Toxicity of Biodiesel

8/20/2019 Toxicity of Biodiesel

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DEGRADATION AND

PHYTOTOXI ITY

OF

IODIESEL

OIL

Contract Ref: CSA 2614

Caroline Birchall, Jonathan R. Newman & Michael P. Greaves

lnsritute of Arable Crops Research

Long Ashton Research Station,

CENTRE FOR AQUATIC PLANT MANAGEMENT

Broadmoor Lane,

Sonning-on-Thames,

Reading. RG4 OTH.

Tel.: (01734) 690072Fax.: (01754) 441730


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CONTENTS

CONTENTS

LIST OF TABLES

Page

i

ii

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

EXECUTIVE SUMMARY

INTRODUCTION

2.1 Background2.2 The Research

TOXlCirY TO ALGAE

3.1 Materials and Methods

3.2 Results and Discussion

TCXlCl-iY TO FLOATING MACROPHYTES



T0XlCll-Y TO SUBMERGED MACROPHYTES



TOXICITY TO INVERTEBRATES



TOXICITY IN AQUATIC MICROCOSMS

7.1 Materials and Methods7.2 Results and Discussion

BIODE,GRADATION AND FATE



GENERAL CONCLUSIONS

REFERENCES

APPENDICES

APPENDIX 1. Temperature, pH and Dissolved Oxygen

Concentrations in Aquatic Microcosms

APPENDIX 2. Eiodiesel Spectra and Ion Chromatograms

3

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4

5

8

8

9

12

1213

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1.

2.

3.

4.

5.

6.

7.

a.

9.

I O.

11.

12. The effect of biodiesel and marine diesel on Lymnaea peregra.

13.

14.

15.

A PPEN D IX 3 . Diesel Spectra and Ion Chromatograms 40

APPEND IX 4. Biodiesel and Diesei Ion Spectra Library Searches 44

A PPEN D IX 5. Mass Spectrometer and Gas Chromatography Instruments 4 9

APPENDIX 6. List of Suppliers 50

LIST OF TABLES

The effect of biodiesel and marine diesel on the growth of freshwater algae 6

The effect of biodiesel and marine diesel on specific growth rates of freshwater algae

The effect of biodiesel and marine diesel on growth of Lemna minor and Lemna minuta

The effect of biodiesel and marine diesel on growth of Lemna minor and Lemna minuta

The growth measured as the increase in the number of green fronds, of Lemna minor

and Lemna minufa on 7 day old medium contaminated with biodiesel and marine diesel

The effect of biodiesel and marine diesel applied to the water suriace on the growth

of Elodea canadensis and Myriophyllum spicatum.

The effect of biodiesel and marine diesel applied to the water surface on the growth

of Eiodea canadensis and Myriophyllum spicatum.

The effect of biodiesel and marine diesel applied to the hydrosol on the growth

of Eiodea canadensis and Myriophyllum spicatum.

The effect of biodiesel and marine diesel applied to the hydrosol on the growth

of Elodea canadensis and Myriophyllum spicatum.

The effect of biodiesel and marine diesel on Daphnia magna.

The effect of biodiesel and marine diesel on Gammarus pulex.

The mean10s

of weight of fish in aquatic microcosms contaminated with biodiesel

and marine diesel oil.

Partition of biodiesel and marine diesel into aquatic microcosm components. 26

Persistence of biodiesel and marine diesel in microcosms. 27

7

9

70

11

13

14

15

ia

19

19

23

ii


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1. EXECUTIVE SUMMARY

. .

A short pre iminary examination has been made of the comparative toxicity of rape methyl ester (biodiesel)

and marine diesel to a range of aquatic species. Partitioning and persistence of the oils in aquatic

microcosms have also been examined. The oils were tested at a range of dose rates calculated to represent

light, medium and severe spillages.

Bearing in mind that the data from short duration acute toxicity tests with small numbers of replicates are

not unequivocal the following general conclusions appear warranted. -

1 Biodiesel is considerably less toxic to microalgae (green and blue-green species) than marine dieseleven at high dose rates.

2 The floating plant, Lemna minor (Duckweed) was affected equally by both oiis, growth being reduced

by 65% at the highest doses. The related Lemna minuta, however, was significantly more

susceptibie to marine diesel, the highest dose killing the plant whereas biodiesel merely reduced its

growth by 60%. The susceptibility of both species was increased somewhat when nutrient

concentrations were reduced but still grew in the highest doses of biodiesel but not in marine diesel.

Submerged macrophytes grew erratically in the tests and produce imprecise data. Even so

Myriophyllum spicarum (Water milfoii) was clearly much more susceptible to marine diesel than

biodiese!. Nodea canadensis (Canadian pondweed) appeared to be severely affected by low doses

of biodiesel but less so by higher doses. Marine diesel was severely toxic at moderate to high

doses.

When biodiesel was applied to the sediments it appeared to stimulate growth of E. canadensis the

effect increasing with dose. Marine diesel was increasingly toxic to this species with increasing

dose. M. spicatum unusually was inhibited by both low and high doses of both oils but unaffected

by medium doses.

4 The invertebrates Daphnia magna (water flea), Gammarus puiex (water louse) and Lymnea peregra(water snai) were highly sensitive to marine diesel, all animals being killed relatively quickly at all

doses. D. magna and L. peregra were much more tolerant of biodiesel, effects only being severe

at the highest dose. G. pulex was more sensitive, mortality being high even at relatively low doses.

5 P.ssays of toxicity to mixed species were inconclusive, mainly due to the effects of the unusually high

ambient temperatures raising water temperatures during the tests. Observations suggest thatrainbow trout, Onchorhynchus mykill, were more severely affected by marine diesel than biodiesel.

Body weight loss was greater and the fish showed more severe behavioural symptoms, loss of

baiance, muscular spasms and erratic fin and gill movements.

6

Biodiesel forms discrete globules on the water sunace whereas marine diesei produced a continuous

slick that is potentially more damaging to invertebrates moving or breathing at the water suriace.

Eoth oils entered the water column from surface deposits, and contaminated plants and sediments,

very quickly in the microcosms. This is likely to be aided by the water agitation resulting from forced

ae:ation which may, to some extent, mimic the result of boat propeller action.

In the conditions of the test biodiesel formed waxy deposits on leaf sunaces, possibly by interaction

with calcium carbonates produced at the leaf surface during the elevated respiration induced by high

water temperatures.


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Biodiesel. as measured by GC/MS analysis of a major characteristic methyl ester ion, disappeared

from the water body, plants and sediments significantly more quickly than marine diesel. There was

no evidence of persistent intermediate compounds and it is likely that all the oii was rapidly

degraded to CO,.

7 The foregoing suggests that biodiesel is generally less toxic than marine diesel and persists less.

The toxicity it does exert may, especially at high contamination rates, cause some shifts in species

balance for a short time. Overall, however, biodiesel does appear to offer considerable

environmental advantage over marine diesel as a boat fuel, especially in iniand waterJJays of

conservation value. This conclusion needs confirmation by more detailed and precise examinations

of ecotoxicity, persistence, and fare.

2


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2. INTRODUCTION

2.7 Background

Agriculture has the potential to produce a large number of renewable fuels. The most obvious is

wood, but any plant tissue can be used, either directly or after processing, as a biofuel

in recent years there has been growing interest in Europe in the use of modified Rapeseed oil as

a diesel substitute. Rapeseed Oil Methyl Ester (RME), commonly known in Britain as biodiesel, is

produced by a simple esterification process:

5ooc

RAPESEED OIL + methanol p diester (RME) + glycerol

NaOH

A new approach to RME production is currently under investigation to determine if methanol, of

fossil fuel origin, can be replaced by ethanol, produced from biomass. This would result in a

biofuel produced entirely from renewable resources.

Using the whole of the UK’s set-aside land 1/6th of the arable land in the UK) to produce

rapeseed would supply only 6 of the UK current diesel fuel requirements, so there is little

possibility of biodiesel completely replacing conventional diesel. However, because vegetable oils

such as rapeseed oil are more readily biodegradable than mineral oils, biodiesei could give

environmental benefits in niche fuel markets such as National Parks and inland waterways. There

is a particular concern that water pollution by conventional diesel fuel is causing severe

environmental problems in many inland waterways, especially those such as the Norfolk Broads

which are exposed to heavy boat trafiic.

2.2 The Research

This study was established as a prelininary examination of the comparative toxicity of diesel and

biodiesel fuels to a range of aquatic species at doses which might result from spillages from boats.The partition of the fuels in the aquatic system and their degradation are also examined.

Two specific objectives are addressed:

A. To assess the toxicity to algae, macrophytes and invertebrates, using single-species

toxicity tests

B To determine the partition and degradation in water, sediment and plants determined in

aquatic microcosms


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3.2. Results and Discussion

Growth was calculated as the difference in cell density between the initial (T,) value and that at the end of

the 96 h incubation period (T,). All data was subject to analysis of variance. The values obtained are

shown in Table1.

Mean specific growth rates were also calculated Fable 2).

The growth of all the species tested was reduced by both diesel and biodiesel oils, though each species

shows different levels of response to the fue s.

Selenasirum capricomufum, the standard OECD test species, was particularly sensitive to marine diesel.

Even the lowest dose (1 g/l) was lethal to this species. Biodiesel, however, was considerably less toxic,

causing only a slight, non-significant, reduction in growth at the lowest dose and still permitting some growth

at the highest dose.

Effects on C.vulgaris

hf

aeruginosa and N. coccoides were somewhat similar. Biodiesel significantly

reduced growth at all concentrations though the effect at the lowest dose rate was relatively small. At the

highest dose rate growth was still measurable except withM.

aeruginosa, a blue-green species, which was

killed.

The other blue-green species, A. spiroides showed highly significant growth stimulation by biodiesel at all

dose rates. A similar effect was found with marine diesel at the two lower doses although at 100 g/l diesel

was signiiicantly inhibitory.

These results show clearly that biodiesei is significantly less toxic than marine diesel to a diverse range of

freshwater aigae, although it is toxic to ail but one of the species tested.

The results obtained with S. capricornutum show an extreme sensitivity to the diesel compared to the

majority of the algae tested. On the other hand the magnitude of its response to biodiesel was more

representative. This differential in the response of this species to two toxicants, compared to that of other

freshwater algal species, raises doubts about its suitability as a standard test organism that is supposedly

representative of a range of algal species.


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Table The ff t of biodiesel and marine diesel on the growth of freshwater algae.Values are means of 3 replicates with Standard Errors in parentheses.

Species Dose

Rate

Growth

Control Biodiesel Diesel

Chlorella vul ris 0 4.16 (0.22)

2 2.58 (0.27) f (0.32)

20 1.71 (0.23) 1.29 (0.40)

200 1.52 (0.52) -0.40 (0.21)

Mi c r ocys r i s 0 32. 1 ( 8. 2)

a e r u g i n o s a

1

16. 00 ( 3. 20) 12. 00 ( 0. 65)

10 1.72 ( 0. 28) 1.75 ( 0. 32)

10 0 - 0. 13 ( 0. 05) 6. 2 0 ( 0. 02)

n b en

0 1.59 ( 0. 31) spiroides

1 11.5 ( 4. 00) 1 7 . 8 0 ( 7. 40) 10 10. 40 ( 3. 5) 6. 76 ( 3. 20)

1 0 0 8. 31 ( 3. 0) 0. 42 ( 0. 11)

e l e n s t r u m

c a p r i c o r n u t u m

0 3.32 (0.77)

1 3.09 (0.50) ‘J.32 (0.04)10 0.79 ( 0. 30) - 0. 35 ( 0. 03)

100 0.36 (0.25) -1.33 (0.02)

nnochloris

c oc c oi c i e s

0 12. 8 ( 0. 55)

1 9.92 (0.50) 2.71 (0.33)10 1.86 (0.12) 1.37(0.08)

100 1.01 (0.16) 0.59 (0.05)


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Table 2 The effect of biodiesel and marine diesel on specific growth rates of

freshwater algae

Species

Dose

Rate

g/1

Specific Growth Rate

Control El

iodiesel Di esel

Chlorella vulgaris 0 0.496

2 0. 427 0. 337

20 0. 346 0. 296

200 0. 324 - 0. 308

Microcystis aeruginosa 0 1. 173

1 1. 002 0. 930

10 0. 479 0. 483

100 - 0. 144 - 0. 286

kabaena

spiroides 0 1. 224

1 1. 155 1. 265

10 1. 130 1. 025

100 1. 076 0. 383

Seienastrumcapricornutum

0 0.432

1 0. 471 a 148

10 0. 186 XI . 170

100 0. 102 -0. 163

Nannochioris coccoides 0 0.893

1 0.831 0.530 10 0. 449 0. 387

100 0. 330 0. 239


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4. TOXICITY TO FLOATING MACROPHYTES: Lemna minor and Lemna miwra.

4.1. Materials and Methods

Toxicity to L. minor and L.minfla

was determined through a series of modified toxicity tests (ASTM,

1991).

Lemna plants were grown in 15 ml of a prepared culture medium (3 partsJMl

and part soil extract)

in60

mm diameter Petri dishes. Plants from a stock culture were assigned randomly, 6 green fronds to

each dish. The dishes were incubated in a controlled environment room at a temperature of20”

II’C

and a light intensity of160

9

mol photonsTn.*

s”.

After an establishment period of 24 hours,oil

was added to the dishes to achieve dose rates ofI IO

and 100 g/l. Each dose rate and the controls were replicated three times. The dishes were returned tothe controlled environment room for a further 7 days and the number of green fronds in each dish and

their appearance were recorded daily.

Biodiesel is immiscible with water and, at low dose rates, tends to form discrete globules allowing the

plants to grow around the edges of the test dish without contact with the biodiesel. Attempts to

eliminate this problem, by emulsifying the biodiesel, were made using 5 emulsifiers. These were tested

for toxicity against the blue green alga Microcysfis aeruginosa in a standard 4 day bioassay on

Jaworski’s medium(JMI).

(See ‘3. Toxicity to Alga e, Materials and Methods’ for method) at dose

rates of 0.001, 0.01 and 0.1 g/l. This failed to find a non toxic emulsifier and the approach wasabandoned.

In

a further attempt to minimise the problem of biodiesel immiscibility, the experiment was

redesigned using 200 ml of growth medium (10% JMIv/v)

in 500 ml jars. Approximately half the volume

of medium was rapidly mixed with the appropriate dose using a Waring blender for 30 seconds and

transferred to the test jar. The blender was washed with the remaining100

ml of medium for 30

seconds. Control (oil free) solutions were treated similarly and oil treatments were prepared in the

sequence lowest to highest dose rates. The blender wascleaned

with detergent, thoroughly rinsed and

autoctaved between use for the two oils. Five dose rates 0.0125, 0.125,1.25,

12.5 and 125 g/l were

used. The minimum dose rate required to give complete surface cover was 12.5 g/l.

After blending, the medium was left for 24 hours before adding the plants which were taken from

laboratory stock cultures grown on the same medium (10%JMl)

in the same controlled environment

(temperature203C

/l”C, light intensity 160 p moi photonm“

s”) for at least 8 weeks. Plants were added

to each chamber randomly until all jars contained 3-5 plants each consisting of 3-4 fronds with a totalof15

fronds in each jar. The composition of fronds/plant in each jar was noted.

Change in plant colour, break-up of plants, destruction of roots and ihe number of fronds, were recorded

daily for 7 days. Every frond that visibly projected beyond the edge of the parent frond was counted

as ase,parate

frond. After 7 days a subsample (8 ml) of the medium in each jar was taken from about

half way down the jar andpiaced

in a60

mm glass Petri dish. Fresh plants from the stock culture were

added to the dishes, as described above, to give 15 fronds per dish and obsemations and number of

fronds were recorded daiiy for a further 7 days.


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Growth was calculated as the mean increase in the number of green fronds in each jar after 7 days.These values are presented in Tables 3, 4 and 5.

The results of the tests carried out in Petri dishes Fable 3) show clearly that both biodiesel and marine

diesel exhibit toxicity to Lemna minor at similar levels, the effect being non-significant at the lowest dose

rate but increasing markedly with dose. Growth at the highest dose rat6 was only about 35% of that

in the control.

Table 3 The effect of biodiesel and marine diesel on the growth of Lemna minor and Lemnam i x r t Values are means of 3 replicates with Standard Errors in parentheses.Petri dish Experiments.

Species Dose rate

g/1

Growth (increase in number of green fronds)


L. minor 7.3 (0.3)

1 5.7 (1.4) 6.7 (1.2)

10 4.0 (0.6) 3.3 (0.3)

100 2.7 (1.2) 2.3 (0.9)

L. minuta 0 18.7 (2.2)

1 14.3 (2.0) 13.0 (1.1)

10

11.0 (0.6) 3.3 (1.3)

10 0 7.3 (1.2) 0.0

Lsmna minuta, which had a faster growth rate than L. minor in this test, was significantly less affectedby biodiesel than by marine diesel, especially at the highest dose where diesel completely inhibited

growth but biodiesel permitted growth at approximately 40% of growth in control experiments.

i minuta grew less well in the large jars, only doubling frond numbers in 7 days compared to a three-

fold increase in the Petri dish experiment (Table 4). The growth of L. minor was the same in both

experiments, even though the growth mediium was considerably more dilute in the jars (10% JMl) then

in the Petri dishes (75% JMl). Considering this change in medium concentration it is, perhaps, not

surprising that both L. minor and L. minufa

were more susceptible to the growth-inhibiting effects of both

oiis in the jars than in the Petri dishes. As noted before, however, the effect of both oils was similar on

_.

minor but diesel was significantly more toxic to L. minuta than biodiesel. indeed, diesel at 1.25 g/l

prevented growth of L. mintia whereas in biodiesel at the same dose this species maintained more than

60% of the growth on the control.


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Table 4. The effect of biodiesel and marine diesel on the growth of Lemna minor and Len m

minuta Values are means of 3 replicates with Standard Errors in parentheses.

Experiment in jars.

Species Dose

rate

Q l

Growth (increase in number of green fronds)


L. minor 0.0000 19.7 (0.3)

0.0125 5.7 (2.2) 2.7 (0.9)

0.1250 3.0 (1.0) 6.0 (1.5)

1.2500 0.7 (1.9) -0.7 (0.9)

12.5000 6.0 (1.7) -6.0 (1.1)

125.0000 -1.7 (1.3) -7.6 (0.9)

L. minuta 0.0000 16.3 (3.6)

0.0125 11.7 (2.0) 15.3 (1.1)

0.1250 14.0 (0.6) 10.3 (3.3)

1.2500 10.3 (2.7) -2.3 (1.2)

12.5000 9.0 (1.7) -8.7 (0.3)

125.0000 5.3 (3.7) -11.7 (0.3)

It must be borne in mind that these tests were done using a nutrient medium to support growth of the

plants. The nutrient concentrations used in these experiments are greater than those usually foundunder natural conditions in the field. The degree of toxicity exhibited by the test species will be related

to the nutrient status of the receiving waters. Both biodiesel and marine diesel will have a greater impact

on plant health in oiigotrophic (low nutrient) waters than in eutrophic (high) or mesotrophic (medium)

waters. Results from these experiments are more likely to reflect toxicity in mesotrophic or eutrophic

waterbodies. Care should be exercised when extrapolating these data to natural scenarios.

The effect of blending the oil in the nutrient medium before introducing the plants could be said to

simulate the effect of boat propellers. It could be that, by dispersing the oil throughout the solution for

a period, it has allowed the release of water miscible components which may have contributed to the

increased toxicity found in the jar experiment.

This suggestion is supported by the results from the experiment in which plants were grown in nutrient

medium which had been in contact with the oiis for 7 days before planting. (Table 5). The susceptibility

of both species to both oils, but especially to biodiesel, was significantly increased. Thus, whereas 12.5

g/l of biodiesel permitted some growth (c. 30 to 50 of control) when it was introduced to the medium

at about the same time as the plants, the same dose caused some mortality of both species when the

oii had been aged in the medium for 7 days before plants were introduced. Undoubtedly, some

degradation had occurred in this time (s ee later) and may have increased the toxic response of theplants. Had the eariier experiment been continued for a funher 7 days, this additional toxicity may have

been seen. However, this effect was not seen in other toxicity tests. An alternative explanation is that

degradation reduced the viscosity of the oil and allowed a film to spread over the water surface, thus

increasing contact with the plant fronds. However, the toxicity of marine diesel, which forms a film onthe water in its raw state, is also increased by ageing. It is, therefore, uniikeiy that fiim formation is a

likely factor.

The magnitude of the toxicity effects found in these tests must be viewed with some caution.

Macrophytes are oft en very variable in their growth, resulting in large standard errors as found here.

Future experiments should involve much larger numbers of replicates.

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Table 5. The growth measured as the increase in the number of green fronds, of en ma

minor

and Lemna minuta on 7 day old medium contaminated with biodiesel and

marine diesel. Values are means of 3 replicates with Standard Errors ii

parentheses.

Species Dose

rate

g/1

Control

Mean average_ growth

(number of green fronds)

Biodiesel Diesel

L. minor 0.0000 5.0 (1.0)

0.0125 3.7 (0.3) 2.3 (0.3)

0.1250 1.3 (1.2) 0.0 (1.7)

1.2500 -0.7 (0.9) -4.3 (3.2)

12.5000 -4.7 (0.3) -5.3 (1.4)

125.0000 -5.3 (0.9) -10.7 (0.9)

L. minuta 0.0000 11.7 (0.9)

0.0125 12.0 (1.5) 4.0 (1.1)

0.1250 11.3 (1.2) 1.3 (1.2)

1.2500 6.3 (1.4) -0.7 (1.8)

12.5000 11.0 (1.5) -5.3 (1.8)

125.0000 -8.3 (0.9) -11 .o (0.6)

One problem noted here, but applicable in all the experiments, was that even at high dose rates, the

amount of oil to be added to each test unit was very small. For example, even in the large volumes in

the jars, doses of 0.125 g/l require that only 25 ~1 oil to be pipetted into the jar. The accuracy of

pipetting such small volumes, even with automatic micropipettes, is compromised by the viscous nature

of the oils, especially of biodiesel.

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5.

TOXICITY TO SUBMERGED MACROPHYTES: i? a

cmadensis and Myriophyllum spicaf~rrr


The toxicity of biodiesel and marine diesel to Elodea canadensis and Myriophyllum spicatum was assessed

using

14 day toxicity tests on individual plants grown in 3 litre glass jars.

Sediment, consisting of 500 g sieved soil under 250 g silver sand overlaid with 300 g washed gravel was

placed in each jar. Healthy single stems (IO cm long) were cut from plants gathered from an outdoor stock

pond. Each stem had a terminal meristem, no branches or roots and all were of a similar weight. Threestems were weighed and planted in the jar. The jars were filled with 2 litres of ::rater taken from a natural

bore hole source and the initial water level marked on each. The planted jars were randomised and kept

indoors at ambient temperature and light level for 48 hours, after which they were placed in a greenhouse

(25/15OC day/night: 16 h photo period) and oils were applied to the sunace of the water. The dose rates

used were 0.0125, 0.125, 1.25, 12.5 and 125 g/l. Three replications of each dose rate and of controls were

prepared.

Water losses due to evaporation were corrected each day using bore hole water and observations such as

changes in coiour and breakdown of plants were recorded. After 14 days the length and weight of each

plant in each jar was recorded.

A second experiment was established to investigate the toxicity of biodiesel and marine diesel as

contaminant in the hydrosol (hydrosol refers to the sediment component of the experimental system whenfully saturated with water). The experiment was set up as above but the oils were added to the soil before

the macrophytes were planted. The oil and the soil were mixed thoroughly for 5 minutes using a clean giass

rod and taking care to ensure mixing was similar in all the jars. After the oil and soil had been mixed

together, the sand and gravel were added and the macrophyte stems were planted. ?ars were incubated

as above for 14 days.

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5 .2 Resul ts and Discussion

The results presented in Tables 6 to 9 indicate that the growth of the two test species was erratic. The

values for weights of control plants vary by a factor of 2 or 3 between the two experiments and those for

steml ength by a f actor of 2 for M spicarum and by 7 for E. canacfensis. Standard errors were frequently

high, sometimes being as large as, or larger than, the mean values. in further experiments, in order to

improve the precision of the data obtained, large numbers of replicates will be required and efforts should

be made to use genetically homogenous plant material. The erratic results may reflect accurately the

situation in natural populations, where some individuals may be less susceptible than others. No figures can

be assigned to predicted population toxicity.

Table 6. The effect of biodiesel and marine diesel applied to the water surface on the growthof lodea canadensis and ylriophyilum sp ica tum

Speci es Dose

rate

g/1

Control

Growth* (g)

Biodiesel Diesel

E. canadensis 0.0000 0.16 (0. 07)

0. 0125 -0.09 (0. 11) - 0. 02 (0. 04)

0. 1250 -0.16 (0.04) - 0. 18 (0. 03)

1. 2500 -0.08 (0.05) 0. 05 (0. 13)72. 5000 0. 73 (0.06) 0. 11 (0.07)

125.0000 0. 18 (0. 06) 0. 16 (0. 03)

M. spicatum 0.0000 0.25 (0. 10)

0. 0125 0. 65 (0. 14) 0.01 (0. 11)

0.7250 0. 25 (0. 45) - 0. 25 (0. 06)

1. 2500 0. 22 (0. 44) - 0. 40 (0. 01)

72. 5000 0. 43 (0. 12) 0 71 (0.29)

125.0000 -0. 04 (0.37) 0 44 (0.32)

* Growth is expressed as increase in fresh weight during 14 days.

Fi gures in ) are standard errors of the means.

Beari ng in mind the erratic and imprecise nature of the data it is difficult to draw firm conclusions about

the effects of the oil treatments on the test species. Nonetheless some trends do appear in the data.

The response of M. spicarum to oils applied to the water surface appears to some extent to be similar to

that of other plant species such as algae and Lemna. Growth is relatively unaffected at all dose rates of

biodiesel except the very highest (725 g/ I ) where it severely inhibits growth expressed as fresh weight

gain or as increase in stem length. The effect of marine diesel is much more pronounced on growth

expressed as weight gain, doses as low as 0.0725 g/l causing virtual cessation of growth while at all

higher doses weight loss occurs, indicating plant death and decay. In contrast, the effect on growth

expressed as increase in stem length is less pronounced. Stem length is probably not a good measure

of plant response to toxicants such as oils as it can be markedly affected by shading caused by the oil

film on the water surface. This would be particularly marked with coloured marine diesel, especially at

the high doses which gave relatively thick surface layers. The effect of this shading increases stemlength, thus masking reduction in growth resul ti ng from toxi ci ty.

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Table 7. The effect of biodiesel and marine diesel applied to the water surface on the growth

ofEicdea

canadensis andh-fyriophyihm

spicatum

Species Dose

rate

g/1

Control

Growth* (cm)

Biodiesel Diesel

E. canadensis 0.0000 1.76 (2.44)0.0125 -1.37 (1.20) -0.32 (1.9:)

0.1250 -1 Xl (1.51) -2.90 (1.55)

1.2500 0.60 (2.23) 1.69 (2.53)

12.5000 2.88 (1.95) 1.90 (2.54)

125.0000 2.54 (2.35) 2.77 (1.37)

M. spicarum 0.0000 8.50 (1.65)

0.0125 15.78 (6.99) 3.39 (4.55)

0.1250 10.44 (4.76) 2.22 (3.42)

1.2500 10.89 (5.07) 1.89 (1.10)

12.5000 Il.41 (4.27) 0.67 (1.84)

125.0000 5.02 (3.74) 0.83 (0.87)

Growth is expressed as increase in length during 14 days.

Figures in ) are standard errors of the means.

Results withE.

canadensis were curious. Very low dose rates of biodiesel or marine diesel appeared to

completely inhibit growth measured as weight change or as stem length increase. In contrast higher

doses caused no discernable effect, other than the expected increase in stem length due to shading

effects.

Application of theok

via the hydrosol had somewhat different effects on E. canadensisFables

8 and

9). Low doses of biodiesel had a small inibitory effect on growth, both as weight gain and stem length

increase. This effect on plant weight was not measurable at 1.25 g/l and at higher doses plant weights

were significantly higher than control values. Stem lengths were inhibited to a similar extent at all doses

except 12.5 g/l when no significant effect was found. It is likely that this value is a spurious result,although the standard error is not particularly large.

Marine diesel affectedE.

canadensis in a more expected and progressive way. Eoth weight and stem

length were unaffected by doses up to 1.25 g/l but thereafter were progressive y and severely inhibited.

M. spicatum growth was inhibited by both biodiesel and marine diesel applied to the hydrosol (Tables 8

and9),

the data showing an unexpected pat-tern for both weight and stem length. Inhibition was

greatest at the lowest dose, reducing as dose increased until at 12.5 g/l of biodiesei growth was similar

to that in the control. At the highest dose, inhibition was again evident. A similar trend was found with

diesel though the least inhibition was found at 1.25 g/l.

14


18/53

Table 8. The effect of biodiesel and marine diesel applied to the hydrosol on growth of Eiodea

canadens ik

andMy iophy i l um

spicatum.

Species

E. canadensis

Dose

rate

g/t

0.0000

0.0125

0.1250

1.2500

12.5000

Control

0.38 (0.02)

Growth* (g)

Biodiesel

0.21 (0.04)

0.17 (0.06)

0.43 (0.18)

0.69 (0.08)

Diesel

0.38 (0.06)

0.47 (0.06)

0.56 (0.12)

0.17 (0.12)

125.0000 0.67 (0.12) 0.010 05j

M. spicatum 0.0000 0.89 (0.17)

0.0125 -0.26 (0.18) -0.23

0.1250

(0.25)

-0.08 (0.18) 0.01

1.2500

(0.10)

0.14 (0.19) 0.68

12.5000

(0.41)

0.89 (0.42) 0.03

125.0000

(0.17)

0.05 (0.09) -0.20 (0.03)

l

Growth is expressed as increase in fresh weight during 14 days.Figures in ( are standard errors of the means.

Table 9. The effect of biodiesel and marine diesel applied to the hydrosol on growth of

E f cdea canadensis and h yr iophy um spicatum.

Species

E. canadensk

Dose

rate

g/t

0.0000

0.0125

0.1250

1.2500

12.5000

125.0000

Control

12.22 (2.46)

Growth* ( cm)

Biodiesei

6.80 (1.41)

4.84 (2.57)

8.96 (3.31)

10.50 (1.12)

6.48 (2.04)

Diesel

13.28 (2.36)

12.54 (1.41)

11.41 (3.08)

8.92 (3.52)

0.47 (1.75)

M. sp i c a turn 0.0000 19.28 (2.86)

0.0125 5.72 (2.39) 5.23

0.1250

(3.22)

8.18 (4.08) 9.76

1.2500

(2.97)

11.24 (4.00) 15.99

12.5000

(4.85)

17.92 (5.00) 6.73

125.0000

(1.98)

5.89 (1.95) 1.33 (0.73)

Growth is expressed as increase in length during 14 days.Figures in are standard errors of the means.

15


19/53

Further experiments on the effect of contaminated sediment should concentrate on the effect on root

function. This could be measured by nutrient uptake studies.

As was found withLen-ma

and with the microalgae, the effects of the diesel oils varies according to

species, M. spicatum being more tolerant than E. canadensis in general. Also, further differences occur

depending on whether the oil is present on the water surface or in the hydrosol, the former exerting

more toxic effect than the latter. Overall, the results suggest that biodiesel is less toxic to these

submerged plant species, especially M. spicalum , than marine diesel. These differences in susceptibility

may be a factor in altering species balance and diversity in contaminated waters, especially with marine

diesel.

Toxicity tests on macrophytes are of necessity long term, especially if effects on flowering, seed

production and vegetative reproduction are to be assessed. Even in the simple preliminary test reported

here,14

days was required to obtain measurable effects. During such extended periods there may well

be indirect effects due to the oils. For example, they may have differential effects on diffusion of oxygen

and carbon dioxide into the water and between water and hydrosoil. This would be most likely at the

higher doses where cohesive films of oil are farmed on the water surface or where gross contamination

of sediments occur. Comprehensive detaiied tests need to recognise this and water and sedimentpH

and oxygen content should be monitored.

During the experiments it was noted that the leaves of the plants in the jars contaminated with diesel

darkened and turned black. A similar effect occurred in the biodiesel treatments but was generally limited

to the tips of the leaves. Some breakdown of parts of, or whole plants, occurred. This was moreevident in the diesel treatments and when the oil was applied in the hydrosol rather than to the water

surface. There was no distinct dose effect on this symptom.

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6.

TOXlClTY TO INVERTEBRATES:Daphnia

magna,Gammarus

pulew

and Lymnaea peregm

6.1

Materials and Methods

Acute toxicity to the water flea Daphnia magna assessed in a standard (OECD. 1984) acute

immobilisation test using dose rates of 1, 10 and 100 g/l.

Oils were added to 100 ml bore hole water in 250 ml flasks using a micropipette with four replications of

each dose. Treatments and controls were mixed on a rotamix for minute at 100rpm and were incubatedat 20 1°C and a light intensity of 160 p mol photon m“ s” for 24 hours before introducing invertebrates.

The Daphnia used in these experiments were reared in bore hole water in the controlled environment. Young

(24 h) Daphnia were chosen and randomly assigned to flasks until each flask contained 5 animals. Flasks

were covered with aluminium foil, stoppered with a foam bung and returned to the controlled environment

room.

The number of Daphnia trapped at the water surface or immobilised were counted at 24 and 48 hours.

Immobile is defined as ’ those animals unable to swim within 15 seconds after gentle agitation of the test

chamber ‘. The test was considered valid if<

10% of the Daphnia in the controls were immobiie or trapped

at the water surface.

The results of a preliminary test showed a large increase in immobility between treatments of 1 and 10 g/lso the test was repeated using 8 dose rates from 0.5gl*’

increasing by a factor of 0.6 up to 13.422gIW’.

Gammaruspuiex

and Lymnaea peregra were collected from a local stream and acclimatised in tanks of

borehole water for at least 48 hours.

Large (500 ml) glass jars were filled with 709 silver sand and 25Oml bore hole water. immature Gammarus

of similar sizes and colour were chosen from the sample population and randomly assigned to jars until all

the jars contained 5 animals. The jars were illuminated at an intensity of 125 p mol photon me2 se’ in a 8:16

hour light:dark regime at ambient temperature (21:l

l°C day/night).

After 24 hours, lgl-‘, 10 gl-‘, or 100 gl“ doses of biodiesel or marine diesel were applied by micropipette

to the surface of the water in the jars. Four replications of each dose rate and control were prepared, using

a random assignment of dose rates to jars.

At 24, 48, 72 and 96 hours after exposure to oii, the number of dead or affected animals were recorded.

Death and immobiiisation were defined as ‘a lack of movement and a lack of response to gentle prodding”.

Other observations such as erratic swimming, excitability, loss of reflex, disclouration, cessation of burrowing

and behavioural changes were also recorded. The test was considered to be valid if<

10% of animals in the

controls were deadafter

96 hours. Acute toxicity to Lymnaea peregra was assessed in the same way.

17


21/53


The values of mean percentage immobilisation of Daphnia magna at each dose rate (Table 10) and of mean

percentage death Fable 11) show cleariy that marine diesel is highly tokic toD.

magna, all animals being

immobilised at the lowest dose (0.5 g/l-‘) within 24 h. In contrast, although biodiesel was toxic, complete

immobility was only recorded at the highest dose (i3.4 g/l“) after 48 hours. At lower doses between 40 and

55% of the population tested showed no discernible effect of treatment. In all cases where immobilisation

occurred, it was followed by death, as determined by observation of heart beat. It is not known whether

transfer of immobiiised but living animals to clean water would allow recovery.

Table IO. The effect of biodiesel and marine diesel on Daphnia magna.

Dose

rate

9/l

24h

immobiiisation ( )

48h

Control Biodiesel Diesel Control Biodiesel Diesel

Experiment I.

0 7 (4.9) 10(5.2)

1 20 (8.2) 100 (0.0) 35 (5.0) 100 (0.0)10 80 (8.2) 100 (0.0) 95 (5.0) 100 (0.0)

100 85

(5.0) 100 (0.0) 100 (5.0) 100 (0.0)

Experiment 2.

0.000 2 (1.8) 3 (2.6)

0.500 20 (0.0) 100 (0.0) 45 (5.0) 100 (0.0)

0.800 35 (5.0) 100 (0.0) 50 (5.8) 100 (0.0)

1.280 25 (9.6) 100 (0.0) 25 (9.6) 100 (0.0)

2.048 30 (5.8) 100 (0.0) 30 (5.8) 100 (0.0)

3.277 40 (0.0) 100 (0.0) 50 (5.8) 100 (0.0)

5.243 25 (9.6) 100 (0.0) 50 (5.8) 100 (0.0)8.389 55 (9.6) 100 (0.0) 60 (8.2) 100 (0.0)

13.422 65 (5.0) 100 (0.0) 100 (0.0) 100 (0.0)

Data are mean ?L of animals immobilized with standard errors in ( ).

G.pulex

was more sensitive to the biodiese! thanD.

magna, a high proportion being killed within 24 hoursof exposure at reiatively low doses (Tabis 11). Even the lowest dose tested (1

g/r’)

caused5046

mortalityafter 72 hours, though there was no increase thereafter. As with D. magr;a, marine diesel was highly toxic,

h lowest dose causing 100% mortaiity in 24 hours.

18


22/53

Table 11. Effect of biodiesel and marine diesel on Gamman~s pulex.

Dose

rate

l

Control

24h

Biodiesel

Mortality )

Diesel Control

48h

Biodiesel Diesel

0

0

(0.0)

0 (0.0)

10 (5.8) 100 (0.0) 25 (0.0) 100 (0.0)

10

65 (5.0) 100 (0.0) 95 (0.0) 100 (0.0)

100 90 (5.8) 100 (0.0) 100 (0.0) 100 (0.0)

72h 96h

0 5 (5.0)

1 50 (23.8) 100 (0.0)

10

100 (0.0) 100 (0.0)

10 0 100 (0.0) 100 (0.0)

Figures in are standard errors of the means.

5 (5.0)

50 (23.8) 100 (0.0)

100 (0.0) 100 (0.0)100 (0.0) 100 (0.0)

Similar effects of marine diesel were found with L. peregra (Table 12), with 95% mortality at 1 g/I-’ in 24

hours and 100% mortality at 72 hours. Biodiesel, in contrast, exerted toxic effects only at 100 g/l ‘; mortality

increasing from 15 at 43 hours to 90% at 96 hours.

Table 12. Effect of biodiesel and marine diesel on Lyrnnaea peregra

Dose

rate

g/f

24h

Mortality (“A)

48h

Control Biodiesel Diesel Control Biodiesel Diesel

0 0(0.0) 5 (5.0)

1 0 (0.0) 90 (10.0) 0 (0.0) 95(5.0)10 0 (0.0) 95 (5.0) 0 (0.0) 95 (5.0)

100 0 (0.0) 95 (5.0) 15 (9.6) 100 (0.0)

72h

96h

~~

0 5 (5.0) 5 (5.0)

1 0 (0.0) 100 (0.0) 5 (5.0) 100 (0.0)

10 0 (0.0) 100 (0.0) 9 (0.0) 100 (0.0)100 50 (5.8) 100 (0.0)

Figures in ) are standard errors of the means.

90 (10.0) 100

o oj

19


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Marine diesei was highly toxic to all species at all On entratiOnS the effects becoming apparent very

quickly. Biodiesel was much less toxic especially at the lowest dose, representative of a small spillage.

There were, as with plants, different responses from different species.L.

peregra was very tolerant of

biodiesel whereas G. pulex and D. magna were tolerant Only Of low doses. Again, as was found for plants,

it seems that pollution of water with biodiesel may cause some shift in species balance in the habitat.

20


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7.

TOXICITY IN AQUATIC MICROCOSMS


Microcosms were established in small (0.6 x 0.3 x 0.3 m) aquaria containing a basal layer of 6 kg loam soil,

overlaid with 3 kg silver sand and then 3 kg of washed gravel. Tanks were filled with 40 I of water from a

bore hole and aerated with an air diffusing stone at 4 air minute-‘.

Stems of Elodea canadensis and Myriophyllum spicatum were carefuily selected, as in section 5, to have

stem weights of 26i 1 g or 82 1 g respectively. Plants were weighed accurately and they were planted into

the hydrosol. Twenty plants of each species were planted in separate but adjacent blocks so that not more

than 25% of the water body was occupied by plants.

Planted tanks were incubated under lights at 125 p mol photons m“ s-’ with an 8:16 hours light:dark regime

for 6 weeks before fish and invertebrates were introduced. All the animals had been kept in stock tanks,

under the same conditions as the microcosms, for at least three weeks before being used in the experiment.

Daphnia magna were placed in the microcosms in small fine-mesh enclosures to prevent predation by the

fish. Each enclosure containing 20 young (24 hr) animals was maintained at a depth of 20 cm in the water.

Lymnaea peregra were added directly to the aquaria such that each contained 3 large >3.5 cm long shells),

5

medium (2 to 3 5 cm) and 2 small ~2 cm) snails. The collective weight of the 10 snails added to each

tank was recorded.

Fingerling (3 inch) Rainbow trout (Oncorhynchusmykiss

) were weighed and three placed in each tm

The populated microcosms were then left to stabilize for one week before adding the oils. Throughout the

experiment the trout in each tank were fed 1.5 g of proprietary trout peilets each day and the evaporative

losses from the tanks made up with fresh borehole water.

Oils were added to the tanks using micropipettes to give dose rates of 0.002, 0.02 and 0.2 litresm”.

These

dose rates were calculated from the assumption that spillage of the complete contents of the average

pleasure boat fuel tank (50 gallons or 227 litres) would be restricted to an area of 1000 m*. This worst case

scenario would produce 0.2 I m’.

Due to constraints of space, available tanks and time, the doses were not replicated but three control +ankswere set up. Microcosms were observed daily for 3 weeks and any dead fish removed using a separate net

for each ‘tank. While every care was taken in this operation it is unavoidable to trap some of the surface oii

on the net, especially at the higher doses. The use of very long forceps can minimise this problem but even

then some oil is removed from the tank at each operation. Dissolved oxygen, pH and water temperature

were monitored throughout the experiment.

21


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7.2 Resutts and Discussion

Ambient temperatures during the experimental period were very high, frequently reaching 27-29’C. As a

result, water temperatures rose rapidiy (from the optimum18’C)

to25OC

and remained at that level

throughout the experiment. Although the elevated temperature had little effect on the dissolved oxygen

content of the water, it affected plant respiration and, as a consequence, pH rose to levels around 8.8.

Before the fish and animals were added to the tanks the pH was brought down to c. 7.0 by adding 0.1 m

HCl and 5.25 mol mJ HEPES

buffer to each tank. Subsequently pH values stayed relatively constant

berween 7 and 8 (Appendix 1).

The increase in water temperature adversely affected all the members of the microcosm community. Plants

became sickly and failed to grow in the controls as well as in the treatments. Similarly, invertebrate

behaviour was atypical and the mortality rate in the controls was high enough (> 10%) to invalidate the

tests. Fish were also affected and by two days after their introduction were gathering by the air-stones or

gulping air at the surface even though the dissolved oxygen content was close to 100% saturation. The

gulping of air at the surface may have led to intake of oil, especially marine diesel (see comments on oil

behaviour below), and contamination of the gills. The fish showed progressive darkening, a typical symptom

of stress, in both control and treated tanks. In the control tanks about half the fish died 5 or 6 days after

the treatment date, the remaining fish surviving until at least 9 days after treatment.

This test is invalid as a long term microcosm study but it is worth recording some observations of fish

behaviour, as these do indicate effects induced by the oils.

In the two iowest dose rates of biodiesel fish were initially little affected, showing only the stress symptoms

noted above. Ereathing and fin movements were no different to those in fish remaining in very large stock

tanks where conditions were optimal for growth. However, on day 2 after treatment all these fish died. Fish

in the highest biodiesel treatment all died 1 day after treatment, after showing erratic breathing, cessation

of fin movement and hovering at the water surface.

Low dose rates of marine diesel caused similar effects to biodiesel. Fish behaviour was normal on the first

day after treatment but on day 2 all the fish but one died. The survivor (at 0.02 Im’)

remained stationary

on the sediment until day 9 when it came to the surface, breathing erratically and showing severe loss of

balance. Fish exposed to the highest dose of diesel showed signs of stress within one or two hours of

treatment. They began to swim with their bodies in the horizontal plane, with complete loss of balance and

gulping air at the surface continuously. Toward the end of the first 24 hours symptoms were extreme loss

of balance and of coordination of movement. One fish was showing frequent spasms every 5 seconds.411

fish had died at the end of the first day after treatment.

All the fish were weighed at death and the weight losses are shown in Table 13. It is apparent that the

marine diesel treatment is associated with much greater weight loss than either the biodiesel treatment or

control which showed similar but lower losses. This would indicate that, as in all other tests, the marine

diesel is considerably more toxic than biodiesel. This is supported by the appearance of much greater

stress symptoms, such as loss of balance, erratic breathing and development of spasms, in fish in the

marine diesel treatments.

22


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Table 13. The mean loss of weight of fish in aquatic microcosms contaminated with biodiesel

and marine diesel oil.

Dose Rate g/I

0.002

0.0200.200

Control

7.57

3.925.10

Average Loss of Weight of Fish (g)

Biodiesel Diesel

4.71 8.27

8.11 13.363.12 28.72

The toxicity of biodiesel to fish and other members of microcosm populations needs to be established

properly in valid tests. This will require some modifications of the method used in this preliminary study.

Larger tanks, with at least 1m3

capacity, or smaller fish species should be used. Suitable species would

include 3-spined stickleback Gasterosteus aculeatus or fat-head minnow Pimephales promeks .

Fish

stocking rates should be below 50 gm’3.

Further, some provision for water-cooling should be made or tests

restricted to periods when ambient temperatures do not exceed 78’C. Acute toxicity can of course be

determined with fish kept in isolation (as in OECD guideiines 202,1992

and 204, 1984). However, toxicants

which are immiscible in water, such as biodiesel and marine diesel are probiematic in such tests. Dispersal

of the oils with emulsifiers may help hut this will necessitate screening to find non-toxic emulsifiers. Thedifferent behaviours of the two oils when added to water are important. Marine diesel spreads rapidly

forming a thin cohesive ‘siick’ on the surface. At the lowest dose the ‘slick’ was unable to cover the whoie

water surface but broke up into discrete isiands. Some of these became trapped at the water/glass

interface, this being aided by the water turbulence caused by forced aeration. At the remaining doses the

‘slick’ covered the entire surface. Biodiesel, in marked contrast, formed discrete globules which moved

around the surface with water currents. Many of these were trapped in the meniscus at the water/glass

interface and often coalesced to form larger aggregates. A significant proportion of the biodiesel, after 7

days, had visibly moved down into the water column and formed a white waxy deposit on surfaces of plants

and gravel. Diesel was not seen to move into the water column in this way although the sand layer of the

sediment became discoloured at the highest diesel dose.

23


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BIODEGRADATION AND FATE

8.1

Materials and Methods

The microcosms described in Section 7 were also used to determine the biodegradation and fate of the oilsin the aquatic ecosystem. Samples of water, plant material and sediment were taken from each microcosm

at regular intervals.

Water samples (5.0 ml) taken using a micropipette from a depth of 10 cm at each of 3 sites in eachaquarium, were combined into one bulk sample. The same sites were sampled on each sampling date.

Plant samples were obtained from E. c a na de ns i s by cutting off the terminal 5 cm from one plant in eachmicrocosm.

Sediment was sampled using a 25 ml wide-bore glass pipette inserted below the sand and gravel layers and

drawing up the suspension of soil. Samples were taken from 3 sites equidistant along the centre of the

length of the tank and combined into one bulk sample. Samples were taken from random sites on each

occasion. Samples were taken from each microcosm 1, 7, 14 and 21 days after application of the

treatments. Additional samples for analysis of degradation products were taken from the 0.2 I m2 treatments

only on days 3 and 5.

Plant samples were submerged in concentrated HCI for 2 minutes, to remove surface debris and epiphytes,and then washed gently in distilled water. The washed plant material was ground in a mortar with 1 g of acid washed sand and 15 ml of ethyl acetate. The slurry was centrifuged at 4000 g for 10 minutes and the

supernatant decanted into McCartney bottles, granular sodium sulphate was added to dessicate the solution

until no further ciumps formed and the dried solutions were stored at 4’C until analysed.

Sediment samples and water samples were mixed with 15 ml ethyl acetate and shaken at 60 rpm for 24

hours at room temperature. Thet extracts were allowed to separate for 30 minutes and the ethyl acetate

supernatants transferred to vials with added sodium sulphate granules to dessicate and stored at 4’C until

anaiysed.

All ethyl acetate extracts were analysed by Gas Chromatography/Mass Spectrometry GC/MS) after

concentration by evaporation in a stream of nitrogen at room temperature. Details of the equipment and

operating conditions are given in Appendix 4. Pure samples of both oils were also analysed. After

inspection of preliminary ion chromatograms certain ions were selected as suitable for indicating degradation

of the parer,! oils. Those selected as representative of the methyl esters and biodiesel were ions with

masses m/z - 74 and m/z-

264. The former is the largest ion characteristic of all unsaturated straight chainmethyl esters (C,,,) in biodiesei. Subsequent analyses showed that many peaks on the 74 chromatogram

were not from a biodiesel methyl ester but were contaminants, possibly from plastic tubing used duringconcentration of samples before analysis. Consequently, the fate and degradation of biodiesel was tracked

using only 264 ion chromatograms since 264 is known to come only from the C,,, methyl ester in biodiesel.

The ions initially selected as representative of marine diesel were m/z - 91 and m/z-

57. The former is

characteristic of all benzene-containing compounds and the latter of the homologous series of straight chain

aliphatic hydrocarbons. Subsequent analysis showed that several isomeric compounds repeatedly appeared

on the chromatograms and confused the analysis. As the straight chain saturated hydrocarbon region of

marine diesel peaks at hexadecane (C,,) its molecular ion, m/z- 226 was tracked in the samples instead

of 57 and 91.

The ion chromatograms produced show an absolute value for peak intensity, the values of each peak beingnormaiised

with

respect to the highest peak(-

100%). Thus, changes in absolute value for the selected

24


28/53

peaks can be monitored throughout the incubation period and indicate the rate of partition of the parent oil

into different components of the microcosm and give some indication of degradation.

25


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Analysis of the biodiesel oil showed that the major methyl esters present were (Cl 6:10, Cl 8:10, Cl 8:2, Cl 3and C22:l) which a library search identified as hexadecanoic X6:10), octadecanoic

(Cl lo),

octadecadienoic Cl8:2), octadecatrienoic Cl8:3) and erucic acids (C2.21). The major straight chain

saturated hydrocarbons in marine diesel were simiiariy identified as pentadecane, hexadecane and

heptadecane. (Appendix 3.2).

At one stage in this experiment airstones rose to the water surface where they caused some minor splashing. This contaminated a neighbouring control tank which then gave chromatograms with measurableamounts of m/z - 26-4. This was not used further in the analyses. Airstones were then fixed in position to

prevent recurrence of this probiem.

The absolute values for the selected tracking ions in the different mesocosm components are shown in Table14. The results for biodiesel and marine diesel cannot be compared quantitatively as the values used refer to different ions. Values for an individual oil can be compared and do indicate relative concentrations in thedifferent microcosm components. However, the sampling regime used did not account for the oil floatingon the surface of the microcosm and, so, the results cannot be used to indicate the total extent of degradation of the added oil, merely that degradation is or is not occurring. Analysis to accurate y quantify

degradation would require many more samples, and commensurate large increases in analytical time and

cost. This was not possible within the confines of this preliminary project.

Table 14. Partition of biodiesel and marine diesel into aquatic microcosm components.

Doserate

Time*

(days)

Biodiesel (m/z 264)+

Water Plant Hydrosol

Diesel (m/z - 226)+

Water Plant Hydrosol

0.002

1

7

14

21

0.02 1

7

14

21

0.20 1

3

5

7

14

21

293 24412 1756 412 1560 869

244 430 19 3 78 578 31420 500 406 574 47 99

ND 418 NT 455 71 319

200786 23740 1671168

2641 2349 12206

404 2284 61694 ND 867

1191 883 3594

143 NT 2251306 536 754159 1294 439

1892966 162222 4074291 48256 6487

75694

4085453 716145 3629056 1188 11813 127491022310

344064

2638971 2788 94413 2981924178 209644 233226 519 4018 26804

ND 14642 ND 14 1 2335 555

ND 2989 ND 43 4500 7324

t Time is days after treatmentt

Values given are absolute values; ND is not detected; NT is not tested.

The most obvious feature of ihe results is that both oils very rapidly permeate the whole microcosm.

Significant amounts are found in the water column, on plants and in the sediment only 1 day after application to the water surface. This is almost certainly largely due to the disturbance caused at the water

26


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surface and in the water column by the forced aeration of the system. In the case of biodiesel, the observed

movement of globules onto surfaces where they deposited as a white, waxy, residue (see Section 7) is also

a factor. This was only noticed visually after 7 days but undoubtedly occurred earlier. It would be

reasonable to expect this effect to be greatest at the highest dose rate and, indeed, the very high absolute

values seen in the water column, on plant surfaces and on the sediment might support this.

The second notable feature of the results is the failure to detect biodiesel in most of the samples of the water

column or sediments in the later stages of the experiment. This contrasts with the results for diesel which

was detectable in significant quantities at all sample dates, indicating a slower degradation rate. Dieselappeared to be particularly recalcitrant in the sediment, as was biodiesel on the plant surface. In the case

of biodiesel, the slow loss from plant surfaces is probably related to the formation of white waxy deposit.

A summary of the persistence of the total oils present in the water column, on plants and in the sediment

is shown in Table 1.5. These figures exclude that part of the added oil which floats on the water surface and,

as stated before, it is not possible to draw direct comparisons between values for biodiesel and diesel as

different ions have been measured.

Table 15. Persistence of biodiesel and marine diesel in microcosms. (The data are percentages

of the total ion detected)

Time

days)

iodiesel dose rate I me Diesel/dose rate I m“

0.002 0.02 0.20 0.002 0.02 0.20

l - 7 94.00 99.50 97.48 70.1 61.2 94.1

8 -14 4.60 0.46 2.50 13.1 12.2 2.4

5 -21 1.40 0.04 0.02 16.8 26.6 3.5

These data indicate that the tracking ion for biodiesel enters the microcosm from the surface giobules more

quickly than the diesel ion enters from the surface ‘slick’. Equally, the data show that the biodiesei appears

to disappear (degrade) more quickly than the marine diesel.

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9.

GENERAL CONCLUSlONS

It is important to bear in mind that although, whenever possible, standard toxicity tests have been used, the

constraints of time and resource imposed in this project mean that the results must be regarded with

caution. They represent only a preliminary and superficial survey of the toxicity of biodiesel compared to

that of marine diesel. Furthermore, the adverse environmental conditions that affected the microcosm

experiments detract from the value of data obtained in these experiments. The data for partition and

degradation of the oils will be least affected, the elevated water temperatures .accelerating decomposition

and, probably, being responsible for deposition of biodiesel as white waxy deposits on plant surfaces.

Nonetheless, it looks quite probable that biodiesel does in fact enter the water body more quickly than dieselfrom surface deposits. This, along with theglobular

distribution of the surface biodiese! on the water

surface, will result in less interference with oxygen diffusion into the water, and with surface breathing or

moving invertebrates, than the uniform ‘slicks’ produced by marine diesel.

Rapid entry is associated with more rapid disappearance from the system by biodiesel than marine diesel.

As the biodiesel appears to form few or no intermediate compounds during degradation, but rapidly to

proceed to CO,, it would again seem to have environmental advantage over the more persistent marine

diesel. This may be offset, albeit temporarily, by an increased biological oxygen demand.

This environmental advantage is strengthened by the significantly lower degree of toxicity of biodiesel

towards most of the algae, macrophytes and animals tested. However, it does have some toxic effect and

this may be enough to allow shifts in balance and diversity of aquatic species, especially where

contamination is severe.

Nonetheless, the environmental advantages which seem to be identified in this preliminary examination are

sufficient‘.o

warrant a more detailed and comprehensive examination and an analysis of the potential to use

biodiesel as a fuel for boats on inland waterways, especially those with recognised conservation value.


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10. REFERENCES

ASTM (1991) Standard Guide for Conducting Static Toxicity Tests with Lemna gibba G3’ E 1415 - 91

ASTM (1993) Standards on Aquatic Toxicology and Hazard Evaluation(7,093)

03978-80 1993) Practice for

Algal Growth Potential Testing with Seienasfzrm caprkomlrtum 79.

OECD (1984) Algal Grow&h Inhibition Test, Guideline207

OECD (1984) Daphnia sp. Acute lmmobiiisation Test, Guideline 2M

OECD (1984) Fish, Prolonged Toxicity Test, Guideline204

OECD (1992) Fish, Acute Toxicity Test, Guioeiine 203

THOMPSON, A.S.; RHODES, J.C.&

PEITMAN, (1988) Culture Collection of Algae and Protozoa -

Catalogue of Strains. Published by CCAP, Cumbria, UK ISBN 1 871105 0 3

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APPENDIX 2. Biodiesel Spectra and Ion Chromatograms

1. Biodiesel Spectrum showing the peaks of the major methyl esters.

2.

Ion Chromatogram for 1% biodiesel (centre graph) shows that the ion with a mass 74

peaks at SCAN 850 but also peaks at SCAN 720, emphasising the confusion

encountered during analysis.

3. An example of an Ion Chromatogram produced from the analysis of a sample taken from

a microcosm contaminated with biodiesel.

4. An example of an Ion Chromatogram produced from the analysis of a sample taken from

a control microcosm.


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APPENDIX 3. Diesel Spectra and Ion Chromatograms

1.

Diesel Spectrum.

2.

3.

Ion Chromatogram for 1% Diesei showing ions with masses 57 and ‘31.

An example of an Ion Chromatogram produced from the analysis of a sample taken from amicroccsm contaminated with marine diesel oii.

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APPENDIX 5. Mass Spectrometer and Gas Chromatography Instuments.

GAS CHROMATOGRAPHY INSTRUMENT

COLUMN

film thickness

phase

INJECTION MODE

CARRIER GAS

PRESSURE

GC PROGRAMME

IONlSlNG VOLTAGE

: Carlo Erbahlega

: 25m x 0.22m

: 0.25p

: 3PX70 (specifically apprcpriate for far,y

acid methyl esters)

: spiit/splitless

: Heiium

: 0.5 kgcm-’

:7O”C

for min, upto 240°C a: 6°C per min,

staying a: 240°C for 30 min

: 70 electron volts

MASS SPECTROMETER

Source Temperature: Kratos MSeORFA Medium Easolution

: 200°CInterface Temperature

SCAN: 250°C _

interscan Time

: 500 daltons 0 40 daltons at :s per decade

: 0.25


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APPENDIX 6. List of Suppliers

Giodiesel

Oil Imported from Italy, supplied by Mr. Stephen TUG, Dalgety Oiiseeds Dalgety

Agricultura Ltd., Cheveley House, Fordham Road, Newmarke?, Suffolk CB8 7AH.I

Marine Diesel Oil Supplied by Better Boating Co., Caversham, Zeading, Berks.

Chemicals HEPES buffer supplied by Sigma Chemical Co. Ltd., Fancy road, Poole, DorsetBH17 7BR.

Granular sodium suiphate suppiied by BDH Laboratory Supplies, Merck Ltd., Hunter

Boulevard, Luttemorth, Leicestershire LEI 7 4XN.

Hydrochloric acid and Ethyl acetate supplied by Fisons Scientific Equipment,

Bishop Meadow Road, Loughborough, LeicestershireLE11

ORG.

Toxicity of Biodiesel

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