1

FINAL TECHNICAL REPORT

PART I: PUBLISHABLE FINAL REPORT

 CONTRACT N° : NNE5-2001-00744 

 PROJECT N° : S07.16365 

 ACRONYM : BIOTOX 

 TITLE : An assessment of bio-oil toxicity for safe handling and transportation 

 PROJECT CO-ORDINATOR : Centre de Coopération Internationale en Recherche 

Agronomique pour le Développement (Cirad) 

 PARTNERS : - Aston University 

 - Bundesforschungsanstalt für Forst- und Holzwirtschaft (BFH) 

 REPORTING PERIOD : FROM 01 January 2003 TO 30 June 2005 

 PROJECT START DATE : 01 January 2003 DURATION : 30 Month 

 Date of issue of this report : 

Project funded by the European Community under 

the ‘Energy FP5’ Programme (1998-2002)’ 
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Part I: Publishable Final Report 

Table of contents 

PART I: PUBLISHABLE FINAL REPORT ................................................................................................................. 2

0. EXECUTIVE PUBLISHABLE SUMMARY ................................................................................................4 

I. PUBLISHABLE SYNTHESIS REPORT......................................................................................................7 

I.1. OBJECTIVES AND STRATEGIC ASPECTS ............................................................................................7 

I.1.1 Social and economic objectives of the project. .........................................................................7 

I.1.2 Scientific/technological objectives of the project. ....................................................................7 

I.2. SCIENTIFIC AND TECHNICAL DESCRIPTION OF THE RESULTS............................................................8 

I.2.1 Selection, production and collection of 21 bio-oil samples to be assessed.............................10 

I.2.2 Assessment of the physico-chemical composition and the toxicology of the collected samples

.........................................................................................................................................................11 

I.2.2.1 Bio-oils physico-chemical characterisation .......................................................................12 

a) Chemical and physical characterization ..............................................................................13 

- Physical-chemical analysis.........................................................................................13 

- Gas chromatographic analysis....................................................................................17 

- Reproducibility of pyrolysis reaction and GC-analysis .............................................21 

- PAH analysis ..............................................................................................................22 

-UV/VIS-Spectral Analysis ..........................................................................................25 

b) Correlations between oil properties and production processes............................................26 

I.2.2.2 Bio-oils Toxicological screening tests...............................................................................27

a) Preparation of the test solutions...........................................................................................27 

b) Bacterial strains....................................................................................................................27 

c) Metabolic activation system ................................................................................................27 

d) Mutagenicity experiment .....................................................................................................27 

e) Evaluation of the results ......................................................................................................27 

f) Results..................................................................................................................................28 

I.2.2.3 Bio-oils Ecotoxicological screening tests ..........................................................................29 

a) Algal growth inhibition test .................................................................................................29 

b) Acute toxicity in daphnia magna .........................................................................................30 

c) Results of the ecotoxicological tests:...................................................................................31 

I.2.2.4 Bio-oils Aerobic biodegradability in fresh water...............................................................34 

a) Methodology........................................................................................................................34 

b) Test system ..........................................................................................................................35 

c) Results..................................................................................................................................35 

d) Conclusions..........................................................................................................................37 

I.2.2.5 Conclusions and selection of the representative sample for the full toxicological study ..37 

I.2.3 Full toxicological study of one selected representative sample ..............................................39 

I.2.3.1 Tests A6 Water solubility, A8 Partition coefficient and C2 Acute toxicity to daphnia.....40 

I.2.3.2 -Test A14: Explosive properties ........................................................................................40 

I.2.3.3 -Test B4 : Dermal Irritation In Rabbit ...............................................................................41

a) Methodology........................................................................................................................41 

b) Interpretation of results and classification...........................................................................42 

c) Results..................................................................................................................................43 

d) Conclusions..........................................................................................................................43 

I.2.3.4 –Test B3: Acute Dermal Toxicity in Rat and B5 Eye Irritation in Rabbit ........................44 
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I.2.3.5 –Test B1 tris: Acute Oral Toxicity in Rat..........................................................................44 

a) Methodology........................................................................................................................44 

b) Results..................................................................................................................................45 

c) Conclusions..........................................................................................................................45 

I.2.3.6 –Test B6 : Evaluation of skin sensitization potential in Mice Using LLNA .....................45 

a) Methodology........................................................................................................................45 

b) Results..................................................................................................................................46 

c) Conclusions..........................................................................................................................46 

I.2.3.7 –Test B7: 7-day study oral route in rats.............................................................................47

a) Methodology........................................................................................................................47 

b) Clinical examinations ..........................................................................................................47 

c) Pathology .............................................................................................................................48 

d) Statistical analysis................................................................................................................48 

e) Results..................................................................................................................................48 

f) Conclusions..........................................................................................................................49 

I.2.3.8 –Test MAS In Vivo: Bone marrow micronucleus test by oral route gavage in mice........49 

a) Methodology........................................................................................................................49 

b) Results..................................................................................................................................50 

I.2.3.9 – Test MNV In Vitro: Micronucleus test in L5178 TK+/- mouse lymphoma cells ..........50 

a) Methodology........................................................................................................................51 

b) Results..................................................................................................................................52 

c) Conlusion.............................................................................................................................52 

I.2.3.10 – Conclusions of the full toxicological characterisation of the selected sample .............52 

I.2.4 Recommendations for safety procedures and dissemination of the results.............................52 

I.2.4.1 Dissemination ....................................................................................................................53 

a) Conferences and experts meetings.......................................................................................53 

b) Published articles .................................................................................................................53 

c) Web site: ..............................................................................................................................54 

I.2.4.2 Notification of Bio-oil as a new substance to be placed on the Eu market .......................54 

I.2.4.3 Editions of safety documents based on the obtained results..............................................56 

I.3. ACKNOWLEDGEMENTS..................................................................................................................57 
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0. Executive publishable summary 

1 BACKGROUND AND INTRODUCTION 

Pyrolysis is one of the three main thermochemical routes 

to convert biomass into useful primary energy products. Fast 

pyrolysis has been the subject of active research for 

approximately the last 25 years in order to obtain optimum 

yields of bio-oils which can be used in engines for the 

generation of electricity or after refining in transport. 

Development has now reached a stage where 

commercialisation is being attempted. To successfully 

achieve this, the question of safety procedures for ensuring 

human health and preservation of the environment needs to 

be addressed. Within Biotox project bio-oils from all major 

current producers have been collected to determine factors 

influencing toxicity, eco-toxicity, mutagenicity and 

biodegradability. 

2 SCREENING TESTS 

19 samples were collected from producers utilising fast 

pyrolysis technologies, namely rotating cone, ablative, 

fluidised bed and circulating fluidised bed pyrolysis systems. 

Furthermore, two slow pyrolysis samples were included 

2.1 Summary of tests 

An overview of the areas that were investigated in the 

screening tests of the 21 bio-oil samples is shown in Table I. 

A full description of the test methods will be published in the 

final project report and will also be available on the PyNe 

website. 

Table I: Details of performed screening tests 

Type of study Detailed analysis 

Physico chemical 

(performed by IWC) 

Chemical composition 

Viscosity (at 20 & 50°C) 

pH 

Density 

Stability 

Solids content 

Water insolubles 

PAH 

Elemental Analysis C,H,N & O 

Toxicological (CIT) Bacterial reverse mutation test 

Ecotoxicological (CIT) Algal growth inhibition test 

Acute toxicity in Daphnia

Magna

Biodegradability (Cirad) Modified Sturm test 

2.2 Physical and basic chemical properties of bio-oil 

All fast pyrolysis oil samples are in the typical ranges for 

bio-oil physical and chemical properties. Most measured 

water contents are between 20 and 30%. One sample is 

significantly higher with a water content of 37%, which may 

be due to the pyrolysis technology of the sample provider or 

may just be a sign of high feedstock moisture content. No 

measurement of the biomass moisture content was supplied 

with the oil sample. Solids content varies widely, from 0.03% 

to 3.43%, indicating large variation in char product collection 

efficiency. The density of all samples is close to 1.2 kg/l. 

The viscosity of the samples ranges from 17.5 to 451 cSt 

at 20C. Water insolubles, often referred to as pyrolytic 

lignin, represent between 6% and 25% of the whole oil. 

Stability of the oil samples, as assessed by measuring 

viscosity and water content of the oil again after storage for 

24 hours at 80C, varies greatly, between virtually no change, 

and a tripling of the viscosity measured at 20C, or 

respectively an increase in water content by a factor 1.26. 

2.3 Gas chromatography 

As can be seen in Fig. 2. chemical species quantified by 

gas chromatography show substantial variation between the 

samples. Approximately a quarter of the mass of the wet oil 

is quantified by the employed gas chromatography methods. 

The quantified chemicals typically represent 70-80% of the 

total peak area measured by GC. Several low molecular 

weight chemicals, for example methanol, acetone and 

ethanol, are subsumed in the peak of the solvent (acetone) 

used to dilute the bio-oil in preparation for GC analysis. They 

are therefore neither quantified nor included in the total peak 

area, which excludes the very large solvent peak. 

2.4 Polyaromatic hydrocarbons 

As polyaromatic hydrocarbons are known to represent a 

potential health and safety concern, a method was developed 

for analysing their concentrations in bio-oil. It was found that 

typical concentrations for total PAH are below 10 PPM. PAH 

concentration is temperature and residence time dependent. 

The slow pyrolysis oil samples exhibit much higher values 

for PAH, with one of the two samples slightly exceeding 100 

PPM. Bio-oils produced at temperatures exceeding 550C 

had the highest PAH values for fast pyrolysis samples, 

ranging as high as 23 PPM at 600C. The lowest PAH 

concentration was found in a sample produced at 425C. 

2.5 Mutagenicity Ames test 

For all fast pyrolysis oil samples at least one of the tested 

strains of Salmonella typhimurium showed a sufficient 

increase in reverse mutants that the result would be 

considered a positive indication of mutagenicity according to 

international regulations. Both slow pyrolysis samples 

assessed were too toxic for the bacteria for the test to give a 

result. 

0,0

5,0

10,0

15,0

20,0

25,0

30,0

1234567

Others

Syringols

Sugars

Pyrans

Phenols

Ketones

Guaiacols

Furans

Aromates

Aldehydes

Alcohols

Acids

Figure 2: Selection of 7 gas chromatography results, 

wt% of whole oil, chemical species grouped together 
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2.6 Ecotoxicological screening 

The algal growth test indicates that low concentrations of 

bio-oil may have a small fertilising effect. The potential to 

contribute to eutrophication, however, should be small due to 

the very low nitrogen, and very low to negligible minerals 

content of the bio-oil. At higher concentrations, algal growth 

is slightly inhibited. 

Acute toxicity on Daphnia Magna, even at the highest 

loading rate of 100 mg/l, is negligible. 

Overall the ecotoxicological screening tests indicate that 

all tests fast pyrolysis oils are benign in their effects on algae 

and small water animals. 

2.7 Biodegradation 

The ready biodegradability of the samples, as assessed 

through a modified Sturm test, indicates rapid biodegradation 

of 40-50% of the organic carbon in the oil. These values are 

better than for most mineral oil products. For example, heavy 

fuel oil only gives a value of 11% in the modified Sturm test. 

They are, however, slightly worse than for slow pyrolysis 

derived oil. This is likely due to the fact that bio-oil contains 

a large fraction of incompletely decomposed lignin 

fragments. Lignin is well known to be particularly resistant to 

microbial decomposition, with only a few species capable of 

degrading it aerobically, and little degradation being possible 

anaerobically]. 

3 COMPLETE SET OF TESTS ON 1 SAMPLE 

The preliminary tests indicate little difference between 

different feedstocks (miscanthus, hardwood, softwood and 

forest residues with varying bark content) and fast pyrolysis 

technologies. At higher temperatures PAH is slightly 

elevated. Consequently, it was decided to do the full suite of 

tests on a sample produced under well known conditions at 

Aston University. A temperature of 500C was chosen, as 

this is the optimal yield temperature. Furthermore, much 

lower temperatures, towards 400C, make it difficult to avoid 

blockages in the hot piping due to condensation of bio-oil, 

and temperatures towards 600C require extra, and more 

difficult to supply high temperature, process heat. 

It was also decided to use a fluidised bed system, as this 

is currently the most widely used technology, and as 

repeatability and consistency of results have been particularly 

well established for this reactor system at Aston. 

A softwood was chosen as the feedstock as this is a high 

quality biomass particularly widely available, at reasonable 

cost, in the EU. 

3.1 Physical and basic chemical properties 

In addition to the tests performed for the screening, 

explosive properties (test A14) were assessed with the 

conclusion that bio-oil is not explosive. Water solubility (test 

A6) and partition coefficient (test A8) are difficult to assess 

for bio-oil. A suitable method is still under discussion with 

the relevant authorities. 

3.2 Acute oral toxicity in rats 

At a dosage of 2000 mg/kg no mortality was observed in 

test B1 tris. Piloerection, the rat’s body hair standing on end, 

which is a potential sign of the rat feeling cold or stressed, 

was seen within the first 6 hours after treatment. There was 

also hypoactivity or sedation. No clinical signs were observed 

on day 2 after treatment. 

Test TSR, a seven day study of oral toxicity with the 

highest treatment at 1500 mg/kg/day, was also performed. 

Hypersalivation and, at least an initial reduction in food 

consumption and body weight gain were observed. 

These tests indicate no toxicity of bio-oil by oral 

ingestion. 

Table II: Selection of the feasible test to be carried out in 

the selected bio-oil sample 

Study title 

PHYSICO-CHIMICAL PROPERTIES 

A6 / Water solubility 

A8 / Estimation of the partition 

coefficient 

A14 / Explosive properties 

TOXICOLOGICAL STUDIES 

B1 tris / Oral route - rat 

B3 / Cutaneous - rat 

B4 / Cutaneous - rabbit 

B5 / Eye irritation - rabbit 

B6 / LLNA 

TSR / 7-day study oral routein rats 

MAS / Micronucleus test in vivo 

MNV / Micronucleus test in vitro 

ECOTOXICOLOGICAL STUDY 

C2 / Acute toxicity in daphnia magna 

3.3 Dermal irritation in rabbits 

In this test (B4) the potential for skin irritation in rabbits 

is evaluated. Clinical symptoms, such as discoloration, 

dryness and damage to the skin in the form of lesions or 

swelling, were observed. 

The tested sample therefore has to be classified as 

corrosive when applied topically to rabbits, and is assigned 

the symbol C “corrosive” and risk phrase R34 “causes 

burns”. Test B5 (eye irritation in rabbits) was consequently 

not performed in order to protect the animals. It is also well 

known that bio-oils are irritating to the eye. 

3.4 Evaluation of skin sensitisation potential in mice 

Skin sensitisation potential in mice was assessed through 

test B6 (LLNA or local lymph node assay). This test 

indicated that bio-oil is a moderate skin sensitiser and 

therefore has to be assigned risk phrase R43 “May cause 

sensitisation by skin contact”. 

3.5 Mutagenicity 

Mutagenicity was assessed in two tests (MAS and MNV). 

In the MAS test the potential of bio-oil to induce damage to 

the chromosomes or miotic apparatus in bone marrow cells of 

mice (after 3 oral administrations) is assessed in vivo. In the 

MNV test mutagenicity is assessed in vitro on mouse 

lymphoma cells. 

Together the tests indicate the potential for slight 

mutagenicity which would need confirmation through further 

testing. 
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4 DISCUSSION 

The screening tests indicate a wide variability in chemical 

composition measured by GC and little difference in 

toxicological properties of the bio-oils. It is well known that 

depending on process conditions the relative proportions of 

sugars, acids and aldehydes vary in bio-oil [8]. These groups 

contain the chemicals with the highest mass proportions in 

bio-oil, notably acetic acid, hydroxyacetaldehyde and 

anhydrosugars, which are known to have low toxicity. The 

results of the screening tests indicate that the variations in 

these groups do not materially impact the overall toxicity of 

the bio-oil, which they could in theory through synergistic or 

antagonistic effects. 

There is an indication that the concentration of PAH may 

correlate with toxicity, as the two slow pyrolysis oils had 

both the highest PAH concentrations and showed the greatest 

toxicity. PAH are also well known to contribute to health and 

safety concerns in conventional petroleum derived fuels. 

The results also provide relevant information for the 

labelling, storage and transportation of bio-oils. They indicate 

that the oils do not need special precautions in terms of 

explosive concerns, or toxic or ecotoxic emissions. They are, 

however, corrosive and irritating to skin and therefore require 

appropriate personal protective equipment during handling. 

The data generated in this work can also be used to help 

produce an MSDS sheet and technical dossier for bio-oil as 

required by incoming European legislation on chemicals 

control, as will be discussed in detail in the final project 

report. 

5 CONCLUSIONS 

The results indicate that in spite of substantial variations 

in the proportions of some chemicals contained in different 

bio-oils, all fast pyrolysis processes give bio-oil that is very 

similar in terms of toxicity, eco-toxicity and biodegradability. 

Bio-oil appears to be more benign than slow pyrolysis 

derived tars, although it takes longer to biodegrade. In 

comparison to traditional petroleum derived fuels bio-oil 

biodegrades faster, and is considerably less toxic. 

. 
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I. Publishable synthesis report 

I.1. Objectives and strategic aspects 

I.1.1 Social and economic objectives of the project. 

Pyrolysis is one of the three main thermochemical routes to convert biomass into useful primary 

energy products. It consists in the heating of a raw material in absence of oxygen. As a result of the 

thermal decomposition of the raw material, a gas, a liquid and a solid are formed, which can be used 

directly or further upgraded to give more value-added fuels. Fast pyrolysis at a temperature around 

500°C, at very high heating rates and short vapour residence times (less than 1 second) gives high 

liquid yields of up to 80% weight on a dry feed basis. Fast pyrolysis is therefore unique in that a liquid 

product is produced in high yields in a simple one step process with all the advantages offered by a 

liquid fuel. The White Paper edited by the Commission has described biomass as the most important 

source of renewable energy for the future. In the long term, biomass will undoubtedly play a significant 

role in the supply of energy in many countries. The project intends to assess and minimize the effect of 

a liquid fuel (bio-oils) from fast pyrolysis of biomass, on human health and its impacts on environment, 

before large scale marketing and utilisation, by acting on the production parameters. It aims at 

producing cleaner bio-oils to both at local and global scale. 

The development of the use of bio-oils as fuels will have a positive impact on the reduction of CO2

emissions as they derive from biomass (CO2 neutral). 

More generally speaking, the development of the use of bio-oils will increase the use of European 

forest and agricultural products and by products. From an economic viewpoint, this will induce job 

creations in rural areas, thus developing economic and social welfare. The most promising application 

of bio-oils is electricity production, or combined heat and power, due to their ability to be used in an 

engine without extensive upgrading as well as the ability to decouple the fuel production source from 

the end-use location. Small size (a few MWe) decentralised electricity production can be achieved, 

which will allow the development of activities in less favoured regions. 

An other very attractive option offered by fast pyrolysis is transport fuel application through bio oil 

gasification and Fischer Tropsch reaction as fast pyrolysis offers a unique advantage of decoupling the 

production phase of its use as well as generating standard fuel form dispersed and divers biomasses. 

Adapted health and safety procedures are important and will have running cost reduction implication as 

it will allow appropriate risk evaluation and a better insurance coverage. More over a better 

understanding of operating conditions responsible for more severe toxicity and eco-toxicity will 

improve day-to-day operation and reduce losses. 

I.1.2 Scientific/technological objectives of the project. 

Fast pyrolysis has benefited from active research programme since 1980’s because bio-oils can be 

substituted directly for fuel oil in many static applications or used as a source of renewable chemicals 

and in the longer term it can be upgraded for more demanding applications such as transport fuel. 

Today, different demonstration plants are set up in Europe as well as in North America and significant 

quantities of bio-oils are produced for research and development purposes. Thus, the question of safety 

procedures for human health and environment preservation is raised during production, transport and 

use of the bio-oils. Indeed bio-oils contain 100s of chemicals from different functional groups, such as 

organic acids, aldehydes and ketones, phenolic compounds, aromatics. Their composition mainly 

depends on the feedstock used and the pyrolysis conditions. Thus, the toxicology of the oils also 
8

depend on feedstock and process. No systematic studies were made to relate the different parameters. 

This was mainly due to the high cost of the necessary toxicological and biodegradability studies, which 

prevents their financing by generally SMEs fast pyrolysis companies. 

Moreover with the increasing amount of bio-oils manufactured, transported (imported) and stored 

within the EU market, it was necessary to register (notify) this substance by competent authorities with 

a comprehensive and definitive MSDS and proper preventative and remedial procedures to adopt 

during production, transport and use of bio-oils. 

Base on these observations during the project, the relation between process parameters on one hand 

and chemical composition and toxicity for human health and environment on the other hand were 

investigated. Then, a bio-oil, selected to be representative of those placed in the Eu market, was 

submitted to mandatory tests required by the commission, the objective being the definition of secure 

handling and storage procedures, in order to control the risks related to the product for the population 

and the environment. The effects of different ways of exposure (inhalation, ingestion or skin contact) 

were quantified, as well as the effects of long term exposures. The impacts on the environment was 

also be evaluated by biodegradability, and effects on bio-organisms. 

The aims of this project were: 

- to determine toxicological and eco-toxicological data on bio-oils. The work will concern both acute 

and chronic effects on human health, carcinogenic and mutagenic effects. The environmental 

impacts of bio-oils liquids will also be measured in water and soil. This will allow a comprehensive 

and definitive MSDS to be produced with the proper preventative and remedial procedures to adopt 

during production, transport and use of bio-oils, 

- to determine the best operating conditions to avoid or minimise the formation of toxic products 

from the composition of the bio-oils, 

- to produce fast pyrolysis bio-oils with low impact on human health and environment by optimising 

the production process of bio-oils related to toxicity characteristics, 

- to encourage the active involvement of industry in the development and exploitation of the results 

through an existing network, which includes developers of bio-oils production and application 

technologies. 

- to disseminate information on this activity from the database and results of meetings by 

newsletters, reports, conferences, specific workshops, web site and other publications, to those of 

the network and other interested parties in regional, national and international programmes. 

I.2. Scientific and technical description of the results 

Fast pyrolysis is a high temperature process in which biomass is rapidly heated in the absence of 

oxygen. As a result it decomposes to generate mostly vapours and aerosols and some charcoal. 

Liquid production requires very low vapour residence time to minimise secondary reactions of 

typical 1s, although acceptable yields can be obtained at residence times of up to 5s if the vapour 

temperature is kept below 400°C. After cooling and condensation, a dark brown mobile liquid is 

formed which has a heating value about half that of conventional fuel oil. Moreover, biomass is a 

complex mixture of hemicellulose, cellulose, lignin and minor amounts of other organics which 

pyrolyses or degrades at different temperatures and by different mechanisms and pathways. The rate 

and extent of decomposition of each of these components depends on the process parameters of 

reactor (pyrolysis) temperature, biomass heating rate and pressure. The degree of secondary 

reaction (and hence the product yields and characteristics) of the gas/vapour products depends on 

the time-temperature history to which they are subjected before collection, which includes the 

influence of the reactor configuration. 
9

Pyrolysis, has received considerable creativity and innovation in design reactor systems that provide 

the essential ingredients of high heating rates, moderate temperatures and short vapour product 

residence times for liquids. As a result a large variety of reactor configurations has been developed 

and is used by bio-oil producers. 

Project partners decided to study the impact of the pyrolysis parameters on the nature of the 

obtained oils trough assessment of the physicochemical composition and the toxicological impact of 

oils samples produced under different conditions and in diverse reactors. Goal of this work was to 

give a overview of the characteristics of the oils produced within the Eu market, and based on the 

whole results to select one representative oil to be fully analysed in term of toxicity and ecotoxicity. 

Methodology applied during the project is presented on schema Figure 1. At the beginning of the 

project, 21 selected samples of bio-oils from different processes, temperatures and feedstock were 

gathered, in order to be analysed in screening through physicochemical and toxicological tests. 

Results of these screening tests were collected to correlate bio-oils production parameters to 

compositions and toxicological characteristics, and to select the most representative sample to be 

used for full toxicological analysis through mandatory tests required by the EU legal authority; the 

objective being the definition of secure handling and storage procedures, in order to control the 

risks related to the product for the population and the environment. 

Figure 1: Schematic methodology applied for the project

The whole work carried out within Biotox was shared in four successive phases: 

1. Selection, production and collection of 21 bio-oil samples to be assessed 

2. Assessment of the physico-chemical and the toxicology properties of the collected samples 

3. Full toxicological study of one selected representative sample 

4. Recommendations for safety procedures and dissemination of the results 

Collection of 21 bio-oils

( reactors feedstock t°C)

Bio-oils characterization 

 - physical chemical analysis

 - toxicological screening 

Selection of 1 representative 

bio-oil for notification analysis

Full toxicological analyses of 

the selected bio-oil

- Redaction of the «notification dossier»

- Reports on transport requirement & 

secure handling

Relation between production parameters, 

oil composition and toxicological 

characteristics
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I.2.1 Selection, production and collection of 21 bio-oil samples to be assessed 

As bio-oils producers are the main beneficiary of the results of the project, they were implied into the 

project at the beginning. In order to involve them to the project, they were invited to the first Biotox 

steering committee meeting held in Paris in February 2003. The project was also introduced to the 

European pyrolysis network “PyNe” during the Florence meeting in April 2003. Through these 

meetings industrialists were informed about the objectives of the project and the methodology to reach 

them. Industrialists confirmed their interests and their willingness to take part to the project. All of 

them were favourable to provide bio-oil samples. 

A first series of fourteen samples was collected for the screening tests and based on the first analytical 

results; it has been decided to produce 7 additional samples in order to assess the reproducibility of the 

tests, to further investigate the effect of temperature and assess aging of sample. A total of 21 samples 

provided by different laboratories or companies were collected for physicochemical analysis and 

toxicological assessment. 

As shown on Table 1, different types of pyrolysis processes and biomass feedstock were selected in 

order to provide representative samples for the project and to meet, as well as possible, the 

industrialists’ needs. 

Sample nb° T° Parameters Biomasse 

Biotox - 1 520°C Fluidised Bed Spruce 

Biotox - 2 500°C Circulating Fluidised Bed Beech 

Biotox - 3 500°C Circulating Fluidised Bed Green forest residue 

Biotox - 4 480°C Circulating Fluidised Bed Pine sawdust 

Biotox - 5 460°C Fluidised Bed Pine/Spruce/Fir 

Biotox - 6 560°C Ablative Pine 

Biotox - 7 475°C Slow pyrolysis Residues of soft wood barks 

Biotox - 8 500°C Slow pyrolysis Spruce chips 

Biotox - 9 500°C Fluidised Bed Beech 

Biotox - 10 500°C Fluidised Bed Spruce 

Biotox - 11 425°C Fluidised Bed Spruce 

Biotox - 12 600°C Fluidised Bed Spruce 

Biotox - 13 500°C Fluidised Bed Miscanthus 

Biotox - 14 600°C Ablative Spruce 

Biotox - 15 575°C Fluidised Bed Spruce 

Biotox - 16 425°C Fluidised Bed Spruce 

Biotox - 17 500°C Fluidised Bed Beech 

Biotox - 18 500°C Ablative Beech 

Biotox - 19 500°C Fluidised Bed Spruce 

Biotox - 20 600°C Biotox 17 +Biotox 18 +Biotox 19 Spruce 

Biotox - 21 500°C Fluidised Bed Spruce 

Table 1: List of collected pyrolysis oils sample 
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In the selection of samples for the screening tests, the main methods of achieving pyrolysis were 

represented (Fluid bed, Circulating Fluid Bed, Ablative and slow pyrolysis) 

In order to assess the impact of the nature of the feedstock and the pyrolysis temperature on the 

composition and toxicology of bio-oils these parameters were studied: 

Samples Biotox-10;-11;-12;-15;-16;-21, were produced under the same condition just 

varying the pyrolysis temperature from 425°C to 6000°C. 

Samples Biotox-9;-10;-13;-17;-21, were prepared from different biomass; beech (hard 

wood), spruce (soft wood) and miscanthus (perennial grass). 

To assess the reproducibility of pyrolysis processes and of the physico-chemical analysis two biooil samples (Biotox-9 and -10) were produced in double (respectively Biotox-17 and -21). 

As bio-oils are achieved through thermo-chemical degradation of the macromolecules in biomass 

(hemicelluloses, cellulose and lignin) some obtained compounds in the oils can react during storage 

with the time to give new longer components. To study this effect sample Biotox-10 was analysed 

twice, at 4 and 16 months after its production respectively (Biotox-10 and 16). 

All samples were collected by Cirad before being distributed between BFH, CIT and Cirad for 

analysis. 

For each sample, oil producers had to fill out a sample data sheet, with all the information 

concerning the pyrolysis run. This data sheet was collected by Cirad together with the oil sample. 

Seven of the twenty-one oils were produced by industrialists in their own equipments. 

I.2.2 Assessment of the physico-chemical composition and the toxicology of the collected 

samples

Physico-chemical composition, toxicity and eco-toxicity of the collected samples were assessed by 

basic tests before carrying out a comprehensive full toxicological study on one sample 

Main goals of this work were to: 

obtained complete description of the chemical and physical properties of the 21 bio-oils 

evaluate effects of bi-oils on bio-organisms (fauna and flora) and their persistence in the 

environment 

relate for each oil the production parameters to toxicology. 

to select a representative sample for comprehensive full toxicological studies, based on the 

results of the tests carrying out on the 21 bio-oils, 

All the collected bio-oils were first distributed between BFH, CIT and Cirad for analysis. 

The Institute of wood chemistry (BFH) analysed the chemical and physical composition of bio-oils. 

Toxicological and eco-toxicological test were performed by CIT. This laboratory is an independent 

laboratory which is legally recognizes to carry out environment, health and safety mandatory tests 

in Europe. In order to select relevant test for the screening of bio-oils, a meeting was organized in May 

2003 in CIT headquarter in Evreux (see minute meeting n°2). Based on the bibliography and 

objectives of Biotox, Mrs De Jouffrey from CIT, discussed the different tests which would offer the 

maximum of information during the screening test and suggest to put emphasis on : 

- Toxicological test : mutagenicity ames test, as controversial data appears in bibliography 

- Eco-toxicological tests: acute screening toxicity study in daphnia and algae due to the 

environmental benefit expected from the Oil. 
12

Cirad carried out biodegradability tests. Due to the specificities of Bio-oils (low solubility in water, 

high carbon content and viscosity) a new method was developed to asses their aerobic degradation 

in fresh water. 

Details of all analytical tests performed on the 21 bio-oils is summarised in 

Table 2. 

Type of study Detailed analysis Laboratory 

Physico chemical Chemical composition 

Viscosity (at 20 & 50°C) 

pH 

Density 

Stability 

Solids content 

Water insoluble content 

PAH 

Elemental Analysis C,H,N & O 

BFH

BFH

BFH

BFH

BFH

BFH

BFH

BFH

BFH

Toxicological Bacterial reverse mutation test CIT

Eco-toxicological Algal growth inhibition test 

Acute toxicity in Daphnia Magna

CIT

CIT

Biodegradability study Modified Sturm test Cirad

Table 2: Details of tests performed 

I.2.2.1 Bio-oils physico-chemical characterisation 

BFH was responsible of the physico-chemical analysis Bio-oils. The objectives of this work were: 

to obtained complete description of the bio-oils with chemical and physical properties 

and to determine the range of variation of bio-oils characteristics upon feedstock, 

temperature and reactor technology 

In order to improve and verify the analytical methods used for the physico-chemical 

characterisation of bio-oils, BFH worked in the framework of the project on the development of 

new techniques. In order to verify the analysis methods, a same bio-oil sample was analysed, at the 

beginning of the project (summer 2003) by BFH and Cirad. 

The main tool for the analysis of the bio-oils is gas chromatography (GC).The GC method applied 

in this project was developed at BFH and has been used also for the analysis of liquid smoke 

aromas. It is recommended by the EU Joint Research Centre (Institute for Reference Methods and 

Materials, IRMM) for liquid smoke producers and other related research laboratories. A method to 

analyse PAH was newly developed and agreed with the European Joint Research Centre. The most 

problematic step was the extraction of the PAH’s from the matrix bio-oil. A dedicated GC/MS 

system with single ion monitoring (SIM) was necessary to obtained the necessary low detection 

levels. 
13

a) Chemical and physical characterization 

In the course of project 21 bio-oil samples were fully characterized by the following parameters: 

1. Water (%) 

2. Viscosity 20°C (cSt) 

3. Viscosity 50°C (cSt) 

4. Density 20 °C (g/cm3

) 

5. Solids (%) 

6. water insolubles (pyrolytic lignin) 

7. Stability 

8. Viscosity index 20 °C 

9. Viscosity index 50 °C 

10. Water index 

11. Elemental analysis 

12. GC analysis 

13. PAH determination 

14. UV/Vis spectra 

- Physical-chemical analysis 

The results of the physical-chemical analyses are summarized in Table 3. 
14

 Biotox1 Biotox- 2 Biotox- 3 Biotox- 4 Biotox- 5 Biotox- 6 Biotox- 7 Biotox- 8 Biotox- 9 Biotox- 10 Biotox- 11 Biotox- 12 Biotox- 13 Biotox- 14 Biotox- 15 Biotox- 16 Biotox- 17 Biotox- 18 Biotox- 19 Biotox- 20 Biotox- 21

Water (%) 23.50 28.50 29.10 24.90 29.40 37.00 8.10 28.60 26.80 22.40 26.70 20.30 24.60 22.70 12.12 18.23 25.51 31.79 22.95 26.77 17.57 Viscosity 20°C (cSt) 128.90 118.30 49.30 62.40 47.60 17.50 n/a n/a 29.10 90.30 81.00 451.30 110.20 112.30 n/a 359.10 41.57 52.78 191.25 39.36 226.43 Viscosity 50°C (cSt) 11.60 3.30 9.90 11.00 10.50 4.90 n/a n/a 6.90 14.30 14.60 41.70 14.80 16.40 n/a 39.36 8.46 4.70 23.10 8.45 27.96 Density 20°C (g/cm3) 1.21 1.20 1.20 1.21 1.22 1.19 1.21 1.09 1.16 1.20 1.22 1.21 1.17 1.21 n/a 1.27 1.16 1.14 1.21 1.17 1.25 Solids (%) 0.03 3.43 0.37 0.10 0.03 0.35 2.48 0.03 0.06 0.03 0.52 0.55 0.48 0.40 0.24 0.04 0.74 0.08 0.02 0.09 0.10 Water insoluble 19.7 17.7 12.4 15.2 10.9 5.8 54.4 ? 18.2 19.1 15.6 24.9 20.2 21.1 n/a 14.9 16.2 11.3 20.7 15.2 15.7 Stability  visc. 20°C (cSt) 256.9 n/a 31.1 97.1 44.7 n/a n/a n/a 43.6 262.7 154.0 n/a 190.9 142.0 n/a 621.3 n/a 20.8 332.2 80.1 173.7  visc. 50°C (cSt) 27.4 n/a 10.6 16.8 10.4 n/a n/a n/a 9.0 28.8 19.1 n/a 23.5 36.3 n/a 58.8 n/a 0.6 32.9 12.5 52.2  water (%) 24.4 35.3 31.5 26.5 30.6 41.4 n/a n/a 27.6 24.3 29.2 20.8 25.6 28.7 n/a 19.2 28.4 33.7 24.9 28.8 19.1 Viscosity index 20°C 0.99 n/a -0.37 0.56 -0.06 n/a n/a n/a 0.50 1.91 0.90 n/a 0.73 0.26 n/a 0.73 n/a -0.61 0.74 1.04 -0.23 Viscosity index 50°C 1.36 n/a 0.07 0.53 -0.01 n/a n/a n/a 0.30 1.01 0.31 n/a 0.59 1.21 n/a 0.49 n/a -0.87 0.43 0.47 0.87 Water index 0.04 0.24 0.08 0.06 0.04 0.12 0.03 0.08 0.09 0.02 0.04 0.26 n/a 0.05 0.11 0.06 0.08 0.08 0.09 CHO, total oil as received 

% N 0.00 0.01 0.30 0.01 0.35 0.35 0.25 0.00 0.00 0.00 0.37 0.38 0.22 0.42 0.50 0.04 0.38 0.21 0.05 0.61 0.36 % C 43.24 39.45 39.44 41.27 38.28 32.64 59.38 62.54 41.67 43.66 39.37 45.10 43.13 45.18 47.43 44.05 42.59 37.38 43.14 40.78 44.59 % H 7.76 7.96 8.01 7.79 7.77 8.30 7.46 7.59 7.87 7.67 7.64 7.35 8.14 7.60 6.87 7.10 8.09 8.48 7.64 8.07 7.12 % O 49.00 52.58 52.25 50.93 53.60 58.71 32.91 29.87 50.46 48.67 52.62 47.17 48.51 46.80 45.20 48.81 48.94 53.93 49.17 50.54 47.93 

 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 CHO, organic part of oil 

% N 0.00 0.01 0.42 0.01 0.50 0.56 0.27 0.00 0.00 0.00 0.50 0.48 0.29 0.54 0.57 0.05 0.51 0.31 0.06 0.83 0.44 % C 56.52 55.17 55.63 54.95 54.22 51.81 64.61 87.59 56.93 56.26 53.71 56.59 57.20 58.45 53.97 53.87 57.18 54.80 55.99 55.69 54.09 % H 6.73 6.70 6.74 6.69 6.38 6.65 7.14 6.18 6.68 6.68 6.38 6.39 7.17 6.57 6.29 6.21 7.06 7.25 6.61 6.96 6.27 % O 36.75 38.11 37.21 38.34 38.90 40.99 27.98 6.23 36.39 37.06 39.41 36.54 35.34 34.44 39.17 39.87 35.26 37.64 37.34 36.52 39.20 

 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 n/a not applicable due to sample characteristic 

Table 3: Summary of physical-chemical analyses of all Biotox oils
15

Most of the oils show typical values for fast pyrolysis liquids. 

The variation in water content is visualized in Figure 2. Water content ranges between 8 and 

37 %. The highest water content shows bio-oil 6 and the lowest can be found in Biotox-7. 

0

5

10

15

20

25

30

35

40

BT 01 BT 02 BT 03 BT 04 BT 05 BT 06 BT 07 BT 08 BT 09 BT 10 BT 11 BT 12 BT 13 BT 14 BT 15 BT 16 BT 17 BT 18 BT 

19

BT 20 BT 21

Figure 2: Water content (wt.%) of bio-oil samples

Biotox-6 is from an ablative process which used wood with a moisture content of 14 %. 

Together with possible slight cracking reactions on the hot surface of the reactor this might 

explain the high water content. The low water content of Biotox-7 is probably due to the two 

stage condensation process where the water is condensed in the first stage and most of the 

organics in the second stage. Biotox-18 shows also water content above 30 %. This oil is 

made in a rotating cone reactor. It is important to note, that the water content cannot be used 

as indicator to assess a pyrolysis reactor. The water content in the oil is much more dependent 

on the condensation system and operating conditions. 

Figure 3 demonstrates the viscosity of the bio-oils. Two oils (Biotox-7 and Biotox-8) could 

not be measured with the capillary viscosimeter because of handling difficulties caused by 

very high viscosities at room and elevated temperatures. Most of the oils are in the normal 

range (50-120 cSt) but oil Biotox-12 exhibits extraordinarily high values of 451 cSt without 

clear explication. 
16

0

50

100

150

200

250

300

350

400

450

500

BT 01 BT 02 BT 03 BT 04 BT 05 BT 06 BT 07 BT 08 BT 09 BT 10 BT 11 BT 12 BT 13 BT 14 BT 15 BT 16 BT 17 BT 18 BT 19 BT 20 BT 21

viscosit y 20°C (cSt ) viscosit y 50°C (cSt )

Figure 3: Viscosity at 20 and 50 °C 

Density is for all bio-oils in the normal range of 1.2 gcm-1 while the solid content varies a lot 

between 0.03 to 2.5 %. Solid content is mainly function of the condensation device of the 

pyrolysis unit. 

The samples Biotox-2 and Biotox-7 have very high solids contents, 3.43 and 2.48, 

respectively. Biotox-2 was made in a circulating fluidized bed reactor and some sand might be 

entrained into the condensation system for bio-oil. For Biotox-7 the high solids content can be 

explained by the choice of feedstock (pine bark) which is rich in condensed phenolics which 

might not be completely solubilized in ethanol which is the normal solvent for bio-oils for the 

determination of the solid content. 

The water insoluble part resembles the pyrolytic lignin fraction which describes the 

oligomeric part of the oil derived from lignin. It ranges from 5.8 to 54.4 %. Biotox- 6 from 

ablative pyrolysis has only 5.8 % lignin which could be explained by cracking reactions. This 

finding explains the high water content in this oil. On the other hand, Biotox-7 contains 54.4 

% which can easily be explained by the used feedstock (bark) and the two stage condensation 

system in which lighter molecules are separated in the first stage. 

The water index is a measure of the water increase after the stability test, which comprises the 

treatment of bio-oils for 24 h at 80 °C. The more water is formed, the higher is the index 

number. From 

Figure 4 it can be seen that oil Biotox-14 has the highest water index number, followed by 

Biotox-2, Biotox-16, Biotox-17, and Biotox-11.
17

0,00

0,05

0,10

0,15

0,20

0,25

0,30

BT 

01

BT 

02

BT 

03

BT 

04

BT 

05

BT 

06

BT 

07

BT 

08

BT 

09

BT 

10

BT 

11

BT 

12

BT 

13

BT 

14

BT 

15

BT 

16

BT 

17

BT 

18

 BT 

19

BT 

20

BT 

21

Figure 4: Water index of bio-oils 

- Gas chromatographic analysis 

GC analysis was performed using standard conditions developed at BFH. Separation takes 

place on a medium polar capillary column. The overall results are presented in Table 4. The 

identified single components were clustered into chemically different groups. 

The GC method used in this project will be mandatory for future liquid smoke analysis 

required by the European Joint Research Centre (JRC), Institute for Reference Materials and 

Methods, Geel, Belgium. 
18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Identified peaks % 65.7 68.5 74.0 70.3 75.7 74.2 61.5 70.6 75.4 72.8 78.4 71.7 70.4 72.3 84.9 80.1 93.4 89.3 92.5 92.7 93.9 Unknown peaks % 34.3 31.5 26.0 29.7 24.3 25.8 38.5 29.4 24.6 27.2 21.6 28.3 29.6 27.7 15.1 19.9 6.6 10.7 7.5 7.3 6.1 

Groups (wt. %)*Acids 4.4 8.8 3.8 3.4 3.4 4.2 3.5 4.1 7.2 2.8 3.2 3.0 4.9 5.7 2.3 2.3 6.0 5.1 2.5 5.2 2.2 Alcohols 0.5 0.5 0.2 0.3 0.0 0.0 0.1 0.0 0.6 0.2 0.1 0.3 0.1 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 Aldehydes 6.0 4.0 7.0 6.5 1.4 3.8 2.1 0.9 3.9 7.0 5.1 6.1 5.9 5.3 17.8 7.4 3.7 2.4 3.5 3.6 6.8 Aromates 0.1 0.1 0.3 0.0 0.0 0.0 0.4 0.3 0.3 0.3 0.0 0.0 0.0 0.3 0.0 0.0 0.1 0.0 0.0 0.0 0.0 Furans 1.6 1.1 1.9 1.5 1.5 1.4 1.4 2.3 2.0 2.2 3.0 2.4 2.0 1.4 1.3 1.2 1.2 0.9 1.0 1.0 1.4 Guaiacols 1.9 0.5 3.6 4.3 2.6 1.9 1.7 8.1 1.8 4.2 3.9 3.2 1.8 2.8 2.6 2.3 1.0 1.5 2.2 1.6 2.6 Ketones 3.1 2.7 3.8 3.8 4.0 3.4 2.9 5.5 4.1 3.5 5.8 4.3 4.5 4.6 3.3 3.0 3.2 2.7 2.4 3.0 2.9 Phenols 0.4 0.4 0.3 0.2 0.2 0.3 2.2 2.3 0.3 0.4 0.2 0.6 1.1 0.5 0.4 0.1 0.2 0.2 0.2 0.2 0.2 Pyrans 0.0 0.0 0.1 0.1 0.3 0.0 0.0 0.0 0.1 0.1 0.4 0.5 0.7 0.3 0.3 0.1 0.2 0.1 0.0 0.1 0.1 Sugars 4.0 3.0 5.2 4.2 13.1 6.6 5.7 1.1 3.4 4.6 5.7 5.9 5.3 3.6 2.6 3.0 2.8 2.8 2.9 2.8 2.5 Syringols 0.0 0.3 0.4 0.1 0.0 0.0 1.6 0.0 3.3 0.2 0.0 0.0 1.6 2.2 0.0 0.0 3.3 3.1 0.0 2.5 0.0 Others 0.1 0.0 0.1 0.1 0.9 0.6 0.0 0.5 0.0 0.1 0.6 1.4 0.8 0.6 0.1 0.2 0.1 0.1 0.1 0.1 0.2 Total 22.3 21.5 26.9 24.5 27.5 22.0 21.6 25.1 26.9 25.7 27.8 28.1 28.7 27.8 30.6 19.6 21.8 18.8 14.9 20.2 18.8

* wt.% based on wet oil as received 

Table 4: Summary results of GC analysis 
19

The gas chromatographic results show that maximum 30.6 wt% of the whole could be identified by 

GC corresponding to 70-95 % of the total GC-peak area. 

The amounts of identified and unidentified peak areas are presented in Figure 5. 

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Unknown

peaks (%)

Identified

peaks (%)

Figure 5: Percentage of identified and unidentified peaks based on the total gas 

chromatographic area

Oils Biotox-17 to Biotox-21 exhibit the highest identified amount (92-95 %), whereas oil Biotox-1 

and Biotox-7 have the lowest identified portion of 62-65%. 

Figure 6 shows the distribution of chemical groups in the data set of 21 bio-oils. Predominant 

groups are acids, aldehydes, phenols, and sugars. Variation in of these groups within the oils is quite 

substantial and reflects the different processing parameters such as pyrolysis technique and 

condensation mode. 

0,0

5,0

10,0

15,0

20,0

25,0

30,0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Others

Syringols

Sugars

Pyrans

Phenols

Ketones

Guaiacols

Furans

Aromates

Aldehydes

Alcohols

Acids

Figure 6: Distribution of chemical groups in the bio-oils (wt.% based on wet oil) 
20

Samples Biotox-16 to -21 give 5-10 % less yield mainly due to 2 reasons: 

1. At BFH the sample amount for analysis was adopted by taking into account the different water 

contents. For samples 1-15 always 60 mg of sample were diluted with 1 ml of acetone 

regardless the different water contents. This procedure led to the problem, that peak areas were 

often too high or too small and fell out of the calibration range. Therefore, sample 15-21 were 

weight in such a way, that always the same organic concentration was used for sample dilution, 

taking into account the water content. 

An error in calibration could be excluded as all data were crosschecked. As an example the area 

of the internal standard and hydroxyacetaldehyd is demonstrated in 

Table 5 showing only small variation in the area of the internal standard peak.

Area Area

Biotox RT Hydroxyacetaldehyd Internal Standard

1r1 6.255 682323 305455

1r2 6.264 595758 305985

2r1 6.246 436699 301644

2r2 6.21 460930 312260

3r1 6.066 673015 292142

3r2 6.066 630364 280806

3r3 6.066 649367 283249

4r1 6.264 635401 292520

4r2 6.255 644617 295873

5r1 6.921 119532 295243

5r2 6.885 165268 283354

6r1 6.894 278112 252300

6r2 6.894 308401 253305

7r1 6.039 218146 288248

7r2 6.048 206653 291135

7r3 6.048 207618 285047

8r1 6.066 79120 281034

8r2 6.057 75029 281461

8r3 6.039 75554 287073

9r1 6.246 359548 308607

9r2 6.228 347723 310951

10r1 6.066 620199 289728

10r2 6.066 675975 281066

10r3 6.066 654678 285446

11r1 6.822 377959 253663

11r2 6.813 393844 268642

12r1 6.822 505283 273225

12r2 6.831 502952 274304

13r1 6.813 388624 235927

13r2 6.804 403134 238437

14r1 6.804 383762 230998

14r2 6.804 389723 241084

15r1 6.633 1252482 280470

15r2 6.624 1197549 249190

16r1 6.597 703662 288658

16r2 6.597 655881 282806

17r1 6.57 325925 284788

17r2 6.579 341422 286665

18r1 6.57 249539 281257

18r2 6.579 260484 281991

19r1 6.579 410800 287402

19r2 6.588 482772 289631

20r1 6.588 377092 282448

20r2 6.57 377794 285634

21r1 6.651 567262 287010

21r2 6.651 574567 281342

Table 5: Comparison of internal standard areas and hydroxyacetaldehyde 

2. Another reason for the lower detection of organic components from Biotox-16 to Biotox-21 was 

the higher amount of a quenching liquid detected in the gas chromatograms. This hydrocarbon 

liquid is used in the pyrolysis cooling system and leads to a dilution of the sample. 

Figure 7 shows the results of quantified amount from pyrolytic lignin determination, water 

determination by Karl-Fischer and the GC-quantification. It also shows the GC unknowns which are 
21

detectable by gas chromatography but could not be analyzed due to missing spectral and 

chromatographic data. Nevertheless, the most relevant peaks were quantified, as is shown in Figure 

7. The data from Figure 7 are also presented in 

Table 6. 

It is also worthwhile to note that the amount of unknowns is larger in the sample set 1-15. This is 

because of the larger sample amount used for the preparation of the sample. As a consequence the 

amount of GC unknowns is increasing. 

The large amount of aldehydes of Biotox-15 is due to an extraordinary amount of hydroxylacetaldehyde. 

Table 6: Overall Composition of Biotox- oils

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Biotox Oil Number

wt.%

GC unknown

GC known

Water

Py-Lignin

Figure 7: Quantified amounts of Biotox- samples

- Reproducibility of pyrolysis reaction and GC-analysis 

In order to compare the pyrolytic conditions and the GC-results the following two figures were 

produced as Biotox-9 and Biotox-17 as well as Biotox-10 and Biotox-21 were produced under 

identical conditions. 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Py-Lignin 19.7 17.7 12.4 15.2 10.9 5.8 54.4 n/a 18.2 19.1 15.6 24.9 20.2 21.1 n/a 14.9 16.2 11.3 20.7 15.2 15.7

Water 23.5 28.5 29.1 24.9 29.4 37.0 8.1 28.6 26.8 22.4 26.7 20.3 24.6 22.7 12.1 18.2 25.5 31.8 23.0 26.8 17.6

GC known 22.3 21.5 26.9 24.5 27.5 22.0 21.6 25.1 26.9 25.7 27.8 28.1 28.7 27.8 31.6 19.6 21.8 19.1 15.3 19.9 19.1

GC unknown 11.6 9.9 9.4 10.4 8.9 7.7 13.5 10.5 8.8 9.6 7.7 11.1 12.1 10.6 5.6 4.9 1.5 2.3 1.3 1.6 1.2

Unknown portion 22.9 22.4 22.2 25.1 23.3 27.5 2.4 n/a 19.3 23.3 22.2 15.7 14.4 17.8 n7a 42.4 35.0 35.5 39.8 36.6 46.4
22

Biotox 17

Biotox 9

Biotox 10

Biotox 21

Figure 8: Comparison of Biotox-9 vs. Biotox-17 and Biotox-10 vs. Biotox-21 

It is visible that Biotox-9 and Biotox-10 oils show more peaks which might be attributed to 

differences in the quenching system. Differences in peak intensity can be attributed to the more 

diluted samples Biotox-17 and Biotox-21 due to quenching liquid and the GC solvent acetone. 

- PAH analysis 

The existing PAH method of BFH was slightly modified in consultation with the EU JRC (Joint 

Research Centre, Geel). BFH has participated in a round robin test organized by JRC. The 

following 

Table 7 gives the detailed steps for extraction and clean-up of PAH from pyrolysis oils. 

put 5 g pyrolysis oil in a beaker

add 1 ml internal standard solution with benzo(A)anthracene D12, c = 4 μg/ml

add 1 ml internal standard solution with anthracene D10, c = 4 μg/ml

mix with 10 ml NaOH and pour into separation funnel 

extract with 60 ml cyclohexane

remove NaOH phase

add 10 ml NaOH

extract; remove NaOH phase

dry cyclohexane extract over Na2SO4

condition 2 g Florisil-cartrige (MgO3Si) with 15 ml cyclohexane

add cyclohexane-extract on cartridge

use 50 ml cyclohexane for elution

add keeper (100 μl DMF) to eluate

evaporate solvent to dryness

dissolve in 1ml cyclohexane and fill vial for GC/MS
23

Table 7: Extraction and clean-up of PAH from pyrolysis oils 

The results of the determination are presented in Table 8. Benzo(a)anthracene (BaA) and Benzo-

(a)pyrene (BaP) are in bold as these components are the key substances for legislative limits. For 

comparison, in liquid smoke samples the limit for BaA is 0.01 ppm and for BAP is 0.02 ppm. 

Biotox 1 Biotox 2 Biotox 3 Biotox 4 Biotox 5 Biotox 6 Biotox 7

ppm ppm ppm ppm ppm ppm ppm

Fluorene 4.68 1.90 0.97 0.89 2.18 1.02 39.0

Phenantrene 4.54 1.64 0.76 0.46 2.53 2.09 23.8

Anthracene 1.08 0.27 0.17 0.15 0.79 0.54 14.0

Fluoranthene 0.77 0.42 0.21 0.11 0.36 0.50 6.15

Pyrene 1.38 0.58 0.23 0.18 0.75 0.65 9.53

Benzo(a)anthracene 0.31 0.09 0.02 0.02 0.23 0.14 4.14

Chrysene 0.27 0.09 0.05 0.03 0.15 0.14 3.16

Benzo(A)Fluoranthene 0.10 0.05 0.02 0.03 0.05 0.06 1.04

Benzo(K)Fluoranthene 0.09 0.03 0.01 0.03 0.05 0.07 1.06

Benzo(A)Pyrene 0.24 0.07 0.03 0.04 0.16 0.12 1.32

Indenopyrene 0.14 0.06 0.02 0.01 0.08 0.10 1.44

Dibenzoanthracene n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Benzoperylene 0.11 0.06 0.02 0.02 0.06 0.10 n.d.

TOTAL 13.71 5.28 2.52 1.97 7.40 5.53 104.55

Biotox 8 Biotox 9 Biotox 10 Biotox 11 Biotox 12 Biotox 13 Biotox 14

ppm ppm ppm ppm ppm ppm ppm

Fluorene - 0.90 n.d. n.d. n.d. n.d. 0.39

Phenantrene - 1.07 0.83 3.70 9.63 n.d. 0.75

Anthracene 14.7 0.31 0.24 0.84 2.02 0.26 0.22

Fluoranthene 5.11 0.23 0.21 0.76 0.98 0.13 0.25

Pyrene 8.50 0.46 0.45 1.60 2.17 0.20 0.41

Benzo(a)anthracene 2.16 0.15 0.19 0.81 1.68 0.03 0.16

Chrysene 1.87 0.12 0.13 0.82 1.34 0.01 0.11

Benzo(A)Fluoranthene 0.51 0.06 0.08 0.37 0.78 n.d. 0.08

Benzo(K)Fluoranthene 0.41 0.06 0.07 0.39 0.67 n.d. 0.08

Benzo(A)Pyrene n.d. 0.17 0.20 0.97 1.88 0.04 0.19

Indenopyrene 0.37 0.08 0.16 0.43 0.98 n.d. 0.12

Dibenzoanthracene n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Benzoperylene 0.35 0.08 0.13 0.58 1.28 0.03 0.09

TOTAL 33.95 3.70 2.70 11.28 23.43 0.70 2.86

Biotox 15 Biotox 16 Biotox 17 Biotox 18 Biotox 19 Biotox 20 Biotox 21

ppm ppm ppm ppm ppm ppm ppm

Fluorene 5.03 0.03 n.d. 2.81 0.66 1.23 0.13

Phenantren 5.30 0.05 0.10 0.97 1.26 0.85 0.21

Anthracene 1.40 0.01 0.05 0.29 0.32 0.23 0.06

Fluoranthene 0.17 n.d. 0.01 0.35 0.31 0.23 0.06

Pyrene 1.24 0.02 0.06 0.35 0.63 0.35 0.26

Benzo(a)anthracene 0.50 n.d. 0.02 0.06 0.23 0.11 0.03

Chrysene 0.36 n.d. 0.01 0.10 0.18 0.09 0.02

Benzo(A)Fluoranthene 0.19 n.d. 0.01 0.03 0.09 0.04 0.01

Benzo(K)Fluoranthene 0.17 n.d. 0.01 0.01 0.08 0.03 0.01

Benzo(A)Pyrene 0.84 0.17 0.39 0.47 0.57 0.49 0.30

Indenopyrene 0.15 n.d. 0.01 0.03 0.09 0.04 0.01

Dibenzoanthracene n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Benzoperylene 0.09 n.d. 0.01 0.03 0.08 0.04 0.02

TOTAL 10.41 0.25 0.70 2.68 3.84 2.50 1.01

Table 8: Determination of most relevant PAH's in bio-oils

A visual inspection of the PAH composition is shown in Figure 9. It is clearly demonstrated that the 

Biotox-7 and Biotox-8 oils have the highest amounts of PAH. This was expected and is due to the 

relatively long pyrolysis times compared to the other fast pyrolysis processes. 
24

0

20

40

60

80

100

120

p

p

m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

BIOTOX Oil No.

Benzoperylene

Indenopyrene

Benzo(A)Pyrene

Benzo(k)Fluoranthene

Benzo(A)Fluoranthene

Chrysene

Benzo(A)Anthracene

Pyrene

Fluoranthene

Anthracene

Phenantrene

Fluorene

Figure 9: Graphical comparison of PAH's amounts and composition

In order to improve visualization of the smaller remaining PAH data in Figure 9, Figure 10 was 

produced excluding the data set of Biotox-7 and Biotox-8. 

0

5

10

15

20

25

p

p

m

1 2 3 4 5 6 9 10 11 12 13 14 15 16 17 18 19 20 21

BIOTOX Oil No.

Benzoperylene

Indenopyrene

Benzo(A)Pyrene

Benzo(k)Fluoranthene

Benzo(A)Fluoranthene

Chrysene

Benzo(A)Anthracene

Pyrene

Fluoranthene

Anthracene

Phenantrene

Fluorene

Figure 10: Graphical comparison of PAH's amounts and composition with exclusion of Biotox-7 

and Biotox-8

As Benzo(a)pyrene is considered the most toxic component it amount in the different Biotoxsamples is illustrated in Figure 11. 
25

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Oil Producer

ppm

Benzo(A)pyrene

Figure 11: Benzo(a)pyrene content in Biotox oils 

Interestingly, Biotox-11 and Biotox-12 show relative high values although they are produced by 

fluidized bed pyrolysis. Biotox-12 shows the highest amount as is was produced at 600°C. 

-UV/VIS-Spectral Analysis 

Because some of the CIT laboratory test reported on possible effects of light absorption, the spectra 

in the ultra violet region (190-400 nm) and the visible light region (400-850 nm) were recorded with 

a spectralphotometer. The overall spectra for the whole wavelength region are presented in Figure 

12. 

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

180 280 380 480 580 680 780

nm

Absorbance

Figure 12 : UV/VIS spectra of all 21 Biotox samples

No important differences can be observed. Figure 13 shows the UV wavelength region. Again, no 

significant differences can be observed. The maximum at 200 nm results from the absorption of 
26

carbohydrate derived components and is not very specific. The smaller maximum at 280 nm can be 

assigned to aromatic structures. 

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

180 230 280 330

nm

Absorbance

Figure 13: UV wavelength region of all 21 Biotox samples 

b) Correlations between oil properties and production processes

Correlations can only be done from oil made at Aston as all oils were produced with the same 

equipment under controlled conditions. 

* Nature of feedstock 

Feedstock comparison is possible between Biotox-9 (beech wood) and 10 (spruce wood). The water 

content is rather similar. The spruce oil is has a 3 fold higher viscosity. 

* Temperature 

The effect of temperature can be observed using Biotox-11 (400°C), Biotox- 10 (500°C), Biotox-15 

(575°C), and Biotox-12 (600°C). Water is indifferent. Viscosity is higher at 600°C, pyrolytic raises 

with temperature from 15 to 25 %, and the carbon content raises with temperature from 39 to 45 %. 

* Aging 

Aging can be discussed by comparing Biotox-19 (12 months old) vs. Biotox-21. As expected the 

water content of Biotox-19 is 5 % higher than that of Biotox-21 due to possible recondensation 

reactions during storage. Surprisingly, the viscosity is slightly higher with the "young" oil. The 

pyrolytic lignin amount is also 5 % higher with the old oil and corroborates results of other studies. 
27

I.2.2.2 Bio-oils Toxicological screening tests

As controversial data concerning the bio-oil’s mutagenicity were published in the few toxicological 

studies reported in the literature the project steering comity decided to assess the potential 

mutagenic effect of Bio-oils, via the “Bacterial reverse mutation test” 

The objective of this study is to evaluate the potential of bio-oils to induce reverse mutation in 

Salmonella typhimurium. The bacterial reverse mutation test is able to identify substances that cause 

point mutations, by substitution, addition or deletion of one or a few DNA base-pairs. Mutagenic 

substances can induce reversion in histidine deficient strains, which are then able to grow and form 

colonies in a histidine-limited medium, while non-reverted strains cannot. This test is performed in 

the absence and presence of a rat liver metabolizing system (S9 mix). 

Guideline: Commission directive 2000/32/EC,B13,8 June 2000 and OECD Guideline No. 471, 

21st July 1997 

a) Preparation of the test solutions 

The vehicle for solubilising bio-oil was selected according to results from solubility trials 

performed before the preliminary toxicity test. Dimethylsulfoxide (DMSO) appeared mainly to be 

the best vehicle for bio-oil. 

b) Bacterial strains 

Five strains of Salmonella typhimurium are used to carry out this study: TA 1535, TA 1537, TA 98, 

TA 100 and TA 102. The day before treatment, cultures are inoculated from bacterial suspensions. 

Each strain derived from Salmonella typhimurium LT2 contains one mutation in the histidine 

operon, resulting in a requirement for histidine. 

c) Metabolic activation system 

The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction 

(S9 fraction) and the co-factors necessary for their function. 

d) Mutagenicity experiment 

In one mutagenicity experiment, using three plate/dose-level, each strain are tested, with and 

without S9 mix, with: 

 at least five dose-levels of bio-oil, 

 the vehicle control, 

 the appropriate positive control 

e) Evaluation of the results 

In each experiment, for each strain and for each experimental point, the number of revertants per 

plate is scored. Studies are considered valid if the following criteria are fully met: 

the number of revertants in the vehicle controls is consistent with historical data, 

the number of revertants in the positive controls is higher than that of the vehicle controls 

and is consistent with historical data. 

A 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 

and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain 

at any dose-level and/or evidence of a dose-relationship is considered as a positive result. 
28

f) Results 

Obtained results are recapitulated in following Table 9.

TA 153TA 153 TA 98TA 10T0A 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100A 102

5000

4000 T T T T

3750

3000 T T T

2500 T T T P T T T T T

2000 T T T T T T T

1875 T

1250 T T T T

1000 T

625 T T

250 T

156,3 T T

TA 153TA 153 TA 98TA 10T0A 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100A 102

5000 T

4000 T

3750

3000 T T

2500 T T T T P T P/T P P T T T T T

2000 T

1875

1250 T

1000

625

156,3

TA 153TA 153 TA 98TA 10T0A 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100A 102

5000 T T T T T T T T

4000

3750 T T T T T T T T T T T T T T P/T P/T P/T P/T P T T T T

3000

2500 T T T T T T T

2000

1875 T T

1250 T T T T T T T

1000

625

250

156,3

TA 153TA 153 TA 98TA 10T0A 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10TA 153TA 153 TA 98TA 100TA 102TA 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 10T2A 153TA 153 TA 98TA 100TA 102

5000 T T T T T T T T T T P/T P/T P/T P P T T T T

4000

3750 T T T T T T T T TTTT TTTT T

3000

2500 T T

2000

1875

1250 TTTT

1000

625

156,3

Coloured compartment : the strongest bio-oil dose tested in the study

When the strongest tested dose< 5000 μg/fla P for precipitation and\or T for toxicity in preliminary toxicity tests

result clearly positive (mutagenic effect). no mutagenic effect was noted

ambiguous result with increase of the number of revertants, which seems to depend on the bio-oil dose level tested.

Biotox 10

-

Biotox 11 Biotox 12 Biotox 13 Biotox 14

S9 Biotox 6 Biotox 7

MIX

μg/ 

plate

Biotox 9

+

Biotox 2 Biotox 3 Biotox 4 Biotox 5 Biotox 8 Biotox 9

Biotox 1 Biotox 2 Biotox 10

Biotox 11 Biotox 12

Biotox 5 Biotox 6 Biotox 7 Biotox 8

Biotox 13 Biotox 14

Biotox 1

S9 Biotox 3 Biotox 4

MIX

μg/ 

plate

Biotox 15 Biotox 16

Biotox 15 Biotox 16 Biotox 19 Biotox 20

Biotox 17 Biotox 18

Biotox 17 Biotox 18

+

Biotox 21

Biotox 21

S9 

MIX

μg/ 

plate

-

S9 

MIX

μg/ 

plate

Biotox 19 Biotox 20

Table 9: Results of the mutagenicity ames tests 

In Table 9 for each oil, each strain, with or without S9 mix, the highest concentration tested in the 

study is marked by a coloured compartment. 

Lower concentration than 5000 μg/flat might have been used when : 

- the solubility of the bio oil is limiting the concentration labelled by a P (precipitation) 

- the bio-oil is too toxic for the test to be representative labelled by a T (toxic) 

Levels of concentration to be used are assessed by preliminary toxicity test. 

Compartments are yellow if no mutagenic effect was noted, red colour is used if the result was 

clearly positive (mutagenic effect). Compartments coloured in orange corresponded to ambiguous 

result. In those cases an increase of the number of revertants was observed, which seem to depend 

on the bio-oil dose level tested. But the increased in number of revertants compared with the vehicle 

controls didn’t reach the level specified in international regulations to consider a positive mutagenic 

result. The observation of a dose-relationship being rather subjective; additional tests would be 

necessary to verify the reproducibility. 
29

For all bio-oils, at least one test for one Salmonella typhimurium strain, the increase in number of 

revertants reach the level specified in international regulations to consider a positive mutagenic 

result (red colored compartment). Base on these results all bio-oils are considered to be mutagenic. 

Some oils seem to be more mutagenic than others, but it is not possible to compare the oils 

mutagenicity as the tested concentrations are different. Indeed, in a lot of cases highest oils 

concentrations tested are below 5000 μg/flat, because in preliminary toxicity tests those samples 

appeared to be toxic or not soluble at high concentrations. In such cases the toxicity and/or the low 

solubility of the sample forbid mutagenicity assessment at high concentration. 

However it emerges that oils produced by slow pyrolysis (Biotox-7 & -8) behave differently from 

the other oils. They have a strongly toxic effect on the strains. It limits the study of their 

mutagenicity while, in order to minimize their toxic effect, it is necessary to work on very weak 

bio-oils dose level. 

Interpretations of these results are not easy. It is in fact not possible to compare bio-oils between 

themselves as tested dose levels are variables. 

However, it seems that for all these tests the number of revertants generally increases when the 

quantity of oil increased, up to a certain oil concentration from which the toxicity of oil prevents the 

development of reverted bacteria. 

I.2.2.3 Bio-oils Ecotoxicological screening tests

In order to assess how pyrolysis oils could affect the environment and the organisms living in it, 

ecotoxicological screening tests were carried on with the 21 samples. Ecotoxicology is the basis for 

defining the No-Effect Loading Rate (NOEL) used in risk assessment. The potential ecotoxicity of 

bio-oils was studied on unicellular algal (Algal growth inhibition test) and on small animal (Acute 

toxicity in Daphnia magna STRAUS). 

a) Algal growth inhibition test 

The objective of this study is to assess the effects of bio-oils on the growth of an unicellular green 

algal species Scenedesmus subspicatus (a rapidly growing unicellular species), in a 72-hour static 

test. The criterion measured is the EL50 (Medium Effect Loading rate), a statistically derived 

loading rate of the bio-oil in water which can be expected to cause a reduction of growth or growth 

rate of 50% in the treated algal populations relative to the control. 

This study has been designed to comply with the following guideline 

Guidelines Commission directive 92/69/EEC, C.3, 31st July 1992 

OECD Guideline No. 201, 7th June 1984 

Species Scenedesmus subspicatus 

Pseudokirchneriella subcapitata

Study design Control + generally 3 concentrations: 1, 10 and 100 mg/L 

The WAF methodology is proposed for poorly soluble substances 

(solubility < 100 mg/L 

Results Growth rate inhibition: estimation of the EC50 after 72 hours

Biomass inhibition: estimation of the EC50 after 72 hours 

Table 10: Informations about the algal growth inhibition test
30

Test system 

Tests were carried out by subjecting three replicates of algal culture to bio-oils test solutions 

(loading rates of bio-oil 1, 10 ad 100 mg/l), over a 72-hours period. The number of cells were 

counted at each observation time (T24, T48 and T72 hours) using a Cell Counter. 

The criterion measured was the EL50 (Medium Effect Loading rate), a statistically derived loading 

rate of the bio-oil in water which can be expected to cause a reduction of growth or growth rate of 

50% in the treated algal populations relative to the control.: 

Data evaluation 

The calculated percentages of inhibition of the cell growth rate and growth at each loading rate of 

bio-oil tested are determined following the procedures recommended in EEC and OECD guidelines. 

 The percentage inhibition of the growth rate or growth is based on comparison between the 

growth rate or growth for each loading rate and the growth rate or growth for the control. This 

comparison allows to determine respectively an ErL50 or EbL50 - loading rate of the test item 

resulting in 50% reduction of the specific growth rate or growth with respect to the control. 

Where possible, the 72-hour ErL 50 and the 72-hours EbL 50 are estimated as follows: 

EL50 1 mg/L 

or 1 < EL50 10 mg/L 

or 10 < EL50 100 mg/L 

or EL50 > 100 mg/L 

For the test to be valid, the cell concentration in the control cultures should have increased by a 

factor of at least 16 within three days. 

b) Acute toxicity in daphnia magna 

The objective of this study is to assess the acute toxicity of bio-oil in Daphnia magna STRAUS – 

clone 5 (the most sensitive clone of the species), in a 48-hour static test. Due to the small size of the 

Daphnia, mortality is difficult to determine and therefore the test criterion of acute toxicity used is 

immobilization, expressed as EC50 (Median Effective Concentration, a statistically derived 

concentration of the test item (bio-oil) in water at which 50% of animals are immobilized). An 

animal is defined as immobile when it is incapable of swimming within 15 seconds after gentle 

agitation of the test container. The criterion measured is the EL50 (Median Effect Loading rate), a 

statistically derived loading rate of the test item (bio-oil) in water at which 50% of animals are 

immobilized. 

This study has been designed to comply with the following guidelines: 

Guidelines Commission directive 92/69/EEC,C.2, 31st July 1992 

OECD Guideline No. 202, 4th April 1984 

Species Daphnia magna STRAUS, clone 5 

Study design Control + generally 3 concentrations: 1, 10 and 100 mg/L 

The WAF methodology is proposed for poorly soluble substances 

(solubility < 100 mg/L) 

Results Immobilization: estimation of the EC50 after 24 and 48 hours 

Table 11: Informations about the Acute toxicity in daphnia magna 
31

Test system 

Daphnia (animals) are selected without preference and randomly assigned to test vessels. The test is 

carried out by subjecting 20 Daphnia to a maximum loading rate of 100 mg/l and further groups of 

20 Daphnia to loading rates of 1 and 10 mg/l, over a 48-hour period. The control also contains 20 

Daphnia. 

The test is carried for 48 hours and immobilisation is noted at T0, T24 and T48 hours by gently 

shaking the test vessel. If an animal is incapable of swimming within 15 seconds after it has been 

disturbed, it is considered immobile. 

Data evaluation 

Where possible, the 24 and 48-hour EL50 are estimated as follows: 

EL50 1 mg/L 

or 1 < EL50 10 mg/L 

or 10 < EL50 100 mg/L 

or EL50 > 100 mg/L 

For a test to be valid, the following conditions should be fulfilled: 

The immobilisation in the control should not exceed 10% at the end of the test; 

The dissolved oxygen concentration should remain  60% of the air saturation value 

throughout the test. 

c) Results of the ecotoxicological tests:

Obtained results for the two tests reported in Table 12 and Table 13. 

The 21 pyrolysis-oils can be shared in three categories: 

1. 12 bio-oils with no eco-toxicology (Biotox -3; 5; 6; 7; 11; 12; 14; 16; 18; 19; 20; 21); 

2. 8 bio-oils with very light eco-toxicology (Biotox -1; 2; 4; 9; 10; 13; 15; 17); 

3. and one eco-toxic pyrolysis oils (Biotox 8 slow pyrolysis sample) 
TEST ITEM STUDY RESULTS 

Algal growth inhibition test 

The 0-72h ErL50 was > 100 mg/L The 0-72h EbL50 was > 100 mg/L The 0-72h NOEL was 

 100 mg/L BIOTOX-3 

Acute toxicity in Daphnia magna

The 48h EL50 was > 100 mg/L The 0-48h NOEL was 

 100 mg/L 

Algal growth inhibition tes Like BIOTOX-3 BIOTOX-5 

Daphnia magna ” ” ” ” 

Algal growth inhibition test 

 ” ” ” ” 

BIOTOX-6 Acute toxicity in 

Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

 ” ” ” ” 

BIOTOX-7 Acute toxicity in 

Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

 ” ” ” ” 

BIOTOX-11 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

” ” ” ”

Significant inhibition of algal growth by light absorption at 10 and 100 mg/L BIOTOX-12 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

The 0-72h ErL50 was > 100 mg/L The 0-72h EbL50 was > 100 mg/L The 0-72h NOEL was 10 mg/L BIOTOX-14 

Acute toxicity in Daphnia magna

The 48h EL50 was > 100 mg/L The 0-48h NOEL was 10 mg/L 

TEST ITEM STUDY RESULTS 

Algal growth inhibition test 

” ” ” ” No significant inhibition of algal growth by light absorption up to 100 mg/L BIOTOX-16 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

” ” ” ”No significant inhibition of algal growth by light absorption up to 100 mg/L BIOTOX-18 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

” ” ” ”

Significant inhibition of algal growth by light absorption at 100 mg/L BIOTOX-19 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

The 0-72h ErL50 was > 100 mg/L The 0-72h EbL50 was > 100 mg/L The 0-72h NOEL was 10 mg/L No significant inhibition of algal growth by light absorption up to 100 mg/L 

BIOTOX-20 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Algal growth inhibition test 

” ” ” ”No significant inhibition of algal growth by light absorption up to 100 mg/L BIOTOX-21 

Acute toxicity in Daphnia magna

 ” ” ” ” 

Table 12: Pyrolysis oils without eco-toxicity
LOWEST EL50 CLOSE TO 100 MG/L 

Algal growth 

inhibition test 

 0-72h ErL50 was > 100 mg/L 

0-72h EbL50 estimated close to 100 mg/L 

 0-72h NOEL was 10 mg/L BIOTOX-4 Acute toxicity 

in Daphnia 

magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 

 100 mg/L 

Algal growth 

inhibition test 

 0-72h ErL50 was > 100 mg/L 

0-72h EbL50 to be close to 100 mg/L 

 0-72h NOEL was 10 mg/L BIOTOX-9 Acute toxicity 

in Daphnia 

magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 

 100 mg/L 

Algal growth 

inhibition test 

 0-72h ErL50 was > 100 mg/L 

0-72h EbL50 was estimated close to 100 

mg/L  0-72h NOEL was 10 mg/L No inhibition by light absorption up to 100 mg/L 

BIOTOX-13

Acute toxicity 

in Daphnia 

magna

 48h EL50 > 100 mg/L  0-48h NOEL was 10 mg/L 

Algal growth 

inhibition test 

 0-72h ErL50 > 100 mg/L 

0-72h EbL50 estimated close to 100 mg/L 

 0-72h NOEL was 10 mg/L No inhibition by light absorption up to 100 mg/L BIOTOX-17

Acute toxicity 

in Daphnia 

magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 

 100 mg/L 

LOWEST EL50 BETWEEN 10 AND 100 MG/L

BIOTOX-1 Algal growth inhibition test 

 0-72h ErL50 was > 100 mg/L 

0-72h EbL50 estimated between 10 and 

100 mg/L  0-72h NOEL was 10 mg/L 

Acute toxicity in 

Daphnia magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 

 100 mg/L 

BIOTOX-2 Algal growth inhibition test 0-72h ErL50 was > 100 mg/L 0-72h EbL50 between 10 & 100 mg/L 0-72h NOEL was 10 mg/L 

Acute toxicity in 

Daphnia magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 

 100 mg/L 

BIOTOX-15 Algal growth inhibition test 

0-72h ErL50 to be close to 100 mg/L 

0-72h EbL50 between 10 & 100 mg/L. 

 0-72h NOEL was 10 mg/L No inhibition by light absorption up to 100 mg/L 

Acute toxicity in 

Daphnia magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 10 mg/L 

LOWEST EL50 CLOSE TO 10 MG/LBIOTOX-10 Algal growth inhibition test 0-72h ErL50 > 100 mg/L 0-72h EbL50 estimated close to 10 mg/L 0-72h NOEL was < 1 mg/L 

Acute toxicity in 

Daphnia magna

 48h EL50 was > 100 mg/L  0-48h NOEL was 

 100 mg/L 

LOWEST EL50 BETWEEN 1 AND 10 MG/L

BIOTOX-8 Algal growth inhibition test 0-72h ErL50 between 10 and 100 mg/L 0-72h EbL50 between 1 and 10 mg/L 0-72h NOEL was 1 mg/L 

Acute toxicity in 

Daphnia magna

 48h EL50 between 10 and 100 mg/L The 0-48h NOEL was 10 mg/L 

Table 13: Pyrolysis oils light eco-toxicity (except Biotox 8)
34

Based on these obtained results, it comes into view that fast pyrolysis oils have none or weak ecotoxicological effect. 

The Acute toxicity in daphnia magna study demonstrates that (except the slow pyrolysis oil sample 

Biotox 8) bio-oils have not toxicological effect on small animals. 

The Algal growth inhibition study demonstrated a very rare effect of flash pyrolysis oils on the 

unicellular green algal. Even low concentrations of bio-oils in the medium had a fertilizer effect, 

increasing algal growth. 

This inhibition of algal growth can be due either to a toxic effect of the oil sample or by light 

adsorption which reduces photosynthesis activity (bio-oils are dark black and can slow down light 

diffusion). 

In order to determine the importance of both phenomena, further light absorption tests were 

performed on samples Biotox -12; 13; 15; 16; 17, 18; 19; 20; 21. These tests consisted of studying 

the algal growth in different light filtered area, with and without bio-oils. 

Results obtained for Biotox -12 and -21 shown that samples absorbed a significant amount of light. 

While results obtained for other sample shown that a growth inhibition didn’t come from light 

absorption, and can only be due to a toxic effect of the oil. It seems that both effects (toxicity and 

light absorption) induce the growth observed inhibition effect, and according to samples these 

phenomena can be more or less important 

I.2.2.4 Bio-oils Aerobic biodegradability in fresh water

The aim of the study is to measure the aerobic biodegradability of pyrolysis oils in order to evaluate 

if they could be a local environmental hazard in case of accidental discharges. 

As bio-oils have a low solubility in water and the volatility of some of their components, analyse of 

their biodegradability was difficult. It was necessary in a first time to experiment and to adapt tests 

protocols to measure the biodegradability of bio-oils.. 

a) Methodology

The bio-oils biodegradability was assessed based on the OECD 301B modified Sturm Test. This 

test is applied to a substance that has passed a stringent test for ultimate biodegradability, which is 

internationally accepted as reference test. It was developed to identify substances that would be 

rapidly and extensively biodegraded in the aqueous environment and it’s stringency lead to good 

estimation of an oil product's biodegradability. 

Test Method Official OECD “ready test” procedures 301B Sturm Modified; 

Respirometry CO2 evolution 

Inoculum Activated sludge from sewage treatment plant (32 mg/l SS) 

Bio-oils concentrations 15 mg/l Dissolved Organic Carbon 

Number of flasks Samples, reference substance, and blanks are duplicates 

Reference Substance Sodium acetate 

Measures CO2 is trapped in Ba(OH)2 and is measured by titration of the 

residual OHBlank values CO2 < 40 mg/l 

Test duration 28 days 

Table 14: Informations about the biodegradability modified Sturm test
35

b) Test system 

A controlled volume of inoculated mineral medium, containing a known concentration of the tested 

substance (10-20 mg DOC/l) as the nominal sole source of organic carbon is aerated by a controlled 

flow of carbon dioxide-free air in the dark or in diffuse light conditions. Degradation is followed 

over 28 days by determining the carbon dioxide produced. The CO2 is trapped in barium hydroxide 

and is measured by titration of the residual hydroxide. The amount of carbon dioxide produced from 

the test substance (corrected for that derived from the blank inoculum) is expressed as a percentage 

of theoretical carbon dioxide (ThCO2). The test lasts for 28 days 

Only 15 mg/l of dissolved organic Carbon from bio-oils sample had to be added to the inoculated 

mineral solution. As bio-oils have a high carbon content, Cirad developed a method to easily weigh 

the small amounts of sample to be assessed. Samples were weighed on a non-biodegradable solid 

carrier (glass filter). 

All tests were performed in replicates and obtained values are close (average deviation between 0,5 

and 2,4 %). 

c) Results

In addition to the 21 oils samples, a conventional diesel oil sample was tested for comparison. 

Biodegradability curves measured over the 28 days are presented in following Figure 14. 

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30

Days

% Biodegradability

Biotox 1

Biotox 2

Biotox 3

Biotox 4

Biotox 5

Biotox 6

Biotox 9

Biotox 10

Biotox 11

Biotox 12

Biotox 13

Biotox 14

Biotox 16

Biotox 18

Biotox 19

Biotox 20

Biotox 21

Diesel

Biotox 7

Biotox 8 organic fraction

Biotox 8 aqueous fraction

Figure 14: Biodegradation curves of pyrolysis oils following the 301B OECD test method. 
36

Biodegradation reactions were described by a simple first-order kinetic Eq. (1) and regressionprocessed by the least squares method, minimization of target function applying the Solver 

subprogram of Microsoft Excel 7.0. 

 Eq. (1) 

where: 

DCO2 , Dmax, biodegradation in time t, and maximal (limit) value 

k rate constant (h-1) 

tlag lag phase of biodegradation (days). 

Kinetic data are numerically presented in Table 15.

 Measured Calculated 

Bio-oils DCO2

(28 days) 

Dmax 

(%)

k

(days-1)

t(lag)

(days)

t1/2 

(days)

Biotox - 1 42,66 43,23 0,16 1,14 4,40 

Biotox - 2 44,64 44,75 0,15 1,03 4,69 

Biotox - 3 43,29 43,34 0,15 1,47 4,60 

Biotox - 4 50,11 51,13 0,13 1,04 5,17 

Biotox - 5 42,50 42,85 0,14 1,57 4,94 

Biotox - 6 49,50 49,14 0,17 1,66 4,02 

Biotox - 7 27,50 29,14 0,08 1,08 8,21 

Biotox - 8 30,63 30,73 0,13 1,13 5,20 

Biotox - 9 45,90 46,15 0,14 0,91 5,06 

Biotox - 10 42,77 42,23 0,15 0,94 4,66 

Biotox - 11 49,72 49,27 0,15 1,16 4,59 

Biotox - 12 40,78 40,33 0,14 1,20 4,88 

Biotox - 13 44,25 43,86 0,14 1,09 5,00 

Biotox - 14 31,06 30,69 0,18 0,97 3,74 

Biotox - 15 Not enough sample 

Biotox - 16 43,71 43,37 0,18 0,5 3,78 

Biotox - 17 Not enough sample 

Biotox - 18 41,01 40,91 0,16 1 4,35 

Biotox - 19 35,22 35,06 0,18 0,93 3,83 

Biotox - 20 39,77 40,24 0,14 0,93 4,78 

Biotox - 21 41,58 41,37 0,18 1,1 3,90 

Diesel 24,33 31,67 0,06 2 12,45 

Table 15: Kinetic data calculated for each oils biodegradation 
37

d) Conclusions

The first important information from the reported study is that all pyrolysis oils assessed are 

biodegradable as for each flask, an emission of CO2 was measured. Bio-oil biodegradation starts 

immediately and no lag phases are observed at the beginning of a test. That indicates a very fast 

biological response (i.e., microbial growth, enzyme activity/synthesis). It means that some 

compounds in pyrolysis oils are immediately degraded by competent degraders from a conventional 

inoculum source: a sludge freshly collected from the aeration tank of a sewage treatment plant. 

No lag phase at the beginning of tests (Tlag~1day) also reflects a rapid mass transfer of oil from the 

glass filters to the solution, which validates the method used to introduce oil samples in flasks. 

Biodegradation is also immediately observed at the beginning of the tests due to the acid nature of 

pyrolysis oils. In acidified aqueous solution, CO2 is not dissolved and is directly removed as gas. 

This was verified by the evolution of the percentage of biodegradation at day 29, as the addition of 

1ml acid solution in the medium engender a large emission of CO2 only for the reference samples 

(which are not acid) and not for the oil samples. 

All biodegradation curves have similar shapes and the values from replicates are close with an 

average deviation of degradability percentage between 0.5 and 2.4 %. Biodegradation curves show 

rapid degradation kinetics during the first 8 days (the growth phase) to reach a plateau (stationary 

phase) highlighting that pyrolysis oils biodegradation has virtually stopped between 22 to 26 days. 

Based on the results presented, it appears that flash pyrolysis oils with biodegradation percentages 

between 32 and 50 % are slightly more biodegradable than gas oils (around 24 %) and have a much 

higher biodegradable potential than heavy fuel (11%). 

I.2.2.5 Conclusions and selection of the representative sample for the full toxicological 

study

Assessment of the physico-chemical and the toxicology of the 21 collected samples gave a lot of 

information on Bio-oils. 

These results reveal that despite being produced under differences conditions all the collected Biooils show similar properties, with minor variations in their physico-chemical composition and very 

similar toxicity (all mutagenic), ecotoxicity (no or light ecotoxicity) and same type of 

biodegradation (between 32 and 50 % and no lag phase) 

Results of the characterisation of samples produced by Aston shown that 

o The spruce oil is has a 3 fold higher viscosity than beech oil. 

o The highest the pyrolysis temperature, the highest are the carbon content and the 

viscosity. 

o Aging induce higher water content 

o The highest the pyrolysis temperature, the highest is the PAH content. 

A rigorous work was done to try to correlate oils productions parameters, to physico-chemical 

characteristics and to the toxicity, eco-toxicity and biodegradability. Three partners meetings were 

devoted to this work. 
38

It clearly appeared that the number of potential influent parameters was too high, and variations of 

the results to low and despite all the collected data it was impossible to correlates all the results and 

in several cases just possible to elaborate hypothesis. 

A goal of the engaged work was to investigate all the results of the tests done, in order to select the 

best parameters to produce a representative (standard) bio-oil sample. 

No real bio-oil shows typical properties (except biotox 7 and 8), based on this statement the 

consortium selected the parameters to be used to produce the sample for the full toxicological and 

ecotoxicological tests: 

a temperature of 500°C : based on the results of the chemical analysis which allowed to 

determined the best pyrolysis temperature around 500°C to minimise the PAH level, it is 

also the temperature which maximize the liquid yield and will therefore be not appropriate 

for large scale production. 

spruce feedstock : soft wood is a typical European biomass 

fluidised bed process : this is the common industrial process used for bio-oil production 

Three kilograms of this bio-oil were produced by Aston University. All the full toxicological and 

ecotoxicological tests were done by CIT.
39

I.2.3 Full toxicological study of one selected representative sample 

One objective of the project was to notify bio-oils as a new substance to be placed on the EU 

market, following the actual notification way recommended nowadays within ELINCS (Eu List of 

Notified Chemical Substances). 

For the notification a technical dossier has to be set up, containing data concerning physicochemical; toxicological and ecotoxicological properties of the substance to be notified. Nature of 

these tests depends on the quantity of substance which is placed on the EU market. Procedures to be 

used are those set in Annex V of the Directive 92/32/CEE. 

Based on the quantity of pyrolysis oils produced today in Europe, the steering comity selected to 

follow the BASET VII A of the Annex V of the Directive 92/32/CEE, valuable for quantity 

manufactured between 1 and 10 tonnes. 

PHYSICO-CHEMICAL PROPERTIES 

A1 Freezing point (if > -20°C) 

A1 Melting point 

A2 Boiling point 

A3 Relative density 

A4 Vapour pressure - Static or gas saturation method 

A5 Surface tension 

A6 Water solubility - Flask or elution column method 

A8 Partition coefficient n-octanol/water 

A9 Flash point (liquids) 

A10 Flammability (solids) 

A12 Flammability : contact with water 

A13 Autoflammability 

A14 Explosive properties : 3 types 

A15 Self-ignition temperature for a liquid 

A16 Self-ignition temperature for a solid 

A17 Oxidising properties (solids) 

TOXICOLOGY STUDIES 

B1 tris Acute oral toxicity (rat) - Acute toxic class method 

B3 Acute dermal toxicity (rat) for a limit test at 2000 mg/kg 

B4 Skin irritation (rabbit) 

B5 Eye irritation (rabbit) 

B6 Local lymph node assay : LLNA 

B7 28-day oral toxicity study by gavage (rat) 

7-day range-finding study for the 28-day study 

B14 Gene mutation in bacteria (Ames test) 

B10 In-vitro chromosomal aberration in human lymphocyte 

ECOTOXICOLOGY STUDIES 

C1 Acute toxicity to fish (trout) according to the WAF method 

C2 Acute toxicity to daphnia according to the WAF method 

C3 Algal growth inhibition according to the WAF method 

C4 Biodegradability (modified Sturm test) 

including activated sludge respiration inhibition test 

C7 Hydrolysis as a function of pH for a highly hydrolysable substance 

Table 16: Notification of a new substance - base set - VII A 

Commission Directive 92/32/EEC (Quantity > 1 tonne and < 10 tonnes) 
40

Steering comity reviewed all the tests requirements from Annex VII A of the notification of a new 

substance. Based on the results of the screening tests and of CIT experience and due to their 

characteristics some tests are not valid for bio-oils. Based on this discussion, a selection of the tests 

to be done is presented in Table 17 

PHYSICO-CHEMICAL PROPERTIES 

A6 Water solubility - Flask or elution column method 

A8 Partition coefficient n-octanol/water 

A14 Explosive properties : 3 types 

TOXICOLOGY STUDIES

B1 tris Acute oral toxicity (rat) - Acute toxic class method 

B3 Acute dermal toxicity (rat) for a limit test at 2000 mg/kg 

B4 Skin irritation (rabbit) 

B5 Eye irritation (rabbit) 

B6 Local lymph node assay : LLNA 

B7 28-day oral toxicity study by gavage (rat) 

7-day range-finding study for the 28-day study 

B14 Gene mutation in bacteria (Ames test) 

B10 In-vitro chromosomal aberration in human lymphocyte 

ECOTOXICOLOGY STUDIES

C2 Acute toxicity to daphnia according to the WAF method 

Table 17: Selection of the feasible test to be carried out in the selected 

bio-oil sample

I.2.3.1 Tests A6 Water solubility, A8 Partition coefficient n-octanol/water and C2 Acute 

toxicity to daphnia 

Tests A6 Water solubility, A8 Partition coefficient n-octanol/water and C2 Acute toxicity to 

daphnia require further chemical analysis to estimate the evolution of the test item concentrations in 

solution during the tests. But due to the nature of bio-oils, which are a mixture of more than 500 

different compounds and not a purr substance, it is not possible to assess the evolution of it’s 

concentration in solution. 

Competent authorities (INERIS) were contacted in order to discuss the relevance of these tests for 

Bio-oils . It refused to give his opinion onto the necessity of leading the A8, A6 and C2 tests as 

REACH has not still officially started. 

In dialogue with the experts of CIT we decided to stop these tests. These tests without chemical 

analyses to follow the evolution of the tested substances in the time are senseless. 

I.2.3.2 -Test A14: Explosive properties 

The objective of this study was to evaluate the potential of the test item Biotox-21 presents a danger 

of explosion when subjected to heat (heat sensitivity) or shock (mechanical sensitivity). Two types 

of tests were carried out: 

Safety-in-handling tests: 

The objective is to establish safe conditions for the performance of the assays of sensitivity in the 

main tests. Exposure of a very small sample (between 10 and 40 mm3) of bio-oil 

- by heating, without any confinement, the sample directly with the flame of 

a gas burner, 

- by subjecting the sample to impact (shock) in suitable apparatus, 
41

The main tests concern a test of heat sensitivity by heating bio-oil in a stainless-steel tube with 

different degrees of confinement being provided by nozzle plates with holes of different diameters 

and a test of mechanical sensitivity (shock) by exposure of the bio-oil to the shock of a falling 

hammer on a steel anvil. 

Heat sensitivity test (flame test) 

Bio-oil was loaded into the tube to a height of 60 mm. The threaded collar was slipped onto the tube 

from below, the appropriate orifice plate was inserted and the nut tightened. The heating of tubes 

was provided by butane / propane, fitted with a pressure regulator, through a meter and evenly 

distributed by a manifold to four burners. The rate of gas is approximately 3.2 L/min. A test 

resulting in the fragmentation of the tube into three or more pieces, which in some cases may be 

connected to each other by narrow strips of metal, is evaluated as giving an explosion. A test 

resulting in fewer fragments or no fragmentation is regarded as not giving an explosion. Two series 

of three tests are mandatory: the first series using a nozzle plate with a hole of 6 mm diameter, the 

second using a hole of 2 mm diameter. 

Six negative assays were recorded: no heat sensitivity was noted with the test item. 

Mechanical sensitivity (shock) 

The test item is considered as presenting a danger of explosion if: 

- an explosion occurs (the tube bursts into three or more fragments) within the fixed number 

of tests for heat sensitivity or, 

- an explosion (bursting into flame and/or a report equivalent to explosion) occurs at least 

once in six tests of mechanical sensitivity (shock). 

For the main test a volume of bio-oil to reach 1 mm height in the cylinder was approximately 100 

μL. Six negative assays were recorded. No shock sensitivity was noted with the bi-oil. 

Conclusions

The Bio-oil sample is not considered to have explosive properties (heat and mechanical sensitivity) 

according to the experimental conditions. 

I.2.3.3 -Test B4 : Dermal Irritation In Rabbit

The objective of this study was to evaluate the potential of the test item Biotox-21 to induce skin 

irritation following a single topical application to rabbits. 

In the assessment of the toxic characteristics of a test item, determination of the irritant and/or 

corrosive effects on the skin of mammals is an important initial step. Information derived from this 

test serves to indicate the possible hazards likely to arise from exposure of the skin to the test item. 

This study was conducted in compliance with: 

• OECD guideline No. 404, 24th April 2002, 

• Directive 2004/73/EC, B.4, 29th April 2004.

a) Methodology 

The test item was applied undiluted on male New Zealand White rabbit. The day before treatment, 

both flanks of the animal were clipped using electric clippers and the skin of the animal was 

examined in order to check the absence of any signs of skin irritation. Bio-oil was first evaluated on 

a single animal. The durations of exposure were 3 minutes, 1 hour and 4 hours. Since the test item 
42

showed corrosive properties on this first animal, the study was considered complete and the test 

item was not evaluated on other animals. 

Doses of 0.5 mL of the undiluted test item were placed on a dry gauze pad, which was then applied 

to an area of approximately 6 cm2 of the anterior left flank (application for 3 minutes), the anterior 

right flank (application for 1 hour) or the posterior right flank (application for 4 hours) of the 

animal. The untreated skin served as control. 

After removal of the dressing, any residual test item was wiped off by means of a moistened cotton 

pad. 

The skin was examined approximately 1 hour, 24, 48 and 72 hours after removal of the dressing. As 

severe irritant effects were observed, the animal was killed on day 4 (after the reading), for ethical 

reasons. 

Dermal irritation was evaluated for each animal according to the following scoring scale: 

Erythema and eschar formation: 

. no erythema........................................................................................................... 0 

. very slight erythema (barely perceptible) ............................................................. 1 

. well-defined erythema........................................................................................... 2 

. moderate to severe erythema................................................................................. 3 

. severe erythema (beet redness) to slight eschar formation (injuries in depth)...... 4 

Oedema formation 

. no oedema ............................................................................................................. 0 

. very slight oedema (barely perceptible)................................................................. 1 

. slight oedema (edges of area well-defined by definite raising) ............................ 2 

. moderate oedema (raised approximately 1 millimetre) ........................................ 3 

. severe oedema (raised more than 1 millimetre and extending beyond area 

of exposure)............................................................................................................. 4 

Any other lesions were noted.

b) Interpretation of results and classification 

The results obtained were evaluated in conjunction with the nature and the reversibility of the 

findings observed. Classification of the test item is based on the criteria laid down in Council 

Directive 67/548/EEC (on the approximation of the laws, regulations and administrative provisions 

relating to the classification, packaging and labelling of dangerous substances. 

Criteria for irritation: A substance or a preparation is considered to be irritating to the skin if, 

when it is applied to healthy intact animal skin for up to 4 hours, significant inflammation is caused 

and which persists for 24 hours or more after the end of the exposure period. 

Criteria for corrosion :A substance or a preparation is considered to be corrosive if, when it is 

applied to healthy intact animal skin, it produces full thickness destruction of skin tissue on at least 

one animal during the test for skin irritation, or if the result can be predicted (for example: from 

strongly acid or alkaline reactions). 

Classification: 

Irritant substances 

- symbol Xi, indication of danger "irritant", 
43

- phrases indicating the nature of special risks: R 38: "Irritating to skin" Inflammation is 

significant if: the mean value of the scores is two or more for either erythema and eschar 

formation or oedema formation. The same will be the case where the test has been completed 

using three animals if the score for either erythema and eschar formation or oedema formation 

observed in two or more animals is equivalent to the value of two or more, it persists in at least 

two animals at the end of the observation period. Specific effects such as hyperplasia, 

desquamation, discolouration, fissures, eschar and alopecia should be taken into account. 

Corrosive substances 

- symbol C, indication of danger: "corrosive", 

- phrases indicating the nature of special risks:. 

R 34: "Causes burns" If, when applied to healthy intact animal skin, full thickness 

destruction of skin tissue occurs as a result of up to 4 hours exposure, or if this 

result can be predicted.. 

R 35: "Causes severe burns"If, when applied to healthy intact animal skin, full 

thickness destruction of skin tissue occurs as a result of up to 3 minutes exposure, 

or if this result can be predicted.

c) Results

After a 3-minute exposure:

A very slight erythema (grade 1) was observed on days 2 and 3. 

No other cutaneous reactions were noted. 

A beige coloration of the skin, due to the test item, was noted from day 1; it persisted up to the end 

of the observation period (day 4). 

Mean scores over 24, 48 and 72 hours were 0.7 for erythema and 0.0 for oedema. 

After a 1-hour exposure:

A well-defined erythema (grade 2; days 1, 2 and 3) then a severe erythema (grade 4; day 4) 

associated with a brownish area similar to a severe burn of the skin, were noted. 

A slight oedema (grade 2) was noted from day 2 up to day 4. 

A beige coloration of the skin, due to the test item, was noted from day 1 up to day 3. 

Mean scores over 24, 48 and 72 hours were 2.7 for erythema and 2.0 for oedema. 

After a 4-hour exposure: 

A well-defined erythema (grade 2; days 1, 2 and 3) then a marked erythema (grade 3; day 4) were 

observed. 

A very slight oedema (grade 1) was observed on days 3 and 4. 

A dryness of the skin was recorded on day 4. 

A beige coloration of the skin, due to the test item, was noted from day 1 up to day 4. 

Mean scores over 24, 48 and 72 hours were 2.3 for erythema and 0.7 for oedema. 

d) Conclusions

Under the experimental conditions, the test Bo-oil Biotox-21 is corrosive when applied topically to 

rabbits. According to the classification criteria laid down in Council Directive 67/548/EEC on the 

approximation of the laws, regulations and administrative provisions relating to the classification, 

packaging and labeling of dangerous substances, the bio-oil Biotox-21 should be classified as 

corrosive and assigned the symbol C, the indication of danger "Corrosive" and the risk phrase R 34: 

"Causes burns".
44

I.2.3.4 –Test B3: Acute Dermal Toxicity in Rat and B5 Eye Irritation in Rabbit 

As the “B4 Dermal Irritation In Rabbit” test shown that bio-oils are corrosives, tests B3 Acute 

Dermal Toxicity In Rat and B5 Eye Irritation In Rabbit were cancelled for ethic reasons as 

recommended by the Commission Directive 92/32/EEC. 

I.2.3.5 –Test B1 tris: Acute Oral Toxicity in Rat

The objective of this study was to evaluate the toxicity of the test item BIOTOX-21 following a 

single oral administration in rats. In the assessment of the toxic characteristics of a test item, 

determination of acute oral toxicity is an initial step. It provides information on health hazards 

likely to arise following a short-term exposure by the oral route in humans and enables the test item 

to be ranked in different classification systems. 

The acute toxic class method is a stepwise procedure. The test item is administered orally to one 

group of animals at one of the defined dose-levels, each step using three females. Absence or 

presence of compound-related mortality of the animals dosed at one step determines the next step, 

i.e.: 

• no further testing is needed, 

• the next step is performed with the same dose, 

• the next step is performed at the next higher or the next lower dose-level. 

The study was conducted in compliance with: 

• EC Directive 2004/73/EC, B.1 tris, 29th April 2004, 

• OECD Guideline No. 423, 17th December 2001. 

a) Methodology 

Bio-oil was prepared at the chosen concentration in the vehicle (propylene glycol). 

The animals were fasted for an overnight period of approximately 18 hours before dosing, but had 

free access to water. Food was given back approximately 4 hours after administration of the test 

item. Three females were used for each step. The dose-level used as the starting dose was selected 

from one of four fixed levels, 5, 50, 300 or 2000 mg/kg body weight. As the information on the 

toxic potential of the test item suggested that mortality was unlikely at the highest dose-level, a 

limit test was performed, using the starting dose-level of 2000 mg/kg with three animals. As no 

deaths occurred, the results were then confirmed in three other females. 

The study design was as follows: 

Dose(mg/kg) Volume(mL/kg) Female 

2000 10 3 

2000 10 3 

The dosage form preparations were administered to the animals under a volume of 10 mL/kg. The 

administration was performed in a single dose by oral route using a metal gavage tube fitted to a 2 

mL plastic syringe (0.1 mL graduations). The volume administered to each animal was adjusted 

according to body weight determined on the day of treatment. The single administration was 

performed in the morning of day 1; it was followed by a 14-day observation period. 
45

The animals were observed frequently during the hours following administration of the test item, 

for detection of possible treatment-related clinical signs. The animals were weighed individually 

just before administration of the test item on day 1 and then on days 8 and 15. The body weight gain 

of the treated animals was compared to that of CIT control animals with the same initial body 

weight.

b) Results

No mortality was noted during the study. 

Hypoactivity or sedation, piloerection and dyspnea (all animals); unsteady gait (3/6 animals), were 

observed within 6 hours of treatment. 

No more clinical signs persisted on day 2, up to the end of the study. 

The overall body weight gain of the treated animals was similar to that of CIT historical control 

animals. Macroscopic examination of the main organs of the animals revealed no apparent 

abnormalities. 

c) Conclusions

Under the experimental conditions, the oral LD50 of the test item BIOTOX-21 is higher than 2000 

mg/kg in rats.

I.2.3.6 –Test B6 : Evaluation of skin sensitization potential in Mice Using Local Lymph 

Node Assay (LLNA)° 

The aim of this study was to evaluate the potential of the test item BIOTOX-21 to induce delayed 

contact hypersensitivity, using the murine Local Lymph Node Assay (LLNA 

The study has been designed to comply with the following guidelines: 

. OECD Guideline No. 429, 24th April 2002, 

. EC Directive No. 2004/73/EC, part B.42, 29th April 2004.

a) Methodology 

A preliminary test was first performed in order to define the concentrations of test item to be used in 

the main test. 

In the main test, twenty-eight female CBA/J mice were allocated to seven groups: 

• five treated groups of four animals receiving the test item BIOTOX-21 at the concentration of 

0.5, 1, 2.5, 5 or 10%, 

• one negative control group of four animals receiving the vehicle (dimethylformamide =DMF), 

• one positive control group of four animals receiving the reference item, -hexylcinnamaldehyde 

(HCA), a moderate sensitizer, at the concentration of 25%. 

During the induction phase, bio-oil, vehicle or reference item was applied over the ears (25 μL per 

ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of 

lymphocytes in the lymph node draining the application site was measured by incorporation of 
46

tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices 

(SI). 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on 

days 1, 2, 3 and 6.

b) Results 

The administration of the undiluted test item was not possible. The undiluted form of the test item 

could not pass through a micropipette. 

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive 

oil (4/1, v/v)), DMF was chosen among the other proposed vehicles. 

A homogeneous dosage form preparation was obtained at the maximal concentration of 50%. 

Consequently, the concentrations selected for the preliminary test were 5, 10, 25 and 50%. 

Since the test item was considered as irritant in the preliminary test at the concentrations of 25 and 

50%, the highest tested concentration retained for the main test was 10%. 

Systemic clinical signs and mortality: No mortality and no clinical signs were observed during the 

study. 

Local irritation: No cutaneous reactions and no noteworthy increase of ear thickness were observed 

in the animals of the treated groups.

Proliferation assay: A significant lymphoproliferation (SI > 3) was noted at the tested 

concentrations of 5 and 10% (SI = 6.35 and 14.58, respectively), as well as in the positive control 

group given HCA (SI = 3.99). 

The results are presented in table Table 18.

Table 18: Results of the “Evaluation of skin sensitization potential in Mice Using LLNA” test°

In the absence of local irritation, the observed lymphoproliferative responses were attributed to 

delayed contact hypersensitivity. The EC3 value for the test item BIOTOX-21 is equal to 3.19%. 

c) Conclusions 

Under the experimental conditions, the test item BIOTOX-21 induces delayed contact 

hypersensitivity in the murine Local Lymph Node Assay. 
47

According to the EC3 value obtained in this experiment and to the categorization of contact 

allergens by Kimber I. and al. (2003), the test item BIOTOX-21 should be considered as a moderate 

sensitizer.

I.2.3.7 –Test B7: 7-day study oral route in rats 

The objective of this study was to evaluate the potential toxicity of the test item, BIOTOX-21, 

following daily oral administration (gavage) to rats for 7 days. 

The rat was chosen because it is a rodent species commonly accepted by regulatory authorities for 

this type of study. The oral route was selected since it is a route of exposure which is requested by 

the authorities. The dose-levels were selected in agreement with the Sponsor on the basis of the 

results of an acute toxicity study by oral route performed in the same species (Test B1 tris: Acute 

Oral Toxicity in Rat). 

This study was based on the following guidelines: 

. OECD Guideline No. 407, 27th July 1995, 

. EEC Directive No. 96/54, B7, 30th September 1996.

a) Methodology 

Three groups of three male and three female Sprague-Dawley rats received the test item, BIOTOX21, daily, by oral (gavage) administration, for 7 days, at dose-levels of 150, 500 or 1500 mg/kg/day. 

Another group of three males and three females received the vehicle, propylene glycol, under the 

same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg. 

Clinical signs and mortality were checked daily. Body weight was recorded three times and food 

consumption twice during the dosing period. On completion of the dosing period, the animals were 

sacrificed and a complete macroscopic examination was performed. Selected organs were weighed 

and any macroscopic lesions were preserved.

b) Clinical examinations 

Morbidity and mortality: Each animal was checked for mortality or signs of morbidity at least twice 

a day during the treatment period, including weekends and public holidays, and at least once a day 

during the acclimation period. 

Clinical signs: Each animal was observed at least once a day, at approximately the same time, for 

the recording of clinical signs. 

Body weight: The body weight of each animal was recorded at least once before group allocation, 

on the first day of treatment, and on days 4 and 7. 

Food consumption: The quantity of food consumed by each animal was recorded twice, over 3- or 

4-day periods, during the treatment period. 
48

c) Pathology 

Sacrifice: On completion of the treatment period, after at least 14 hours fasting, all animals were 

sacrificed by carbon dioxide inhalation and exsanguination. 

Organ weights: The body weights of all animals sacrificed at the end of the treatment period were 

recorded before sacrifice. The adrenals, brain, heart, testes and epididymides, kidneys, liver, lungs, 

ovaries, spleen and thymus were weighed wet as soon as possible after dissection. The ratio of 

organ weight to body weight (recorded immediately before sacrifice) was calculated. 

Macroscopic post-mortem examination: A complete macroscopic post-mortem examination was 

performed on all study animals. This included examination of the external surfaces, all orifices, 

the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities 

with their associated organs and tissues and the neck with its associated organs and tissues. 

Preservation of tissues: For all study animals, macroscopic lesions were preserved in 10% buffered 

formalin. 

d) Statistical analysis 

The specific sequence was used for the statistical analyses of body weight, food consumption, and 

organ weight data.

e) Results

There were no premature deaths during the study. 

All animals given 1500 mg/kg/day had hypersalivation throughout the treatment period and one 

female also had loud breathing and piloerection. No clinical signs were observed at 500 or 150 

mg/kg/day. 

There was a dosage-related reduction in mean body weight gain for females given 150, 500 or 1500 

mg/kg/day and for males given 500 or 1500 mg/kg/day between day 1 and day 4 of dosing. From 

day 4 of dosing all groups had body weight gains comparable with the controls except the males 

given 1500 mg/kg/day who continued to have reduced body weight gains and statistically 

significantly reduced body weights. 

Males given 1500 mg/kg/day had reduced food consumption throughout the study while females 

given 1500 mg/kg/day and males given 500 mg/kg/day had reduced food consumption just for the 

first half of the dosing period. There was no effect at 150 mg/kg/day or for females given 500 

mg/kg/day. 

At necropsy, all animals given 1500 mg/kg/day had yellowish-colored, thickened forestomachs and 

most had dilated duodenums. One male given 500 mg/kg/day had a dilated duodenum. 

The mean absolute and relative spleen weights were reduced for both males and females at 1500 

mg/kg/day. The mean absolute and relative liver and ovary weights were increased for females 

given 1500 mg/kg/day. The mean absolute and relative thymus weights were reduced for males 

given 1500 mg/kg/day. 
49

f) Conclusions

Hypersalivation was observed for all animals at 1500 mg/kg/day with at least an initial reduction in 

food consumption and body weight gain and, for the males, statistically significantly reduced body 

weights. At necropsy, yellowish-colored, thickened forestomach were observed with dilated 

duodenums. Spleen weights were reduced for males and females, thymus weights were reduced for 

the males and liver and ovary weights were increased for the females. 

At 500 mg/kg/day, there were no clinical signs, but there was a reduction in body weight gain and, 

for males, a reduction in food consumption. One male had dilatation of the duodenum. 

At 150 mg/kg/day, there were no clinical signs of toxicity, a slight reduction in the body weight 

gain of the females and no effect on food consumption. 

No macroscopic abnormalities were observed. 

I.2.3.8 –Test MAS In Vivo: Bone marrow micronucleus test by oral route gavage in mice 

The objective of this study is to evaluate the potential of the test item to induce damage to the 

chromosomes or the mitotic apparatus in bone marrow cells of mice. Apart from detecting 

chromosome breakage events (clastogenesis), the micronucleus test is capable of detecting 

chemicals which induce whole chromosome loss (aneuploidy) in the absence of clastogenic activity. 

In the bone marrow of mice exposed to a chemical which induces cytogenetic damage, 

chromosomal fragments or entire chromosomes which are left behind at cell division will not be 

incorporated into the nuclei of daughter cells. Most of these fragments condense and form one or 

more micronuclei in the cytoplasm. The visualization of micronuclei is facilitated in erythrocytes 

because their nucleus is extruded during erythropoiesis. Accordingly, the basis of this test is an 

evaluation of the increase in the number of micronucleated polychromatic erythrocytes (MPE). 

Substances which inhibit either proliferation or maturation of erythroblasts and those which 

are toxic for nucleated cells, decrease the proportion of immature erythrocytes (polychromatic, PE) 

when compared to mature erythrocytes (normochromatic, NE). Thus, the cytotoxicity of a substance 

can be evaluated by a decrease in the PE/NE ratio. 

This study has been designed to comply with the following guidelines: 

· OECD guideline No. 474, adopted on 21st July 1997. 

· Commission Directive No. 2000/32/EC, B12, 8 June 2000.

a) Methodology 

Three dose-levels, administered orally twice separated by 24 hours, were used with one single 

sampling time (see Table 19) . 
50

. 

Table 19: Methodology applied for the” Bone marrow micronucleus test by oral route gavage in 

mice” test

Blood samples for the determination of plasma levels of the test item were taken following the 

second treatment.

At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs were 

removed and the bone marrow were flushed out with fetal calf serum. After centrifugation, the 

supernatant were removed and the cells in the sediment were resuspended by shaking. A drop of 

this cell suspension was placed and spread on a slide. The slides were coded so that the scorer is 

unaware of the treatment group of the slide under evaluation (“blind” scoring).

For each animal, the number of micronucleated polychromatic erythrocytes (MPE) were counted, 

the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a 

total of 1000 erythrocytes (PE + NE). 

The analysis of the slides was performed at CIT or at Microptic, cytogenetic services (2 Langland 

Close Mumbles, Swansea SA3 4LY, UK). 

b) Results 

No significant increase in the frequency of micronucleated cells was observed in animals treated 

with the bio-oil sample. Analyses on blood samples would be necessary to confirm inactivity of the 

bio-oil sample. 

I.2.3.9 – Test MNV In Vitro: Micronucleus test in L5178 TK+/- mouse lymphoma cells

The objective of this study is to evaluate the potential of the test item to induce any increase in the 

frequency of micronucleated cells. The micronuclei observed in the cytoplasm of interphase cells 

may originate from acentric fragments (chromosome fragments lacking a centromere) or whole 

chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of 

cell division. The assay thus has the potential to detect the activity of both clastogenic and 
51

aneugenic chemicals. This test will be performed in the absence and presence of a rat liver 

metabolising system (S9 mix). 

This study has been designed to comply with the following guidelines: 

· OECD guideline No. 487, draft dated June 14, 2004.

Preliminary toxicity test : 

a) Methodology 

To assess the cytotoxicity of the bio-oil sample, six dose-levels (one culture/dose-level) were tested 

both with and without metabolic activation. Assessment of cytotoxicity was performed by 

evaluation of population doubling (PD) (e). 

The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the 

starting count (N0), divided by the log of 2. 

PD = [log (N/N0)]/log 2. 

Mutagenicity experiments : 

In two independent experiments, four dose-levels of bio-oils (two cultures/doselevel) were tested 

both with and without metabolic activation, using treatment duration as follows: 

Each treatment was performed in duplicate as follows: 

For treatment, approximately 3 x 105 cells/mL (final concentration = N0) in RPMI 1640 medium 

containing 5% inactivated horse serum with or without 5% S9 mix, were exposed to the bio-oil or 

control item. 

At the end of the treatment period which will be performed at 37°C in a humidified atmosphere of 

5% CO2/95% air, the cells were washed twice. Cells were suspended in RPMI 1640 medium 

containing 5% inactivated horse serum and the plates were incubated, at 37°C in a humidified 

atmosphere of 5% CO2/95% air, for the recovery period, if any.

At the end of the recovery period, if any, the cells were washed with RPMI 1640 medium 

containing 10% inactivated horse serum containing 1% pluronic acid. The cells were suspended in a 

medium with 49.5% RPMI 1640 medium containing 10% inactivated horse serum, 50% PBS and 

0.5% pluronic acid, before being fixed. 

Depending on the observation at the end of the recovery period (presence or absence of precipitate 

and/or cytotoxicity), at least three dose-levels of bio-oil treated cultures were selected for spreading 

of slides in the view of slide analysis. Cells were dropped onto clean glass slides. Each treatment 

wascoupled to an assessment of cytotoxicity at the same dose-levels. 

Cytotoxicity was evaluated by determining the PD of cells and was also take into account the 

quality of the cells on the slides.

The slides were coded before analysis performed under a microscope (1000 x magnification).
52

b) Results

Without S9 mix: 

 

In the second experiment (24-hour treatment), a dose-related tendency is observed in the frequency 

of micronucleated cells with a significant difference at 15.63 μg/mL. These results come in addition 

to the significant result (although not dose-related) noted in the first experiment (3-hours 

treatment).It is necessary to perform confirmatory assays, ideally for the two experimental 

conditions (3-hour and 24-h treatments) in order to judge of the relevance of the observed results 

With S9 mix: 

In the second experiment, a dose-related tendency is observed in the frequency of micronucleated 

cells with a significant difference at 75 μg/mL. The equivalent of this dose-level of 75 μg/mL could 

not be analyzed in the first experiment, and therefore it is difficult to judge of the reproducibility of 

this effect, all the more since only at this dose that the decrease of doubling population reachs the 

50% level. It would be consequently necessary to perform a confirmatory experiment, using a closer 

range of dose-levels. 

c) Conlusion

Under the experimental conditions, the test item Biotox-21 presented a light mutagenic activity, 

which has to be confirm through further experiment 

I.2.3.10 – Conclusions of the full toxicological characterisation of the selected sample 

All the planned tests were carried out without problems, it appears that bio-oil is: 

 not explosive when subjected to heat or shock (A14 test),

 should be classified as “corrosive” and assigned the symbol C, the indication of danger 

“Corrosive” and risk phrase R34: “ Causes Burns” (B4 test),

 “Moderate sensitizer” and assigned the risk phrase R 43: “May cause sensitization by skin 

contact“ (B6 test),

 not toxic by oral route (B1 tris test) and by 7-days oral gavage (B7 test),

 not mutagenic via In Vivo MAS test but shown a light mutagenic effect via In Vitro MNV test. 

Studies B3 Acute Dermal Toxicity In Rat & B5 Eye Irritation In Rabbit were cancelled, for ethic 

reason, due to the corrosive behaviour of bio-oils substance 

I.2.4 Recommendations for safety procedures and dissemination of the results 

Main objective of Biotox is to ensure that all obtained results within the project will benefit to the 

flash pyrolysis oil community. Three main works were planned: dissemination of the results, 

editions of safety documents based on the obtained results, and notification of Bio-oil as a new 

substance to be placed on the Eu market. 
53

I.2.4.1 Dissemination 

In a first time bio-oils producers were informed and consulted (to choose samples to be collected 

and relevant analyses to be carried out) concerning the projects trough experts meetings and 

conferences (see chapter management). They were invited to take part to the project by producing 

oils sample for the projects. 

All the results were presented several times through several conferences and publications: 

a) Conferences and experts meetings 

Conference: Pyrolysis and gasification of biomass and waste, in Strasbourg, 30 September-1 

October 2002, Poster presentation and proceeding Pyrolysis and Gasification of Biomass 

and Waste, A. V. Bridgwater, CPL press, p155; 

PyNe Expert meeting in Florence, 2-6 April 2003. Oral presentation and proceeding; 

PyNe Expert meeting in Bruges, 15-18 April 2004. Oral presentation and proceeding; 

Conference: Science in Thermal and chemical Biomass Conversion Conference in Victoria

(30 august to 2 september 2004). Poster presentation. 

Proceeding: J. Blin, G. Volle, N. Maghnaoui & P. Girard; “Biodegradability of fast 

pyrolysis” Science in thermal and chemical biomass conversion – Vancouver Island – 

Canada –Sept. 2004, A. V. Bridgwater, CPL press, in press; 

Conference: Science in Thermal and chemical Biomass Conversion Conference in Victoria

(30 august to 2 September 2004). Oral presentation. 

Proceeding: Girard P. Blin J. “Bio-oil toxicity for safe handling and transportation “ Science 

in thermal and chemical biomass conversion – Vancouver Island – Canada –Sept. 2004; A.

V. Bridgwater, CPL press, in press; 

PyNe Expert meeting in Innsbrück, 27-30 September 2005. Oral presentation; 

Results of the project as been selected for an oral presentation in the international conference: 

“The 14th European Conference and Technology Exhibition on Biomass for Energy, 

Industry and Climate Protection” scheduled to take place from the 17th to 21st October 2005 

in Paris. 

b) Published articles 

P. Girard & J. Blin. “Environment, Health and Safety Aspects Related to Pyrolysis oils“. 

2005; PyNe Handbook Volume 3, chap 4, P 61-70, A. V. Bridgwater, CPL press, UK 

J. Blin, P. Girard, G. Volle. “Biodegradability of Fast Pyrolysis Oil“. 2005, PyNe Handbook 

Volume 3, chap 5, P 71-94, A. V. Bridgwater, CPL press, UK. 

J. Blin, P. Girard, G. Volle.“Bio-oil toxicity assessment versus pyrolysis parameters“. 2005, 

PyNe Handbook Volume 3, chap 6, P95-104, A. V. Bridgwater, CPL press, UK. 
54

A complete paper presenting Biodegradability work carried out within Biotox has been 

submited to be published in the international academic journal Chemosphere: Aerobic 

Biodegradability of Biomass Pyrolysis Oils; Joël Blin, Ghislaine Volle, Philippe Girard, 

Anthony Bridgwater, Dietrich Meier, Draft for Chemosphere.

Biotox was also introduced in March 2003 in the Pyne newletter: Paper in issue N° 15 available to 

download in PDF format http://www.pyne.co.uk/docs/PyNews%2015.pdf ; 

A book containing all the results of the project, with details on all analytical methods is under 

preparation to be published in the form of a “fast pyrolysis handbook n°4. 

c) Web site: 

A web pages hosted by the PyNe site is dedicated to Biotox http://www.pyne.co.uk/?_id=29. 

This web page will be used in following months to disseminate all the publishable results of the project 

(publishable report, report on transport requirements and guidelines, MSDS, ..), as accepted by the 

commission. 

I.2.4.2 Notification of Bio-oil as a new substance to be placed on the Eu market 

According to EU legislation since 1981, news substances introduced in the internal market must 

undergo an in-depth risk assessment to examine the risks posed to humans and the environment 

New subsatnces are then notified in ELINCS (Eu List of Notified Chemical Substances 4000 

entries). Risk assessment follows the framework set out in Regulation 1488/94 and Directive 93/67. 

Before, substances introduced between 1971 and 1981 were listed in the EINECS (EU Inventory of 

Existing Commercial Chemical Substances 100 196 entries). 

The present system for general industrial chemicals distinguishes between "existing substances in 

EINECS " i.e. all chemicals declared to be on the market in September 1981, and "new substances 

in ELINCS " i.e. those placed on the market since that date. 

At the beginning of the project, members of the flash pyrolysis thought that Bi-oils were not 

registred as an existing substances witch can be transported and manufactured within the EU 

market. Several bibliographic work and consultations of competent Eu authorities confirmed this 

observation. To reply on oils manufacturers’ request, steering comity decided within Biotox to set 

up the technical dossier to be submitted to the European Competent Authorities to notify bio-oil as a 

new chemical substance. This choice influenced the selection of the tests carried out for the full 

toxicological characterisation of the representative selected oil (see chapter II.2.4). 

But during the first year of the project, investigations on the data list of the European Chemicals 

Bureau (ECB) allowed to find that products obtained by pyrolysis of wood were registered in the 

EINECS data list, which definition fit well with fast pyrolysis bio-oil. 

 Name:Wood, hydropyrolysed 

 Definition: A complex combination of organic compounds obtained from the thermal 

decomposition of wood

 Cas n° : 94114-43-9

 Einecs n° : 302-678-6
55

Examination of the EINECS notification dossier, reveal that pyrolysis oils were only registered with 

a name, a full definition, an EINECS number and CAS number. However, neither other data 

(characterisations, requirement and guidelines for transport and storage) nor MSDS are available. 

Competent authorities (European Chemicals Bureau and INERIS) were contacted in order to know 

if it is necessary to notify Bio-oils as a new substance in ELINCS as it is registered in the existing 

substances list EINECS. They confirmed that because pyrolysis oil is described in the EINECS data 

list with EINECS and CAS numbers, it is not mandatory to do a new notification. These two 

numbers are the only required data for legal commercialisation of products in Europe today. 

In addition, since the project started, the legislation for production, transport and storages of 

substances within the Eu market has been planned to change in the coming years. 

Today, there are only 2,700 new substances (in ELINCS). Testing and assessing their risks to 

human health and the environment according to Directive 67/548 are required before marketing in 

volumes above 10 kg. Indeed, following EU legislation, Directive 93/67/EEC and Commission 

Regulation (EC) N°. 1488/94, the testing requirements are tiered according to the volume placed on 

the market. The lowest volume triggering the need for testing amounts to 10 kg. More extensive 

testing is required when the volume reaches 100 kg, 1 t, 10 t, 100 t and 1,000 t, respectively. 

Generally, testing requirements at the lower volumes (10 kg to 1 t) focus on acute hazards, 

immediate or slightly delayed effects after short term exposure, while those at the higher tonnage 

include more expensive studies on the effects of sub- chronic exposure, on reproductive toxicity and 

on carcinogenicity. The testing package at 1 t is termed ‘base set’ while those triggered by higher 

tonnage are called Level 1 (100 t) and Level 2 (1,000 t). 

Existing substances (in EINECS) amounting for more than 99% of the total volume of all 

substances on the market are not subject to the same testing requirements. The number of existing 

substances reported in 1981 was 100 106, the current number of existing substances marketed in 

volumes above 1 tonne is estimated at 30 000. Some 140 of these substances have been identified as 

priority substances and are subject to comprehensive risk assessment carried out by Member State 

authorities. 

In contrast to new substances, existing substances have never been subjected to systematic testing 

regime. When the requirement for testing and notification of new substances was introduced in 

1981, substances already on the market were exempted. 

The gap in knowledge about intrinsic properties for existing substances should be closed to ensure 

that equivalent information to that on new substances is available. Therefore, the Commission 

proposes that existing and new substances should in the future, following the phasing in of existing 

substances until 2012, be subject to the same procedure under a single system. In October 2003 the 

European Commission adopted a legislative proposal for implementing a new EU chemicals policy. 

The new regulatory system, known as REACH (Registration, Evaluation and Authorization of 

Chemicals), comprises four procedures. 

1. Registration of chemicals, documenting that risk is adequately controlled. Manufacturers 

and importers of chemicals in the EU must register those substances they produce or market in 

quantities above 1 tonne per year (tpa). Phase-in periods are proposed for some 30000 existing 

substances, with substances manufactured or imported in tonnage above 1000 tpa being registered 

first, followed by 100 tpa and 1tpa. In order to avoid multiple testing of the same substance, 
56

REACH aims to encourage industries to form consortia and gather information into a central 

database. 

2. Evaluation of the registration dossiers, considering mainly testing proposals. Member-state 

authorities will evaluate testing proposals for all substances manufactured or imported in quantities 

above 100 tpa (dossier evaluation) and any other prioritized substances if deemed necessary (known 

as a substance evaluation). 

3. Authorization of substances of very high concern. These include substances that are 

carcinogenic, mutagenic or toxic to reproduction and substances with a potential for persistence and 

bioaccumulation combined with high (eco-)toxicity or very persistent and very bio accumulative 

substances. 

4. Restriction of substances at the level of the European Community when risk reduction 

measures proposed by industry are not sufficient. Restrictions can be considered as a “safety net” 

allowing EU member states and/or the Commission to address risks that are not managed 

adequately by other parts of the REACH system. 

REACH will implement the objectives set out in the “White paper: Strategy for a Future Chemicals 

Policy”].It will give industry the responsibility for ensuring and demonstrating the safe 

manufacture, use and disposal of chemicals. This represents a shift of responsibility to demonstrate 

and manage risk(s) associated with the use of chemicals from authorities to the actors in the 

chemical supply chain. As a result, manufacturers and importers of chemicals are required not only 

to control risks present during those stages of a substance’s life cycle under their direct control but 

also to give guidance for use by downstream users on the safe handling and use of the substance. 

So with REACH, it is planned that EINECS registration will be examined to identify the missing 

data for safe handling, transportation and used. However, because REACH is under discussion and 

will probably not be operational before 2006, it is not possible to enrich the existing EINECS data 

base. 

The competent authority suggested us to compile all collected data to set up a full dossier base on 

ELINCS requirement, the SNIF summary (SUMMARY NOTIFICATION INFORMATION 

FORMAT) , with MSDS in order to prepare REACH. 

I.2.4.3 Editions of safety documents based on the obtained results 

Based on the obtained results from the physico-chemical and screening toxicological tests of the 21 oils 

Care set up by a full report requirements for “transport, storage and handling of biomass derived fast 

pyrolysis liquids; compliance with all international modes of transport” ( free available on the Pyne 

Web site). 

This report addresses the legislative requirements and regulations for the safe transport and 

labelling of pyrolysis liquids. 

An article on “Environment, Health and Saftey Aspects Related to Pyrolysis oils“ was published in 

PyNe Handbook Volume 3, (P. Girard & J. Blin. 2005; chap 4, P 61-70, A. V. Bridgwater, CPL 
57

press, UK). This paper review the Risks involved in bio-oil production and use, and the 

environmental risk assessment in the Eu context. 

An other article intituled “Biodegradability of Fast Pyrolysis Oil” was published PyNe Handbook 

Volume 3 (J. Blin, P. Girard, G. Volle, 2005, chap 5, P 71-94, A. V. Bridgwater, CPL press). In this 

report the methodology to be applied to assess bio-oils biodegradability is described as well as the 

results obtained within Biotox. 

A complete MSDS with all data collected within the project is available on the Pyne Web site. 

All these documents on secure handling of bio-oils will be registered on PyNe web site with free 

access and download when the commission will have validated the final report. 

I.3. Acknowledgements 

Biotox is a RTD project funded through the European Commission's Energy, environment and 

sustainable development Programme under Framework Programme. The Biotox Partnership is very 

grateful to the European Union for providing financial support (under contract number NNE5-2001-

00744). The Biotox Partnership thanks the project officer from the Commission, Mr. José Riesgo 

Villanueva, for his support over the period of the grant.