Toxicity comparison of biosurfactants and synthetic surfactants used in oil spill remediation to two estuarine species

Marine Pollution Bulletin 46 (2003) 1309–1316 www.elsevier.com/locate/marpolbul

Toxicity comparison of biosurfactants and synthetic surfactants used in oil spill remediation to two estuarine species Katherine R. Edwards a, Joe Eugene Lepo a

a,*

, Michael A. Lewis

b

Center for Environmental Diagnostics and Bioremediation, University of West Florida, 11000 University Parkway, Pensacola, FL 32514, USA b US EPA, NHEERL Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561, USA

Abstract The relative environmental toxicities of synthetic and biogenic surfactants used in oil spill remediation efforts are not well understood. Acute and chronic toxicities of three synthetic surfactants and three microbiologically produced surfactants were determined and compared in this study for the estuarine epibenthic invertebrate, Mysidopsis bahia and the inland silverside, Menidia beryllina. The toxicities of the surfactant were determined in standard laboratory static and static-renewal tests of 4–7 d duration. Results were specific to the surfactant, response parameter and test species. The LC50 values (nominal concentrations) for M. bahia ranged from 3.3 mg/l (Triton X-100) to >1000 mg/l (PES-61) and 2.5 mg/l (Triton X-100) to 413.6 mg/l (PES-61) for M. beryllina. Chronic first-effect concentrations (mg/l) for the six surfactants ranged from 2.3 to 465.0 (M. beryllina) and 1.0 to >1000.0 (M. bahia) based on reductions in growth and fecundity. Few generalizations could be made concerning the results due to their variability but M. bahia was generally the more sensitive species and the toxicities of the biosurfactants were intermediate to those of the synthetic surfactants. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Biosurfactants; Synthetic surfactants; Toxicity; Estuarine fish; Invertebrate

1. Introduction Approximately five million metric tons of crude and refined oil enter the environment each year as a result of anthropogenic sources such as oil spills (Johnston, 1984). Detrimental effects of offshore oil spills usually occur on shoreline and shallow subtidal areas (NelsonSmith, 1978). Several bioremedial approaches have been applied to contaminated shoreline to reduce such impacts (Swannell et al., 1994). These include stimulation of indigenous oil-degrading microbiota by the addition of fertilizers high in nitrogen and phosphorus (Fox, 1989; Pritchard et al., 1992), seeding of the oil-fouled areas with hydrocarbon-degrading microorganisms (Rambeloarisoa et al., 1984; Chakrabarty, 1985) and,

*

Corresponding author. E-mail address: [email protected] (J.E. Lepo).

0025-326X/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0025-326X(03)00238-8

the focus of this study, application of surfactants to the oil-contaminated zone (Rasiah and Voroney, 1993). Surfactants can either be chemically synthesized (synthetic) or microbially produced (biosurfactant). Synthetic surfactants are of petrochemical origin whereas biosurfactants or biogenic surfactants are produced by bacteria, yeast, and fungi. Synthetic surfactants may be cationic, anionic, nonionic or amphoteric although only anionic and nonionic surfactants have been used as oil dispersants. Biosurfactants are usually classified based on their biochemical nature and the microbial species producing them. Five major classes of biosurfactants are: (1) glycolipids, (2) phospholipids and fatty acids, (3) lipopeptide/lipoproteins, (4) polymeric surfactants, and (5) particulate surfactants (Parra et al., 1989; Desai and Desai, 1993; Nabholz et al., 1993). The common presence of synthetic surfactants in the aquatic environment, primarily as a result of detergent and household cleaner use, has resulted in extensive toxicity testing during the past 30 years. Consequently, an extensive laboratory toxicity database and published

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risk assessments exist for many commercial surfactants and test species, and data summaries are available (Lewis, 1991, 1992; Tiehm, 1994). Also, environmental toxicities events due to biosurfactants have been reported less frequently, and for fewer species, particularly when based on the results of standard toxicity tests and on chronic effects to animal test species common to the Atlantic and Gulf of Mexico coasts. It remains to be determined if the environmental hazards of these surfactants are similar to those for synthetic surfactants based on the response of phylogenetically diverse organisms. The objectives of our report are to (1) provide a toxicity data comparison for synthetic surfactants and biosurfactants and two estuarine species indigenous to the eastern and southern US coastlines and (2) compare results to those previously reported for other surfactant and oil spill dispersants.

2. Materials and methods 2.1. Surfactants and dilution water The three biosurfactants were Bio-EMâ , Emulsan and PES-51â . The synthetic surfactants were Corexitâ 9500, PES-61â and Triton X-100 (Table 1, Fig. 1); these were supplied by their manufacturers, Emulsan Products Division, Hartford, CT (Emulsan), Petrogen, Inc., Arlington Heights, IL (Bio-EMâ ), Petroleum Environmental Services, San Antonio, TX (PES-51â and PES61â ), Falco-Exxon Energy Chemicals, Sugar Land, TX (Corexit 9500) and Sigma Chemical Co., St. Louis, MO (Triton X-100). Additional information on PES-51, PES-61 and Corexit is available from production technical bulletins (Corexit 9500, 1995; PES-51, 1995; PES61, 1995). Seawater from Santa Rosa Sound (Florida) was used as the dilution water in all toxicity tests. This was filtered and had a mean salinity of 20 (standard deviation ±2) ppt. Temperature (°C), dissolved oxygen (mg/l), pH and salinity (ppt) of the test waters (dilution water contain-

ing surfactant) were determined in one replicate per treatment before and after the daily renewal of the test. 2.2. Test species and methodologies Menidia beryllina and Mysidopsis bahia were the test species. M. beryllina, the inland silverside minnow, is common to estuarine waters, Mexico to Massachusetts (McKenney, 1986) and is an important forage fish for striped bass, bluefish, spotted seatrout (Middaugh, 1985). It is a recommended species for toxicity testing of chemicals and effluents (USEPA, 1993, 1994). M. bahia, an epibenthic mysid shrimp, lives in estuarine waters along the Gulf of Mexico, southwest Florida to Mexico. The species is sensitive to a wide range of contaminants and also a recommended test species for toxicity testing (USEPA, 1993). The anionic surfactant, sodium dodecyl sulfate (SDS), was used as a reference toxicant to ensure that the two test species, obtained from either a commercial supplier (M. beryllina) or a USEPA laboratory culture (M. bahia), were of uniform sensitivity before use. The acute toxicities of the six surfactants were determined in static toxicity tests conducted for 96 h following USEPA procedures (USEPA, 1993). Five test organisms in each of two replicate 400 ml beakers were exposed to five surfactant test concentrations in a 0.6 dilution series. Definitive test concentrations were determined using preliminary range-finding toxicity tests. The LC50 value (concentration lethal to 50% of the test species) was calculated based on survival following the 96-h exposure period. The chronic toxicity test methods for both species were those of the USEPA (USEPA, 1994); experimental details are shown in Table 2. We exposed ten 7–11 d old larvae to five test concentrations of the surfactants in four replicate 1.0 l test chambers. These contained 750 ml of test solution (M. beryllina) or in 150 ml of test solution contained in 400 ml test chambers (M. bahia). Survival, growth (dry weight) and fecundity (% of females with eggs in oviduct or brood pouch) were de-

Table 1 Surfactants used in the toxicity tests Test compound

Use

Structure

Biosurfactants BioEM Emulsan

Emulsifier Emulsion stabilizer

PES-51

Biological cleanser

Glycolipid surfactant (produced by Pseudomonas aeruginosa SB 30) (see Fig. 1) Extracellular form of a polyanionic heteropolysaccharide biopolymer (see Fig. 1) (Produced by Acinetobactec alcoaceticus RAG-1) Mixture of D -limnoene and bacteria fermentation by-products

Synthetic surfactants PES-61 Corexit 9500 Triton X-100

Wetting agent/Biological cleanser Oil spill dispersant (antiemulsifier) Emulsifier/Dispersing agent

Mixture of orthosilicate polymer and similar bacterial fermentation by-products as PES-51 Blend of fatty esters, glycol ethers and oxyalkylates in a parafinic solvent Nonionic ethoxylated alkylphenol surfactant (See Fig. 1)

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Fig. 1. Structures of three of six surfactants used as test compounds. Structures for others are proprietary.

Table 2 Experimental conditions for the chronic toxicity tests conducted with the epibenthic invertebrates M. bahia, and the Inland Silverside, M. beryllina Parameter

M. bahia

M. beryllina

Test type Test duration Salinity Temperature Photoperiod Light intensity Test chamber Test solution volume Renewal of test solution Age of test organisms Test concentrations Number of organisms per test beaker Number of replicate beakers per treatment Source of food Feeding regime

Static renewal 7d 20±2 ppt 26±1 °C 16 h light, 8 h dark 10–20 lE/m2 /s 400 ml glass beakers 150 ml per replicate beaker Daily 7d Five and a control 5 8 Artemia nauplii Fed 150 48 d old nauplii per mysid daily after renewal None Filtered natural seawater Survival, growth, fecundity

Static renewal 7d 20±2 ppt 25±1 °C 16 h light, 8 h dark 10–20 lE/m2 /s 1000 ml glass beakers 750 ml per replicate beaker Daily 7–11 d post hatch Five and a control 10 4 Artemia nauplii 0.05 g (+0), 0.08 g wet weight of 48 d old nauplii after renewal None Filtered natural seawater Survival and growth

Aeration Dilution water Effects measured Adapted from USEPA (1993, 1994).

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termined for M. bahia. Larval survival and growth were analyzed for M. beryllina after the 7 d exposures. The 7 d LC50 value was determined based on survival as well as the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for each response parameter. The first effect concentration (FEC) was calculated as the geometric mean of the NOEC and LOEC. All of the effect- and no effect-results reported in this study are based on nominal test concentrations due to the lack of specific analytical methods or to proprietary considerations. 2.3. Statistical analysis LC50 values were determined after the 4 and 7 d exposures following the methods described by Stephan (1977). Chronic toxicity results were analyzed using Toxstat 3.3 (Gulley et al., 1991) to determine the NOEC and LOEC for each surfactant based on measurements of survival, growth, and fecundity.

Table 3 LC50 values and associated 95% confidence limits for M. bahia and M. beryllina determined after 4 and 7 d exposure Test species

Surfactant

LC50 4d

7d

M. bahia

BioEM Emulsan PES-51

18.7 (13.0–21.6) >200.0 20.0 (17.6–23.0)

>20.0a >200.0 15.4 (13.5–17.5)

PES-61 Corexit 9500 Triton X-100

>1000 20.9 (18.6–23.5)

>1000 13.4 (11.4–15.6)

3.8 (3.5–4.2)

3.3 (3.0–4.0)

BioEM

14.7 (14.0–15.5)

14.2 (13.3–15.1)

Emulsan PES-51

345.3 (302.5–394.1) 21.7 (16.8–28)

300.0 (277–348.9) 20.3 (19.2–21.5)

PES-61 Corexit 9500 Triton X-100

413.6 (374.8–446.4) 79.3 (70.5–81.1)

408.0 (366.7–441.8) 75.7 (70.5–81.1)

6.0 (5.6–6.3)

2.5 (2.4–2.7)

M. beryllina

Values represent nominal concentrations in mg/l. a Exceeds maximum test concentration.

3. Results 3.1. Physicochemical parameters of the test waters Physicochemical parameters of the test waters during the toxicity tests were usually within the guidelines of USEPA test methodologies. The exception was temperature, which was exceeded in one toxicity test conducted with M. beryllina. The pH ranged from 7.6 to 8.1 during the toxicity tests with both test species. Water temperatures, were 23.4–27.2 °C; and salinity 18–22 ppt. Dissolved oxygen ranged from 4.4 to 7.8 mg/l. 3.2. Acute toxicities The LC50 values (mg/l) for M. bahia and the six surfactants ranged from 3.8 to >1000 (4 d exposure) and 3.3 to >1000 (7 d exposure) (Table 3). Differences in LC50 values for the same surfactant between two exposure durations were slight which was not unexpected due to the slight duration differences. The LC50 values (mg/l) for M. beryllina ranged from 6.0 to 413.6 (4 d exposure) and 2.5 to 408.0 (7 d exposure). The most toxic surfactant to both species was Triton X-100, the least toxic Emulsan and PES-61. Differences, in LC50 values for the two species after exposure to the same surfactant for the same exposure period was usually less than twofold. There were exceptions. The 4 and 7 d LC50 values for PES-61 and M. beryllina were at least half of those for M. bahia indicating a greater sensitivity. Conversely, LC50 values for Corexit 9500 and the two species differed by approximately fourfold (4 d expo-

sure) and sixfold (7 d exposure) with M. bahia being the more sensitive species. 3.3. Chronic toxicities M. bahia––the first effect concentrations (FEC-mg/l) for M. bahia and the six surfactants exceeded as much as three orders of magnitude, ranging from 2.8 to >1000 (survival), 1.7 to >1000 (growth) and 1.0 to >1000 (fecundity). Differences among the first effect concentrations for the same surfactant based on the three response parameters, when determinable, were usually fivefold or less (Table 4). Fecundity was the most sensitive response parameter in exposures conducted with BioEM, Emulsan and Triton X-100, whereas survival and growth were the more sensitive indicators of the toxicities of PES-51 and Corexit 9500. As observed for acute toxicity, the most toxic surfactant was Triton X-100; PES-51 PES-61 were least toxic. M. beryllina––the FEC values for this species based on reductions in growth exceeded the maximum nominal concentration in all cases (Table 4). First effect concentrations (mg/l) based on survival after 7 d exposure to the surfactants were between 2.3 (Triton X-100) and 464.8 (PES-61). Interspecific differences in the FEC values for the same surfactant based on survival were usually less than twofold (Table 4). The most obvious exception was the almost 20-fold difference in values for Corexit 9500; the FEC values were 4.2 mg/l (M. bahia) and 77.5 mg/l

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Table 4 Toxicities of biosurfactants and synthetic surfactants to M. bahia and M. beryllina after 7 d exposure Surfactant

Response parametera

Calculations M. bahia b

M. beryllina

FEC

NOEC

LOEC

FEC

NOEC

LOEC

S G F

16.8 5.8 3.3

13.0 4.3 2.6

21.6 7.8 4.3

15.5 –c –

12.0 20.0

20.0 >20.0d

Emulsan

S G F

– – 154.9

200 200 120.0

>200 >200 200.0

232.4 – –

180.0 300 –

300.0 >500 –

PES-51

S G F

10.1 10.1 16.8

7.8 7.8 13.0

13.0 13.0 21.6

21.7 –

16.8 16.8

28.0 >16.8

– – –

1000 1000 1000

>1000 >1000 >1000

464.8 –

360.0 1000

600.0 >1000

Biosurfactants BioEM

Synthetic surfactants PES-61 S G F Corexit 9500

S G F

4.2 4.2 15.5

3.2 3.2 12.0

5.4 5.4 20.0

77.5 – –

10.0 100 –

100.0 >100 –

Triton X-100

S G F

2.8 1.7 1.0

2.2 0.8 0.8

3.6 3.6 1.3

2.3 –

1.8 3.0

3.0 >3.0

Effect and no effect concentrations (mg/l) represent nominal concentrations. Fecundity not determined for toxicity tests conducted with M. beryllina. a Effects represent survival (S), growth (G), and fecundity (F). b First effect concentration (FEC)––Geometric mean of NOEC and LOEC. c Not calculable. d Maximum test concentration.

(M. beryllina). M. bahia was more sensitive (lower first effect concentrations) than M. beryllina after exposure to BioEM, PES-51, Corexit 9500, and Triton X-100 based on reductions in weight of surviving organisms.

4. Discussion The toxicity results of our study were specific to the surfactant, response parameter and the test species and findings limited generalizations. Depending on species, LC50 values varied 1–2 orders of magnitude. First effect concentrations for the six surfactants varied as much as three orders of magnitude. Despite these differences, PES-61 (synthetic surfactant) and Emulsan (biosurfactant) were consistently the least toxic surfactants, Triton X-100 (synthetic surfactant) the most toxic. Based primarily on changes in weight after 7 d exposure to many of the surfactants, M. bahia appeared to be more sensitive than M. beryllina. There was no consistent indication that a particular class of surfactants, synthetic or biologically produced, was more toxic than the other (Fig. 2). The toxicities of the biosurfactants, in general

terms, were intermediate to those for the synthetic surfactants. Lang and Wagner (1993) and Poremba et al. (1991a,b), among others, have reported that biosurfactants are generally less toxic than synthetic surfactants based on the responses of other test species. The results of our study are compared to those previously reported for detergent surfactants (Fig. 3) and surfactants used in oil spill remediation and oil spill dispersal (Table 5). Experimental conditions in these toxicity tests were dissimilar and toxicity values varied. Nevertheless, the toxicities of the six surfactants used in this study were usually less than those reported for detergent surfactants (Fig. 3). Furthermore, it is clear that the FEC values (range ¼ 154.9 to >1000 mg/l) and 96 h LC50 values (range ¼ 300 to >1000 mg/l) for Emulsan and PES-61 in this study are greater than the corresponding results reported for other surfactants and oil dispersants (Table 5), which indicates their relatively nontoxic nature. In contrast, the 96 h LC50 values for Triton X-100, the most toxic compound in this study (range ¼ 2.5–6.0 mg/ l), were less than the 96 h LC50 values of 17–163 mg/l (Singer et al., 1991, 1993, 1994) and 8–500 mg/l (Maggi, 1972) reported for oil dispersants.

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Fig. 2. Comparison of the first effect concentrations for synthetic surfactants and biosurfactants based on survival, growth and fecundity of M. bahia and M. beryllina after 7 d exposures. Arrow signifies that the first effect concentration exceeded the maximum test concentration.

Fig. 3. Comparison of the first effect concentrations of the six surfactants used in this study to those previously reported for detergent surfactants. Data adapted from Lewis (1991, 1992).

In summary, the acute and chronic toxicities of the six surfactants used in this study were compound-specific and varied several orders of magnitude. Results suggest that data extrapolations among these and other compounds used in oil spill remediative processes require careful consideration. Furthermore, in terms of a risk assessment, it is unknown to what degree results of this study would be representative of results of natural conditions, as reported by Mearns et al. (1995). In addition, the toxicity results of this study would need to be compared to predicted or measured concentrations of the surfactants in the environment, which are not available in the scientific literature. The magnitude of these concentrations would depend upon site-specific considerations of dilution, biodegradation, and exposure duration and depth. Consequently, research concerning the biological effects of oil spill remediation chemicals should center on determining their environ-

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Table 5 Toxicities (mg/l) of several surfactants and dispersants used in oil spill remediation to marine animals and algae as reported in the scientific literature Surfactant

Effect concentrations

Calculation

Test species

Response parameter

Reference

Linear akylbenzene sulfonate (LAS) LAS LAS

0.05–0.10

First effect

Larval growth

0.04 0.05

NOEC First effect

Crassostrea virginica (oyster) M. bahia (mysid) Mytilus edulis (mussel)

Calabrese and Davis (1967) Kimerle (1989) Granmo (1972)

LAS

0.05–0.50

First effect

LAS

0.003

69% reduction

LAS

0.025

96 h LC50

Sodium dodecyl sulfate

2.6

96 h LC50

1.9

96 h LC50

5.5 19.2 10–50

96 h LC50 First effect First effect

0.05

96 h LC50

17–24

48 h EC50

26–123

48 h EC50

87–102

48 h EC50

14–18

48 h EC50

59–104 120–163

96 h LC50 96 h LC50

Oil spill emulsifiers

>100

Not given

Corexit 7664

1.0 2.1 35–490 13–90 70–500 8–220

24 24 96 96 96 96

44–73 73–96 20–>3000

96 h LC50 48 h EC50 EC50

Oil dispersants

Two oil dispersants

Corexit 9527

Seven oil spill emulsifiers

Two oil spill dispersants Eight synthetic and nine biogenic surfactants a

h h h h h h

Tlm Tlm LC50 LC50 LC50 LC50

Paralichtys olivacens (fish) Limanda yokohomae (fish) Gymnodinium breve (dinoflagellate) Gymnodinium breve Ampelisca abdita (amphipod) Cyprinodon variegatus (fish) M. bahia (mysid) Penaid shrimp Limanda yokohomae (fish) Paralichtys olivacens (fish) Haliotis rufescens (abalone) Holmesimysis costata (mysid) Macrocystis pyrifera (kelp) Haliotis rufescens (abalone) Atherinops affinis (fish) Holmesimysis costata (mysid) Skeletonoma costatum (diatom) Ambassis sp. (fish) Penaeid shrimp Mytilus edulis (mussel) Artemia salina (shrimp) Anguilla anguilla (fish) Penaeus kerathurus (shrimp) A. affinis (fish) M. pyifera (kelp) Dunaliella tertiolecta (alga) Protozoa Bacteria

Survival––NOEC Fertilization, larval development Hatching

Growth

Yasunaga (1976)

Survival

Kutt and Martin (1974) Hitchcock and Martin (1977) Unpublisheda

Hatching

Yasunaga (1976)

Survival, shell deformation Developmental abnormalities Survival

Singer et al. (1993)

Mortality

Growth

Singer et al. (1991)

Survival

Tokuda and Arasaki (1977) Latiff (1971)

Survival

Maggi (1972)

Survival Germ tube length Growth, bacterial bioluminescence

Singer et al. (1994) Poremba et al. (1991b)

Tests conducted at the US EPA Laboratory in Gulf Breeze, FL.

mental concentrations, as well as validating their laboratory-derived toxicities. Acknowledgements Stephen Embry (CSC, Gulf Breeze, FL) prepared the graphics, and Peggy Rogers (NCBA) the manu-

script. Research was conducted at the US EPA research laboratory, Gulf Breeze, Florida. The US Environmental Protection Agency, Office of Research and Development, partially funded and collaborated in the research under Assistance Agreements CR-818998 and CR-822236 to the University of West Florida.

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References Calabrese, A., Davis, J.C., 1967. Effects of soft detergents on embryos and larvae of the American oyster. Proc. Natn. Shellfish Assoc. 57, 11–16. Chakrabarty, A.M., 1985. Genetically-manipulated microorganisms and their products in the oil service industries. Trends Biotechnol. 3, 32–38. Corexit 9500 Product Bulletin, 1995. Nalco/Exxon energy chemicals, L.P., Sugar Land, TX. Desai, J., Desai, A., 1993. Production of biosurfactants. In: Kosaric, N. (Ed.), Biosurfactants, first ed., Production Properties Applications, vol. 48. Marcel Dekker, New York, NY, pp. 65–97. Fox, J., 1989. Native microbes role in Alaskan clean-up. Bio/ Technology 7, 852. Granmo, A., 1972. Development and growth of eggs and larvae of Mytilus edulis exposed to linear dodecylbenzene sulphonate. Mar. Biol. 15, 356–358. Gulley, D.D., Boelter, A.M., Bergman, H.L., 1991. TOXSTAT 3.3. Fish Physiology and Toxicology Laboratory, Department of Zoology and Physiology, University of Wyoming. Hitchcock, W.S., Martin, D.F., 1977. Effects and fate of a surfactant in cultures of the red tide organism. Bull. Environ. Contam. Toxicology 18, 291–296. Johnston, R., 1984. Oil pollution and its management. In: Kinne, O. (Ed.), Marine Ecology Ocean Management. Part 4. Pollution and Protection of the Seas, vol. 5. John Wiley & Sons, Chicester. Kimerle, R., 1989. Aquatic and terrestrial ecotoxicology of linear alkylbenzene sulfonate. Tensides. Surfact. 26, 169–176. Kutt, K., Martin, D., 1974. Effect of settled surfactants on the growth characteristics of Gymnodinium breve. Mar. Biol. 28, 253–259. Lang, S., Wagner, F., 1993. Biological activities of biosurfactants. In: Kosaric, N. (Ed.), Biosurfactants, 1st ed., Production Properties Applications, vol. 48. Marcel Dekker, New York, NY, pp. 251– 268. Latiff, S.A., 1971. The effect of oil slick dispersant, EssoCorexit 7664 and the joint effect with crude oil on the survival of marine glass fish, Ambassis sp. and palaemonid shrimps. Malaipian Agric. J. 48, 13–19. Lewis, M.A., 1991. Chronic and sublethal toxicities of surfactants to aquatic animals: A review and risk assessment. Water Res. 25, 101– 113. Lewis, M.A., 1992. The Effects of mixtures and other environmental modifying factors on the toxicities of surfactants to freshwater and marine life. Water Res. 26, 1013–1023. Maggi, P., 1972. Relative toxicity of even oil-spill emulsifiers. Rev. Trav. Inst. Peches Marit. 36, 121–124. McKenney Jr., C., 1986. Influence of the organophosphate insecticide fenthion on Mysidopsis bahia exposed during a complete life cycle I. Survival, reproduction, and age-specific growth. Dis. Aquat. Org. 1, 131–139. Mearns, A., Doe, K., Fisher, W., Hoff, R., Lee, K., Siron, R., Mueller, C., A. Venosa., 1995. Toxicity trends during an oil spill bioremediation experiment on a sandy shoreline in Delaware, USA. In: Proceedings of the 18th ARCTIC and Marine Oilspill Program, Edmonton, Canada, Environment Canada. Middaugh, D., 1985. Distribution, life cycle, taxonomy and culture methods 4. Silversides (Menidia). In: Peltier, W., Weber, C. (Eds.), Methods for Measuring the Acute Toxicity Effects to Freshwater and Marine Organisms, 3rd ed., US EPA, Environmental Monitoring and Support Laboratory, Cincinnati, OH, pp. 126–137. Nabholz, J.V., Miller, P., Zeeman, M., 1993. Environmental risk assessment of new chemicals under the Toxic Substances Control Act (TSCA). In: Landis, W.G., Hughes, J.S., Lewis, M.A. (Eds.), Section Five: Environmental Toxicology and Risk Assessment.

American Society for Testing and Materials, Philadelphia, PA, pp. 40–55. ASTM STP 1179. Nelson-Smith, A., 1978. Effects of dispersant use on shore life. In: Chemical Dispersants For the Control of Oil Spills. American Society for Testing and Materials, Philadelphia, PA, pp. 253–265. Parra, J., Guinea, J., Manresa, M., Robert, M., Mercade, Comellas, F., Bosch, M., 1989. Chemical characterization and physicochemical behavior of biosurfactants. JOACS 66, 141–145. PES-51â Technical Product Bulletin., 1995. Petroleum Environmental Services, Inc. San Antonio, TX. PES-61TM Technical Product Bulletin., 1995. Petroleum Environmental Services, Inc. San Antonio, TX. Poremba, K., Gunkel, W., Lang, S., Wagner, F., 1991a. Microbiotests with marine organisms for studying the toxic potential of biosurfactants and synthetic surfactants. Kieler Meeresforsch Sonderh. 8, 327–330. Poremba, K., Gunkel, W., Lang, S., Wagner, F., 1991b. Marine biosurfactants, III. Toxicity testing with marine microorganisms and comparison with synthetic surfactants. Z. Naturforsch. 46, 210–216. Pritchard, P.H., Mueller, J.G., Rogers, J.C., Kremer, F.V., Glaser, J.A., 1992. Oil spill bioremediation; experiences, lessons, and results from the Exxon Valdez oil spill in Alaska. Biodegradation 3, 315–335. Rasiah, V., Voroney, R.P., 1993. Assessment of selected surfactants for enhancing C mineralization of an oily waste. Water, Air, Soil Pollut. 71, 347–355. Rambeloarisoa, E., Rontani, J., Giusti, G., Duvnjak, Z., Bertrand, J., 1984. Degradation of crude oil by a mixed population of bacteria isolated from sea-surface foams. Mar. Biol. 83, 69–81. Singer, M.M., Saji, G., Jacobson, S., Lee, I., Tjeerdema, R.S., Sowby, M.L., 1994. Comparative effects of oil dispersants to the early life stages of topsmelt (Atherinops affinis) and kelp (Macrocystis pyrifera). Environ. Toxicol. Chem. 13, 649–655. Singer, M., George, S., Benner, D., Jacobson, S., Tjeerdema, R., Sowby, M., 1993. Comparative toxicity of two oil dispersants to the early life stages of two marine species. Environ. Toxicol. 12, 1855–1863. Singer, M., Smalheer, D.L., Tjeerdema, R.S., Martin, M., 1991. Effects of spiked exposure to an oil dispersant on the early life stages of four marine species. Environ. Toxicol. Chem. 10, 1367–1374. Stephan, C., 1977. Methods for calculating an LC50. In: Mayer, F.L., Hamelink, J.L. (Eds.), Aquatic Toxicology and Hazard Evaluation. American Society of Testing and Materials, Philadelphia, PA, pp. 65–84. Swannell, R.P.J., Tookey, D., McDonaugh, M., 1994. Bioredediation of oil spills: A state of the art review. Warren Spring Laboratory, Abingdon, Oxon, UK, 177 pp, ISBN: 0 85624 868 1. Tiehm, A., 1994. Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants. Appl. Environ. Microbiol. 60, 258–263. Tokuda, H., Arasaki, S., 1977. Fundamental studies of the influence of oil pollution upon marine organisms. Part 1. Lethal concentrations of oil spill emulsifiers for some marine phytoplankton. Bull. Jpn. Soc. Sci. Fish 43, 97–102. USEPA, 1993. Methods for measuring the acute toxicity of effluents to freshwater and marine organisms. Office of Research and Development, Washington, DC. EPA/600/4-90/027F. USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to marine and estuarine organisms. EPA Office of Research and Development, Washington, DC. EPA600-4-91-003. Yasunaga, Y., 1976. The influence of some pollutants on the survival of eggs and larvae of two species of flatfish, Limanda yokoharmae and Paralichtys olivaceus. Bull. Tokei Reg. Fish. Res. Lab. 86, 81–111.