ecotoxicity testing of bioremediation agents for shoreline

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環境毒性学会誌（Jpn. J. Environ. Toxicol.），9（2），115 -131，2006

Ecotoxicity testing of bioremediation agents for shorelineoil-spill clean up

Tomoyasu Hirano1,4,5,*, Rie Masho2, Daisuke Koizumi3, Shigeo Tabata3,6, Kentaro Suzuki3,

Mai Sakabe3, Yoshio Nakano3,7, Nobuyuki Sato3, & Ryuichi Sudo4,5

Industrial Policy Department, Mitsubishi Research Institute,

Otemachi Chiyoda-ku, Tokyo 100-8141, Japan1

Center for Environmental Information Science,

4-7-24 Kudan-minami, Chiyoda-ku, Tokyo 102-0074, Japan2

Institute of Environmental Ecology, IDEA Consultants,

Inc.（former METOCEAN Co., Ltd）, Riemon 1334-5, Oigawa, Shizuoka 412-0212, Japan3

Center for Environmental Science in Saitama, Kami-tanadare 914, Kisai, Saitama 347-0115, Japan4

Department of Environmental Science and Human Engineering, Saitama University5

*Corresponding author.

Present Address: Corporate Marketing Division, Mitsubishi Research Institute,

4-12-2 Higashi-shinagawa, Shinagawa-ku, Tokyo 140-0002, Japan

Tel:+81-3-6711-6896; Fax:+81-3-3277-3431

E-mail address: [email protected]

Present Address:

Okinawa Branch Office, IDEA Consultants, Inc. 6

Tokyo Branch Office, IDEA Consultants, Inc. 7

This study was performed as part of a contract research project with the Japanese Ministry of the

Environment（the Environment Agency）

ABSTRACTWe present the results of a test run done for protocols drafted to evaluate the toxicity

of different bioremediation agents against spilled-oil using marine organisms and crude oil.The aim of the study was to evaluate experimentally the change in toxicity of oil whenmixed with bioremediation agents and fertilizers used on oiled shores to enhance oilbiodegradation under actual environmental conditions prevailing at the oiled site beforeconsidering any large-scale application. The protocols were designed to use three marinespecies at different trophic levels, i.e. diatoms Skeletonema costatum, rotifers Brachionusplicatilis, and red sea bream Pagrus major, and to carry out algal growth inhibition, zoo-plankton acute immobilization, and fish acute toxicity tests. The agents tested are: InipolEAP22TM, oleophilic fertilizer; TerrazymeTM, bioaugmentation agent; and SuperIBTM/LinstarTM, slow-release fertilizer. While Super IBTM/LinstarTM did not enhance the toxicity of oil, clear suppression of diatomcell growth was observed when 100 ppm of Inipol EAP22TM was added to oil. The concen-

1. IntroductionBioremediation is a multidisciplinary strategy

or process that uses microorganisms, plants, or

enzymes to detoxify pollutants in various environ-

mental media. Oil spill bioremediation at shores

embraces biodegradation of oil pollutants using

microorganisms, which is a cleanup technology to

enhance the natural function of the material cycle

and the energy flow of the marine ecosystem

（Skipper & Turco, 1995）. Therefore bioremediation

is regarded as an energy-saving and environmental-

ly sound technology for the ecosystem, compared

with other physical recoveries or chemical treat-

ments of spilled oil, such as washing with pressur-

ized hot-water and dispersant or detergent treat-

ment.

The tanker Exxon Valdez ran aground on Bligh

Reef in the Gulf of Alaska on March 24, 1989,

spilling approximately 41,000 m3 of Alaskan North

Slope crude oil. Extensive efforts were made to

recover the oil, but the conditions and circum-

stances at the seaside resulted in about 2,090km of

coastline being contaminated with oil. Some beach-

es were heavily oiled, particularly those of islands

in Prince William Sound which were directly hit by

spilled-oil in the path of the slick.

Bioremediation was one of several important

shoreline cleanup programs after the Exxon Valdez

oil-spill accident in Prince William Sound in 1989,

involving the application of nitrogen and phospho-

rus contained in fertilizers to stimulate the growth

of indigenous hydorocarbon-degrading microorgan-

isms within the intertidal zone（Pitchard & Costa,

1991）. These microorganisms proved highly effec-

tive in degrading the hydrocarbon component of the

spilled oil（Atlas, 1991; Hoff, 1993; Swannell et al.,

1996）. Application of fertilizer nutrients was shown to

be effective according to Exxon. The oil biodegrada-

tion rate increased by three to five times or more

compared with the previous rate（Bragg et al.,

1992; 1994）. This caused many who heard about

the Exxon report, even in Japan, to place excessive

expectation in bioremediation as being the ultimate

definite recourse in cleanup technology for oil spills

and contaminated seashores.

A Russian tanker, Nakhodka, carrying approxi-

mately 19,000 tons of heavy oil, was torn apart and

wrecked during a winter storm in the Sea of Japan

on January 2, 1997. The bow-half of the ship drifted

toward the east and along the west coast of Japan’s main island for five days before it became strand-

ed at Mikuni, Fukui Prefecture, releasing approxi-

mately 6,000 tons of oil on the way. The rough sea

made the oil surge toward the coast in spite of oil-

fences and intense recovery activities on the water.

The fierce wind and high drift, typical of the condi-

tions in the Sea of Japan during that season,

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Ecotoxicity testing of bioremediation agents

tration is estimated to be of the same range as that of the interstitial water at the ExxonValdez remediation site after the spraying of Inipol EAP22TM. This suggests that the toxiceffect of the agent on some marine microalgae could be greater than the harm done by theoil itself. Effective test methods for evaluating the ecotoxicity of bioremediation agents, therefore,need to allow comparison of the toxicity of the agent with the coexistence of contaminantsand their biodegraded products, reproducing the actual conditions at oiled seashores.

Key words: ecotoxicity, bioremediation, oil-spill, acute toxicity, marine organisms

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brought the oil up above the high tide line. The

black sticky oil covered the soil and vegetation, and

remained there. Due to this accident, approximately

1,200 kilometers of shoreline alongt the Sea of

Japan were contaminated by oil. That is, the coasts

in 10 out of Japan’s 47 prefectures were heavily

impacted by the spilled oil（MDPC, 1997）. The shoreline cleanup operation was performed

by the professionals of the Maritime Disaster

Prevention Center and its contractors according to

their manuals including the use of heavy machin-

ery, whereas many volunteer citizens, virtually

using their own hands, joined the operation. At the

waterfront, the oil was recovered most efficiently by

dippers from the surface and the oil-stained rocks

were wiped to get rid of the covering oil. Since such

cleanup operation by hand was a painstaking and

time-consuming job, and forced many citizens to

work under exposure to oil as well, this operation

was considered unsafe or adverse to their health

（OER, 1998）. Therefore, after the bulk of oil had

been removed, effective and safe shoreline cleanup

measures, requiring less manpower, were eagerly

demanded.

Some bioremediation agents to inoculate oil-

degrading bacteria or to activate indigenous species

for accelerating the natural oil-breakdown process

were used mainly in field tests by the manufactur-

ers of agents and fertilizers in agreement with the

local stakeholders（Harayama, personal communi-

cation; Tsubouchi, personal communication）.Almost all the agents originated outside Japan.

Even though their safety might have been con-

firmed under the regulations of each manufacturing

country, there was no legislation concerning the use

of such agents on the shoreline in Japan. The only

experience of large-scale use of such agents was the

Exxon Valdez accident in Prince William Sound in

1989. Therefore, the Fisheries Agency & the

Environment Agency（1997）, the Japanese govern-

ment, jointly issued a notice as guideline for local

governments on the use of bioremediation agents

and oil dispersants in coastal areas in the after-

math of the Nakhodka accident, which called for

precautionary measures to ensure that they would

not have any harmful effect on human health or on

the environment. The notice virtually worked as a

ban on the use of bioremediation in the oil spill

cleanup operation at the time, because there was no

rule or protocol to follow to ensure the safety of the

bioremediation agents or their innocuousness in the

field.

We believe that a strict framework for the safe

use of any chemical/microbe in the open field is

indispensable. There is no border in the sea to con-

tain applied bioremediation agents or fertilizers.

The coastal areas to which the agents or fertilizers

would be applied are areas of a wide range of

socioeconomic activities and considerable popula-

tions of people will come in contact with the

applied agents or fertilizers. Furthermore, a con-

siderable part of the Japanese diet consists of

seafood from coastal areas, and any agent or fertil-

izer put into the water can possibly contaminate

the coastal food web. Therefore, every precaution

should be taken to prevent any harm to the coastal

environment from the application of bioremediation

agents or fertilizers.

At the same time, we suggest that bioremedia-

tion is one of several potentially environment-friend-

ly oil-cleanup measures. The technology may need

more efficient and safer tests in the field. Before

testing the agent or fertilizer in the Japanese field,

we must know the risks of the agent or fertilizer to

human health and the environment. Toxicity tests

for products have been conducted mainly on mam-

mals and aquatic organisms living in freshwater

（e.g. green algae, Daphnia, zebrafish）, while much

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Fig 1. Schematic model of acute toxicity tests of marine organisms

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fewer appropriate ecotoxicity data are available on

marine organisms（NETAC, 1993; Blenkinsopp et

al., 1995; Kinoshita et al., 1996; Tokoshima, 1997;

Koyama, 1998, Fisheries Agency 1992; 1995; 1999）.We, therefore, designed experimental procedures to

check the ecotoxicity of the agents or fertilizers for

spilled oil bioremediation in the marine environ-

ment, and examined the feasibility of the test proto-

col. Here we present the results of our examina-

tions and discuss the need for the establishment of

ecotoxicity testing on marine organisms.

2. Materials & Methods2.1 Preparation of test seawater

Seawater was taken from the shore of Oigawa-

port, Shizuoka Prefecture, filtrated using a sand fil-

tration apparatus, and stored at room temperature

in the dark before use. Arabian light crude oil was

kindly provided by Nisseki-Mitsubishi Sekiyu K.K.

Negishi Refinery and Showa Shell Sekiyu K.K.

Yokkaichi Refinery. The seawater and the oil were

mixed at a ratio of 9:1, stirred for 20h at 20℃ and

60 rpm using 5 L of closed flask, and left standing

for 6h to allow the water and the oil to dissociate.

The water layer was removed by calm siphoning

and used as the test seawater.

Bioremediation agents were added at the time

when the oil and the seawater were mixed. The test

seawater prepared hereby was considered to reflect

the conditions of the oiled shore and used for the

ecotoxicity tests. As control experiments, we used

the oil-saturated test seawater prepared as

described above without adding bioremediation

agents（oiled control without agents） and the sea-

water without adding oil or bioremediation agents

（oil-free control）. Toxicities of oil and bioremedia-

tion agents or fertilizers were evaluated by compar-

ing the results with two controls: oiled control with-

out agents and oil-free control（Fig. 1）.

2.2 Bioremediation agents and fertilizersInipol EAP22TM and Oppenheimer TerraZymeTM,

which was listed on the US EPA product schedule

and shown to effectively enhance oil biodegradation

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in the field（US EPA, 2005）, were chosen for the

toxicity test as examples of bioremediation agents.

The commercial agricultural fertilizer Super IBTM/

LinstarTM which was dealt with in some case studies

on marine oil spill bioremediation in Japan was

chosen as well（Ishihara et al., 1995; Maki &

Harayama, 1998; Hozumi et al., 2000; Tsutsumi et

al., 2000a; 2000b）. Inipol EAP22TM, an oleophilic fertilizer, was

kindly provided by James W. Lynn of Elf Atochem

North America Inc., Philadelphia USA. It was

stored at 4℃ in the refrigerator until use.

Oppenheimer TerraZymeTM, a microbial culture, was

donated by Masakazu Kono of Oppenheimer

Technology Japan. Fertilizers used were Super IBTM

（35%（w/w）nitrogen content as isobutylidene

diurea（IBDU）blended with a small amount of

formurea; manufactured by Mitsubishi Chemicals,

Japan）, which slowly releases nitrogen; and

LinstarTM 30（35%（w/w）phosphorus content as

calcium and/or magnesium phosphate salts; manu-

factured by Mitsubishi Chemicals, Japan）, which

slowly releases phosphorus. These synthetic fertiliz-

er granules have been shown to effectively stimu-

late microbial degradation of crude oil both in a

beach-simulating microcosm which has tidal action

and in which fresh seawater is continuously sup-

plied（Ishihara et al., 1995） and in a field test in

a coastal area（Maki et al., 2002）. Super IBTM and

LinstarTM were mixed at the ratio of 5:1 and used as

one agent in the tests. TerraZymeTM and Super IBTM

/ LinstarTM were stored at room temperature in the

dark.

2.3 Algal growth inhibition testMarine diatoms, Skeltonema costatum and

NIES-324 were obtained from the National Institute

for Environmental Studies. Diatoms were cultured

in a modified SW II medium containing 72.0 mg /L

KNO3, 4.5g/L KH2PO4, 1.05g/L disodium glyc-

erophosphate, 0.5g/L Fe-EDTA, 0.5g/L tris

（hydroxymethyl） aminomethane, the pH of which

was adjusted to 8.0-8.2（Iwasaki et al., 1961）, sup-

plemented with 60.7g/L Na2SiO3・9H2O as silicon

source. After culturing for three days at 20℃ and

4000 μmole photons・m-2・S-1（14h light / 10h dark）,an algal growth inhibition test was performed.

Prior to the tests, the growth conditions of the

diatom cells were observed under a microscope to

confirm that cells were filled with cytoplasmic mat-

ter.

Fifty milliliters of oil-saturated test seawater

supplemented with the nutrient was filtered using

disk-filter with a pore size of 0.2 μm（Millipore）and transferred to 100mL autoclaved flasks. The

final concentrations of each component in the test

seawaters were: 36.1 g/L KNO3, 2.8g/L K2HPO4,

0.23g/L Fe-EDTA, 60.7g/L Na2SiO3, 0.1g/L MnCl2・4H2O, 0.06g/L Vitamin B1, 0.03mg/L Vitamin B12,

and 0.37g/L Na2-EDTA as complete media. Cultured

diatom cells were inoculated at the final concentra-

tion of 10,000 cells/mL.

Inipol EAP 22 and TerraZymeTM were prepared

in concentrations of 0.1, 1.0, 10, 100 mg/L in the

above mentioned test seawater. Super IBTM /

LinstarTM was tested at 1.0, 10, 100, 1000 mg/L.

Two controls, the oiled and oil-free controls without

bioremediation agents and fertilizers, were taken in

every experimental series.

Algal growth was measured by two methods:

the concentration of chlorophyll a was measured by

luminescence spectroscopy and the cell number was

counted under a microscope. Prior to measurement

of the concentration of chlorophyll a using lumines-

cence microscopy, 50 mL of medium containing

cells were filtered by glassfiber filter（Whatman

GF/F）and extracted with 90% acetone during 2-3h

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reaction.

During the 7-day test period, algal growth was

measured every 24 hours.

2.4 Zooplankton acute immobilization testWe used the rotifers Brachionus plicatilis for the

ecotoxicity test. The rotifers were maintained at

20℃ in a 100L tank filled with a mixture of seawa-

ter and the dechlorinated freshwater mixed at a

ratio of 3:1. The water was continuously agitated by

strong aeration. Before the test, the rotifers were

acclimated to 100% seawater for 7 days at the same

temperature and aeration conditions as those for

the preculture. During the maintenance or cultur-

ing period, a fair amount of chlorella was fed to the

rotifers.

After 7-day culturing in the seawater, the acute

immobilization test was performed. One hundred

fifty milliliters of the oil-saturated test seawater was

put in a 200mL flask. The rotifers were inoculated

at a final concentration of 100 individuals/mL.

Test organisms were selected by sizes of over

100 microns using mesh filters to strain away

smaller individuals. The rotifers for the test condi-

tion were kept at 20℃ and 5000 μmole photons・m-2・S-1（14h light / 10h dark）. No aeration was

done, however, 25mL of pure oxygen was added to

the top of the flasks at 0h and 24h.

Zooplanktons were counted and observed at 0,

24, and 48h. Immobilization was judged based on

the observations of the rotifers' cilia and chrysalis

movement under a microscope. After the observa-

tions, the planktons were fixed with formaldehyde

and their population density was determined.

We tested over the range of 1.0 to 1,000 mg/L

for Super IBTM / LinstarTM. The experimental design

was the same as for the above mentioned algal

growth inhibition test.

In order to check the overall sensitivity of

Brachionus plicatilis against chemical substrates, an

immobilization test with benzene was also per-

formed. The response of organisms to benzene was

reported by Kono and Tabata（1966）. This test

was conducted with the oil-free seawater. Benzene

was prepared in concentrations of 0, 100, 250, 500,

750, and 1,000 mg/L.

2.5 Fish acute toxicity testJuvenile Japanese red sea bream, Pagrus major,

of about 4-5 cm in length and 4-8 g in weight was

used for the toxicity test. The fish were kept in the

seawater for 7 days before the test. During this

period, the seawater was changed once in two or

three days and the fish were fed Eduke-ru #2

（Chubu Shiryo Co.）, feed mixture for juvenile red

sea bream, every day with an amount equivalent to

5% of their body weight.

For the test, 10 fish were placed into 10 L of

the oil-saturated test seawater, and kept at 20℃and 5000 μmole photons・m-2・S-1 14h light/10h dark

without feeding over the test period. Aerated con-

stantly at the rate of 350-400 mL/min.

We tested 1.0-1,000 mg/L of Super IBTM /

LinstarTM using the standard protocol of 48h LC50.

Since no significant change was observed, we

extended the test till 72h. At 72h, body-length, stan-

dard length, and weight of the surviving fish were

measured.

2.6 Oil component analysis of test waterThe constituents of the oil were measured by a

gas chromatograph equipped with mass spectrome-

ter（GC/MS）analysis. GC/MS analysis was car-

ried out on the test seawater with 100 mg/L of

Inipol EAP22TM. About 25 mL of n-hexane was

added to 300mL of the test seawater and vigorously

stirred for 10 min to extract oil components. The

mixture was left standing to allow the water and

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the oil to dissociate. After discarding the water

layer, the remaining n-hexane layer was dehydrated

with sodium sulfate anhydrate. After dehydration,

the n-hexane extract was concentrated under a

nitrogen gas stream. A portion of the concentrated

n-hexane extract was subjected to GC/MS.

3. Results3.1 Algal growth inhibition test

We examined algal growth inhibition of Inipol

EAP22TM, TerraZymeTM and Super IBTM / LinstarTM,

which were used in the bioremediation field tests

on the oiled shores after the Nakhodka accident.

In all the tests, oil inhibited algal growth meas-

ured in terms of the chlorophyll a concentration.

Figure 2 illustrates the results of algal growth inhi-

bition tests of the bioremediation agents and fertil-

izers Inipol EAP22TM, Oppenheimer TerraZymeTM,

and Super IBTM /Linstar in terms of the chlorophyll

a content. The proliferation of diatoms was sup-

pressed in all oiled test groups irrespective of

whether the bioremediation/fertilizer agents were

added or not. Concentrations of chlorophyll a,

except for oil-free controls, decreased up to day 2 to

day 5 and then started to increase. Compared with

the oil-free seawater control, algal growth was sup-

pressed in the oil-saturated seawater with or with-

out bioremediation agents. In all the cases, suppres-

sion became apparent on day 2 of the experiment

when algal growth in the oil-free control recovered

from the usual growth lag on day 1. Suppression of

algal growth in the oiled groups was shown as the

gradual growth curve slopes below that of the oil-

free control in Figure 2. The growth in the oiled

Fig. 2. Algal growth inhibition tests of bioremediationagents and crude using Skeletonema costatumGrowth curves of a diatom, Skeletonema costatum, weretraced by the concentrations of chlorophyll-a. In Fig2a, Inipol EAP22TM was added to seawater andcrude oil, and the water phase of the water-oil mixturewas used for the test to evaluate toxicity（see experimen-tal procedures for details）．Closed boxes: oil-free seawater control without agents;Open boxes: oiled seawater control without agents. Closedtriangles, open diamonds, open and closed circles: oiledseawater supplemented with 0.1, 1.0, 10, and 100mg/L ofInipol, respectively.Fig2b shows the results for TerraZymeTM. The symbolsare the same as for Inipol EAP22TM above.Fig2c shows the results for Super IBTM / LinstarTM.Closed triangles, open diamonds, open and closed circles:oiled seawater supplemented with 1, 10, 100 and 1000mg/L of Super IBTM/LinstarTM, respectively. The introduc-tory notes for the two controls apply analogously.

Fig 2a

Fig 2b

Fig 2c

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groups started to recover after 2 to 5 days.

All the growth curves of experimental groups

with added agents and fertilizers were the same as

the oiled control without the agents or fertilizers,

except for Inipol EAP22TM. The growth curves in

terms of the chlorophyll a content of the tests with

TerraZymeTM and Super IBTM / LinstarTM were identi-

cal to the oiled control without any agents, i.e., no

toxicity due to the bioremediation agents added was

confirmed. Even at a concentration of 100 mg/L,

neither of the agents significantly affect the diatom

proliferation compared with the oiled control.

InipolEAP22TM, however, inhibited algal growth at

100ppm, although, in the lower concentrations of

0.1, 1.0, and 10 mg/L, no difference in growth com-

pared with the oiled control was observed.

Diatom cells in 100 mg/L of Inipol EAP22TM

were smaller in size and contained fewer photosyn-

thetic pigments（Figure 3）, resulting in inhibited

growth as observed by the chlorophyll a content,

though the number of cells was not less than in the

oiled-control.

3.2 Zooplankton inhibition testZooplankton acute immobilization tests using

seawater rotifers, Brachionus plicatilis, were carried

out on Super IBTM / LinstarTM. Since Inipol EAP22TM

at 100 ppm inhibited algal growth considerably, we

decided not to test Inipol EAP22TM in the field tak-

ing into account its strong toxicity. At the same

time, we sensed rather firm opposition to

TerraZymeTM, a bioaugmentation agent including

introduced microorganisms. As we first wanted to

select and check the usability of bioremediation

agents for field tests, we decided to conduct toxicity

tests on of Super IBTM / LinstarTM.

The zooplanktons in the oil-free control prolifer-

ated during the test period of 48 hours. The num-

ber of individuals increased from 100 to 140 organ-

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Fig. 3. Shapes of Skeletonema costatum cells on day 7Diatom cells were photographed using phase differencemicroscopy X 400.The top panel is a photograph of diatom cells in the oil-free seawater control. The center panel shows them inoiled seawater control. The bottom panel shows diatomcells in oil and 100 mg/L Inipol EAP22TM.

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isms/mL in the oil-free control（Figure 4b）. In the

oiled control without agents and in the test series

of Super IBTM / LinstarTM concentrations, the

rotifers proliferated in a similar manner. Neither

significant growth inhibition nor immobilization

was observed in either of the two controls or any of

the experimental series. Super IBTM / LinstarTM

showed no toxicity against the zooplanktons at the

concentrations tested（1, 10, 100, and 1,000

mg/L）. The number of zooplanktons gradually

increased during the 48-hour test period.

The overall sensitivity of rotifers to chemicals

was verified using benzene. At benzene concentra-

tions of 100, 250, and 500 mg/L, the number of

zooplanktons did not change from the start, and

remained at the 100 individuals/mL level during

the 48-hour test period（Figure 4a）. In 750 mg/L

of benzene, the number of individuals decreased to

36 and 8 individuals /mL after 24 and 48h, respec-

tively. In 1000 mg/L of benzene, no living individ-

ual was observed after 24h.

3.3 Fish acute toxicity testFor the same reason as described for the zoo-

plankton test above, the acute toxicity of Super IBTM

/ LinstarTM to fish was tested using juvenile red sea

bream, Pagrus major. Only one out of 10 individuals

died at 1000 mg/L, the highest concentration test-

ed. No lethal or other adverse effect was observed

in four test groups and one control group of 10 fish

each. There was no difference in body length,

weight, or in other observable traits in the surviv-

ing fish between the control and other groups test-

ed with Super IBTM / LinstarTM（Table 1）.

3.4 Degradation of oil during the testFigure 4 shows the results of GC/MS analysis

for the test water in 100 mg/L concentration of

Inipol EAP22TM before（0 days）and after（7 days）the inhibition test. Although n-alkanes species of

small molecular weight were included in the test

water at 0h, alkane molecules smaller than n-tri-

cosane（CH3（CH2）21CH3）almost disappeared

during 7 days of testing（Figure 5a）. Polyaromatic

hydrocarbons（PAHs）and alkylbenzene com-

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4

4

Fig. 4. Number of living individuals of Brachionus pli-catilis individualsBrachionus plicatilis were fixed in by formaldehyde andimmobilization effects were observed. Mobile individualswere counted as alive.Fig 4a shows the results of sensitivity test of Brachionus

plicatilis toagainst benzene as determined in a sen-sitivity test. Closed boxes, open boxes, closed tri-angles, open triangles, open circles, and closed cir-cles: 0, 100, 250, 500, 750, and 1000 mg/L of ben-zene, respectively.

Fig 4b shows the sensitivity against Super IBTM/LinstarTM. Closed boxes are the negative controlwithout any agents or oil. Open boxes are the pos-itive control with soluble part of oil. Closed trian-gles, open diamonds open circles, and closed cir-cles are 1.0, 10, 100, and 1000 mg/L concentra-tions of the agent containing oil, respectively.

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Table 1. Length and weight of testing individuals of Pagrus major individuals used in theSuper IBTM / LinstarTM toxicity test of Super IBTM / LinstarTM.

Ten individuals were used for the tests. The lengths and weights of all fish were measured.

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pounds contained in the test water as water-soluble

fractions of oil also decreased（Figure 5b. 5c）. Half

the naphthalene and most of the toluene were lost

after the test.

4. Discussion4.1 Schemes for testing the ecotoxicity ofbioremediation agents on oiled shores

To justify field testing or further large-scale use

in large-scale after an oil spill, bioremediation

agents and fertilizers should be free from toxicity

against living organisms of different trophic levels

under the application conditions. Protocols using

alga, zooplankton, and fish have been established by

the OECD and JIS（OECD, 1984a; 1984b; 1984c;

JIS, 1992; 1998; 2002） to test the ecotoxicity of

chemicals. However, no ecotoxicity test has been

established for bioremediation agents designed to

accelerate degradation of spilled oil on the coast.

The existing OECD test guidelines and the

Japanese dispersants test protocols（MOT &

JSMQA, 1984）are not suitable for testing bioreme-

diation agents for the following reasons:

- Test methods are designed for freshwater

organisms（e.g. green alga, Daphnia, zebra fish）,and - An evaluation of the agents’toxicity when

mixed with oil has not been stipulated.

There is general consensus, we believe, that eco-

toxicity of bioremediation agents against marine

organisms needs to be checked before they are used

on oiled shores. And since these agents are going to

be used in the presence of oil, their toxicity must

naturally be tested in the presence of oil.

Based on these premises, this study took up the

design of ecotoxicity tests with marine organisms

and test conditions similar to an oiled shore. Our

methods improved on the already established test

procedures for oil dispersants inasmuch as the zoo-

plankton acute immobilization test was added and

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5

5

5

Fig. 5. Concentrations of some hydrocarbons containedin algal growth inhibition test seawaterSeawaters supplemented with 100 mg/L of Inipol EAP22TM were sampled at day 0 and 7 of the test and con-stituents of hydrocarbons were analyzed by GC/MS.Fig5a shows the concentrations of n- alkanes. Fig 5b and5c show those of PAHs and alkylbenzenes, respectively.Bright bars: before the test（0 day）; Dark bars: after thetest（7days）.

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marine fish was used instead of freshwater species.

Our protocols also include procedures for evaluating

the ecotoxicity of bioremediation agents and fertiliz-

ers when mixed with oil.

In addition, for an ecotoxicity test procedure to

be useful, it should be applicable to a variety of

agents and fertilizers. To test the general effective-

ness of our protocols, we carried out ecotoxicity

tests with different kinds of bioremediation agents

and fertilizers, i.e., oleophilic fertilizer（Inipol

EAP22TM）, microbial culture（Oppenheimer

TerraZymeTM）, and slow release fertilizer（Super

IBTM / LinstarTM）. We propose that ecotoxicity test protocols for

oil-spill bioremediation agents be in accord with the

premises mentioned above. Our experimental

schemes were to evaluate toxicities of agents in sat-

urated oil, thus to create entirely novel and unique

protocols. Since our experimental conditions were

designed to closely resemble the actual state of the

application site,, that is, the oiled shore, our proto-

cols may serve as a useful base for the universal

framework of a procedure to objectively evaluate

ecotoxicities of bioremediation agents, oil disper-

sants, and any other chemicals used in the cleanup

of oiled shorelines. For developing standard proto-

cols, it may be necessary to conduct further tests

using species other than what we used here and

smaller and more vulnerable fish, for example,

Pagrus major of less than 1 g in weight.

4.2 Ecotoxicity of the three agents and fertil-izers tested4.2.1 Inipol EAP22TM

Inipol EAP22TM at 100 ppm exerted considerable

algal growth inhibition over the toxicity of oil alone

or oil with the agent at lower concentrations.

Acute toxicities of Inipol EAP22TM had been

reported: 135 ppm（Inipol EAP22TM only）and 125

ppm（Inipol EAP22TM plus #2 fuel oil）in terms

of 96h LC50 for Menidia beryllina; 23 ppm（Inipol

only）and 35 ppm（Inipol EAP22TM plus #2 fuel oil）in terms of 48h LC50 for Mysidopsis bahia（United

States Testing Company, 1995）. This report,

assessed the toxicity of #2 fuel oil itself at 280 ppm

in terms of 96h LC50 for Menidia beryllina and 47

ppm in terms of 48h LC50 for Mysidopsis bahia.

The toxicity data derived from our study, that is,

the suppression of algal growth below 100ppm in

Fig2a, and those of Elf Atochem, manufacturer and

distributor of Inipol EAP22TM, were almost identical.

When assessing the toxicities of bioremediation

agents at the application site we must take in

account the toxicity of oil solubilized by the oil-dis-

persal effect of the applied remediation agents.

Emulsified small-molecular-sized oil droplets can be

more toxic to marine organisms than the solidified

oil mass on the shore. However, our experiments,

excluding the case of 100ppm of Inipol EAP22TM,

did not reveal an intensification of the oil’s toxicitywhen mixed with the agents, which is proof of the

oil-dispersing effect of the agents that allows more

oil to resolve into the aquatic phase and more

severely affect organisms. One hundred ppm of

Inipol EAP22TM did not affect the quantity of

diatom cells but suppressed cell size and pigment

content shown in terms of the Chlorophyll a

amount. This observation provides an interesting

insight into the toxicological mechanisms of the oil

and agents acting on diatoms, which are important

producers in marine ecosystems.

We conclude that part of the problem with oil

dispersants is that their toxicity appears to be

linked to their effectiveness: Effective dispersants

are too toxic, and nontoxic dispersants are ineffec-

tive. Nevertheless, as oil dispersants with less toxici-

ty were recently developed（MDPC 2000）, the

argument of the effectiveness of oil dispersants and

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Hirano et al

their toxicities was not made the focus of our

report. It should be mentioned, however, that biore-

mediation agents themselves or the bacteria they

activate may initiate surfactant activity that in

some cases could intensify the toxicity of oil.

In the bioremediation clean-up project following

the Exxon Valdez accident in Alaska, total nitrogen

concentration measured in interstitial water below

the KN135 site, where Inipol EAP22TM and

Customblen had been sprinkled on the surface, was

10,500 microM-days for 72 days（Bragg et al.,

1993）. As Inipol contains 7.4% of nitrogen, the

equivalent concentration of Inipol EAP22TM in inter-

stitial water was estimated at 27.6 ppm. At the

same time, the nitrogen concentration of the inter-

stitial water exceeded 350 microM on the day the

agent was sprinkled. Under these circumstances,

Inipol EAP22TM equivalent was estimated at more

than 90 ppm. Considering that 100 ppm of Inipol

EAP22TM with oil in our experiment clearly sup-

pressed the cell growth of diatoms, the use of Inipol

EAP22TM to this extent may have led to ecotoxic

effects exceeding those of the spilled oil itself, at

least immediately after sprinkling at the KN135

site.

4.2.2 TerraZymeTM

TerraZymeTM showed no additional toxicity with-

in the range of 100ppm in the algal growth inhibi-

tion test. However, we were unable to examine the

ecotoxicity above this range using our procedures.

TerraZymeTM contains clay as the principal ingredi-

ent（M. Kono, personal communication）, and oil-

degrading microorganisms are said to cling to this

clay. According to the procedures of our study, any

precipitations including clay from TerraZymeTM were

removed during preparation of the test seawater.

The clay from a TerraZymeTM content of over 100

ppm was deposited while the seawater and oil mix-

ture was left standing for dissociation, and we were

unable to determine whether microorganisms and

chemical ingredients contained in TerraZymeTM dis-

sociated into the water-oil mixture or deposited

together with the clay. If a considerable part of

microorganisms and chemicals from TerraZymeTM

tend to deposit with together with the clay, we may

further examine the ecotoxicy of this agent against

benthic organisms.

4.2.3 Super IB TM / Linstar TM

Super IBTM / LinstarTM, slow release fertilizers,

showed no additional toxicity over oil within the

range of 1 to 1,000ppm in any of the tests in terms

of diatom growth, immobilization of rotifers, or

acute toxicity to red sea bream. Studies investigat-

ing the toxicity of these fertilizers against marine

organisms are not available. We, therefore, decided

that field-testing these fertilizers would be appropri-

ate to confirm the efficacy of bioremediation on the

oiled shore.

4. 3 Oil degradationDegradation of oil was observed during the test

period. In GC/MS analysis, hydrocarbon molecules

of oil were shown to become degraded in the test

seawater during the algal test. The smaller hydro-

carbon molecules disappeared from the test water

after day 7 of the test period. This seems to indi-

cate that hydrocarbon-degrading microorganisms

had entered the test system through the seawater.

4. 4 Future studies for bioremediation onoiled shorelines

One of the provisions for use in case of large-

scale oil spill accidents should be that ecotoxicity

tests are completed by the producers or distributors

of bioremediation agents and fertilizers. If the safe-

ty of the agents in ecosystems was tested in

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advance, bioremediation on oiled shores could be

carried out quickly and effectively enough with min-

imum adverse effects in case of an accident.

The fact that only a few case studies of bioreme-

diation on oiled shores have been reported may

explain why there are no established general meth-

ods or guidelines for the use of bioremediation

agents on oiled shores. The problem becomes even

more difficult when we realize that such methods

should be adapted in each case to the site specifici-

ty, the characteristics of the oil that was spilled on

or drifted to the shore, and the environmental con-

ditions. This makes it more necessary to field-test

bioremediation agents under actual conditions at

the oiled shore in order to ensure the effective use

of these agents and thereby a successful cleanup of

oiled shores.

Moreover, field tests to evaluate the effects of

nutrient addition or microbial augmentation may

provide unique opportunities to archive insights on

microbial biodegradation of oil in the field.

The main issue involved in spraying bioremedia-

tion agents on the coast is their potential adverse

effect on marine ecosystems. Especially in Japan,

with its densely populated coastline, people could

face a risk from being exposed to chemicals or bio-

logical agents used on the shores. The socio-eco-

nomic effect on the fisheries industry and other

coastal activities also needs to be considered. That

is why it is so important to establish good guide-

lines, including ecotoxicity test protocols, to ensure

the safety of bioremediation.

Once the health risk has been, ecotoxicity tests

are the most important prerequisite for an agent to

be released into the environment through field

tests. In our study, we designed ecotoxicity test

schemes and presented examples of test protocols

for marine organisms and crude oil.

5. ConclusionThe main issue involved in spraying bioremedia-

tion agents on oiled coasts is their potential adverse

effect on marine ecosystems. To conduct the field

tests needed before any large-scale application in

case of an oil spill, bioremediation agents must

have been tested to prove that they do not have a

significantly toxic effect on living organisms in the

environment when actually applied to oiled

seashores.

Our study proposes ecotoxicity test schemes

and presents examples of test protocols for marine

organisms and crude oil using three bioremediation

agents and fertilizer as typical examples on three

marine species at different trophic levels, i.e. algal

growth inhibition, zooplankton acute immobiliza-

tion, and fish acute toxicity test. As described

above, our test protocols proved the ecotoxicity of

bioremediation agents against marine organisms

and the need for conducting checks before they are

used on oiled shores.

Effective test methods to evaluate the ecotoxici-

ties of bioremediation agents and fertilizers, there-

fore, need to allow a comparison of the toxicity of

the agent or fertilizer with that of contaminants

and of the biodegraded products, which replicate

the actual conditions of the contaminated shore-line

after a large-scale oil spill. Furthermore, as

oleophilic fertilizers for bioremediation and oil-dis-

persing chemicals have nearly overlapping effects

on aquatic organisms, it is hoped that this study

will contribute to unifying the rules with those for

dispersants.

AcknowledgmentsFor their steady advice, patient discussions and

constant encouragement throughout this study, we

wish to express our appreciation to Dr. Jiro

Koyama, Dr. Masakazu Furuki, Dr. Tsuneo

Tsukatani, Dr. Shigeaki Harayama, Dr. Masataka

Watanabe, Dr. Takaaki Yajima, Dr. Hideaki Maki,

Akira Tsubouchi, and Suguru Ogura, all members

of the Committee for Oil Spill Bioremediation on

Seashores, which met at the Center for

Environmental Information Science under the spon-

sorship and supervising of the Environment

Agency. We would also like to thank Akihiko

Ishikawa, Masao Kato, Norihiko Tanaka, Shin-ichiro

Maki, Nobuaki Yoshiguchi, Hiroshi Katsumata,

Yoshimi Matsui, Satoru Murai, Yasunori Yamashita,

and Shinichi Gotoh of the Environment Agency for

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