1, tests of statistical significance were performed with respect to the day of bioassay and spatial distribution of total oil in the exposure tanks. There were no significant differences TABLE 1 Bioassay tank concentration of total oil (CC14 extracts, IR). Tank
(N)
Control Experimental (1) Experimental (2) Experimental (3)
45 45 45 45
Concentration (mg/l) Mean SD 0 0.4 1.0 1.7
0 0.03 0.14 0.21
( P = 0 . 0 5 ) in concentration with respect to the day of sampling either within a single replicate or for all the replicates. For the intermediate concentration, supplementary sampling at 12 vertically and horizontally distributed points in the bioassay tank did not reveal significant differences (P--0.05) in concentrations for depth or horizontal position. These data are relevant to findings by Vaughan (1973) and Eisler (1975) dealing with 'depth' effects. In order to adequately estimate the concentration of waterborne oil to which a test species is exposed in bioassay, Vaughan (1973) proposed the formula: EEC = D1 × 1 6 + / ) 2 × 16+D3 × 16+Dp ×48 96 w h e r e : D1 : analytically determined hydrocarbon con-
centration at V2 in below the water surface; DE = analytically determined hydrocarbon concentration at mid-depth; /)3--analytically determined hydrocarbon concentration 1 in from the tank bottom; and Dp= analytically determined hydrocarbon concentration at the normal vertical location of the test fish so that Dp, depending on the fish species, will be either Dj,/92, or/)3. One can readily see the difficulty in applying such a formula to systems in which the composition is rapidly changing. A major finding of Eisler (1975) indicated a 'depth-protective effect', i.e. the organism is protected from exposure by remaining near the bottom of the tank. Eisler recognized that this effect probably related to toxicant availability. Spatial distribution of toxicant, as produced in this study, greatly enhances control over this major variable. The reduction in LCs0 with increased time indicates that a threshold concentration has not been reached (Sprague, 1970). This is an important finding because from available data (Anderson et aL, 1974) concerning water soluble fraction and oil-in-water dispersions of the same No. 2 fuel oil, one may reach the opposite conclusion. From the data in the present study, we conclude that apparent lethal thresholds reported for static systems are indicative of hydrocarbon loss from those systems rather than threshold resistance levels for the species in question. Thisworkwas conductedby Battelle,PacificNorthwestLaboratories, for the US Atomic Energy Commission (now Energy Research and Development Administration) under contract AT(45-1)-1830. J. R. V A N D E R H O R S T C. I. GIBSON L. J. MOORE Batelle, Pacific Northwest Laboratories Marine Research Laboratory Sequim, Washington, 98382, USA Anderson, J. W., Neff, J. M., Cox, B. A., Tatum, H. E. & Hightower, G. M. (1974). Characteristicsof dispersions and water-soluble extracts of crude and refined oils and their toxicityto estuarine crustaceans and fish. Mar. Biol., 27, 75-88. Eisler, R. (1975). Toxic, sublethal, and latent effects of petroleum on Red Sea macrofauna. Proc. Joint Conf. on Prevention and Control of Oil Spills. API, EPA, USCG. pp. 535-540. Jacobson, S. M. & Boylan, D. B. (1975). Effect of soluble fraction of kerosene on chemotaxis in a marine snail, Nassaris obsoletus. Nature, 241,213-217. 107
Marine Pollution Bulletin Kittredge, J. S. (1971). Effects of the water soluble fraction of oil on chemoreception by crabs. US Dept. of Commerce, AD-73-8-505, 5 pp. McAuliffe, C. D., Smalley, A. E., Groover, R. D., Welsh, W. M., Pickle, W. J. & Jones, G. E. (1975). Chevron main pass block 41 oil spill: Chemical and biological investigations. Proc. Joint Conf. on Prevention and Control o f Oil Spills, San Francisco, Calif. pp. 555-566. Mironov, G. (1970). The effect of oil pollution on flora and fauna of the Black Sea. MarinePollution andSealife, Ed. M. Ruivo, London: Fishing News (Books) pp. 222-224. Morrow, J. E. (1974). Effects of crude oil and some of its components on young coho and sockeye salmon. Office of Research and Development. USEPA, Washington, DC. EPA-660/3-73-018. 37 pp. Rice, S. D., Moles, D. A. & Short, J. M. (1975). The effect of Prudhoe Bay crude oil on survival and growth of eggs alevins, and fry of pink salmon, Oncorhynchus gorbuscha. Proc. Joint Conf. on Prevention and Control o f Oil Spills, San Francisco, Calif. pp. 503-507. Simard, R. G., Hasegawa, I., Bandaruk, W. & Headington, C. (1951). Infrared spectrophotometric determination of oils and phenols in water. A nal. Chem., 23,1384-1387.
Sprague, J. B. (1970). Measurement of pollutant toxicity to fish. If. Utilizingandapplyingbioassayresults. WaterRes., 4,3-32. Swedmark, M., Granmo, A. & Kollberg, S. (1973). Effects of oil dispersants and oil emulsions on marine animals. Water Res., 7, 1649-1672. Tagatz, M. E. (1961). Reduced oxygen tolerance and toxicity of petroleum products to juvenile American shad. Ches. Sci., 2, 65-71. Templeton, W. L. (1974). Study of effects of oil discharges and domestic and industrial wastewaters on the fisheries of Lake Maracaibo, Venezuela. Fate and Effects of Oil, Vol. 2. Battelle, Pacific Northwest Labs, Richland, Wash. 97 pp. plus Appendix. Vanderhorst, J. R., Gibson, C. I. & Moore, L.J. (1975). The role of dispersion in fuel oil bioassay. Bull. Env. Contain. Toxicol., (In press). Vanderhorst, J. R., Gibson, C. I. & Moore, L. J. (1975). Continuous flow apparatus for use in petroleum bioassay. Battelle, Pacific Northwest Labs, Richland, Washington. Vaughan, B. E. (1973). Effects of oil and chemically dispersed oil on selected marinebiota--alaboratory study. API Report No. 4191. p. B3. American Petroleum Institute, Washington, DC.
The Effect of Detergents on Larval D e v e l o p m e n t of a Crab The effect of a mixture of anionic (ethoxylate) and non-ionic (alkylate) detergents on developmental stages of the crab Rhithropanopeus from final embryogenesis to the Megalopa larval stage has been studied. Larval resistance increases with age. Larvae still in the eggshells are only slightly sensitive because of the thick and impermeable chorion. Low concentrations of detergents exert a favourable effect, causing a decrease in larval mortality, but during moults there is an increasing mortality and a lengthening of the developmental period of the larvae. The mixture of detergents is more toxic than single detergents.
Since the 1950s many investigators have been engaged in the estimation of detergent toxicity (Corner et al., 1968; Lemke, 1963; Marchetti, 1965; Hidu, 1965). The aim of these studies was to analyse the effect of high concentrations of detergents over a short period of time (BellanetaL, 1972; Eisler, 1965; Kaim-Malka, 1972a,b,c) and the estimation of the long-term effect of low concentrations of detergents (Arthur, 1970; Wilson, 1968a). The results of these investigations showed that the activity of living organisms is decreased by exposure to detergents, and prolonged exposure usually leads to damage of biological functions and, in consequence, to the paralysis and death of animals. It has been observed that at very low concentrations of detergents organisms display'self-defence' (Sprague & Drury, 1969) manifested as a closing of the shell, for instance, in shellfish and snails. This reaction is gradually inhibited with increasing concentration of detergents because of a limited physiological activity of individuals under study. The effect of detergents on respiratory activity has also been stressed in the literature. There can be no doubt that these agents 108
unfavourably change the gas exchange and injure the respiratory epithelium. Depending on the structure of respiratory organs, they affect the intensity o f respiration. Generally speaking, the effect of detergents on both developmental stages and adult specimens manifests itself in reduced survival, but it should be remembered that these conclusions were drawn from laboratory studies. An important supplement to these conclusions is the discovery that a sublethal effect observed in the laboratory may be a lethal effect under natural conditions when the whole complexity of the ecosystem is taken into consideration.
Material a n d M e t h o d s
The present studies, conducted at the Biological Station of Gdafisk University in G6rki Wschodnie, were undertaken to determine the effect of non-ionic and anionic detergents on larval survival at different developmental stages and on their duration of these stages. We were also interested in changes in physiological function and morphology. The mixture of detergents used is marketed under the commercial name 'Solo' and consists of 19.4°70 Alfenol 8, 4.85 070Olbrotol 18 and 7.25 070Detepan T. The remainder is 67.8°7o water and 0.7°70 complementary additives including odorant oils. Alfenol 8 is a non-ionic surfaceactive agent, ethoxylate alkylophenol, which is soluble in water and organic solvents because of its oxyethylene chain. Olbrotol is also non-ionic and is ethoxylate deacetylate alcohol. It dissolves in water, benzene and fats. Detepan T is a surface-active anionic agent consisting of a solution of the triethanolamine salt of alkyl benzene-