02008R0440 — EN — 26.03.2023 — 009.001


This text is meant purely as a documentation tool and has no legal effect. The Union's institutions do not assume any liability for its contents. The authentic versions of the relevant acts, including their preambles, are those published in the Official Journal of the European Union and available in EUR-Lex. Those official texts are directly accessible through the links embedded in this document

►B

►C1   COMMISSION REGULATION (EC) No 440/2008

of 30 May 2008

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

(Text with EEA relevance)  ◄

(OJ L 142 31.5.2008, p. 1)

Amended by:

 

 

Official Journal

  No

page

date

►M1

COMMISSION REGULATION (EC) No 761/2009 of 23 July 2009

  L 220

1

24.8.2009

 M2

COMMISSION REGULATION (EU) No 1152/2010 of 8 December 2010

  L 324

13

9.12.2010

►M3

COMMISSION REGULATION (EU) No 640/2012 of 6 July 2012

  L 193

1

20.7.2012

►M4

COMMISSION REGULATION (EU) No 260/2014 of 24 January 2014

  L 81

1

19.3.2014

►M5

COMMISSION REGULATION (EU) No 900/2014 of 15 July 2014

  L 247

1

21.8.2014

►M6

COMMISSION REGULATION (EU) 2016/266 of 7 December 2015

  L 54

1

1.3.2016

►M7

COMMISSION REGULATION (EU) 2017/735 of 14 February 2017

  L 112

1

28.4.2017

►M8

COMMISSION REGULATION (EU) 2019/1390 of 31 July 2019

  L 247

1

26.9.2019

►M9

COMMISSION REGULATION (EU) 2023/464 of 3 March 2023

  L 68

37

6.3.2023


Corrected by:

►C1

Corrigendum, OJ L 143, 3.6.2008, p.  55 (440/2008)




▼B

▼C1

COMMISSION REGULATION (EC) No 440/2008

of 30 May 2008

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

(Text with EEA relevance)

▼B



Article 1

The test methods to be applied for the purposes of Regulation 1907/2006/EC are set out in the Annex to this Regulation.

Article 2

The Commission shall review, where appropriate, the test methods contained in this Regulation with a view to replacing, reducing or refining testing on vertebrate animals.

Article 3

All references to Annex V to Directive 67/548/EEC shall be construed as references to this Regulation.

Article 4

This Regulation shall enter into force on the day following its publication in the Official Journal of the European Union.

It shall apply from 1 June 2008.




ANNEX

▼M6

Note:

Before using any of the following test methods to test a multi-constituent substance (MCS), a substance of unknown or variable composition, complex reaction product or biological material (UVCB), or a mixture and where its applicability for the testing of MCS, UVCB, or mixtures is not indicated in the respective test method, it should be considered whether the method is adequate for the intended regulatory purpose.

If the test method is used for the testing of a MCS, UVCB or mixture, sufficient information on its composition should be made available, as far as possible, e.g. by the chemical identity of its constituents, their quantitative occurrence, and relevant properties of the constituents.

▼M9




PART 0

INTERNATIONAL TEST METHODS RECOGNISED AS BEING APPROPRIATE FOR GENERATING INFORMATION ON INTRINSIC PROPERTIES OF SUBSTANCES FOR THE PURPOSES OF REGULATION (EC) No 1907/2006

TABLE 1: TEST METHODS FOR PHYSICOCHEMICAL PROPERTIES OF THE SUBSTANCE



Endpoint

Test method

Corresponding chapter, containing the full description of the test method, in Part A of this Annex (numbers in brackets indicate that the full description of the test method has been deleted from Part A; empty cell: no corresponding test method in Part A of this Annex)

Melting point/freezing point

OECD Test Guideline 102: Melting Point/Melting Range (1995)

A.1.

Boiling point

OECD Test Guideline 103: Boiling point (1995)

A.2.

Density

OECD Test Guideline 109: Density of Liquids and Solids (2012)

(A.3.)

Vapour pressure

OECD Test Guideline 104: Vapour Pressure (2006)

(A.4)

Surface tension

OECD Test Guideline 115: Surface Tension of Aqueous Solutions (1995)

A.5.

Water solubility

OECD Test Guideline 105: Water Solubility (1995)

A.6.

Partition coefficient n-octanol/water

OECD Test Guideline 107: Partition Coefficient (n-octanol/water): Shake-Flask Method (1995)

(A.8.)

OECD Test Guideline 123: Partition Coefficient (1-Octanol/Water): Slow-Stirring Method (2022)

A.23.

OECD Test Guideline 117: Partition Coefficient (n-octanol/water): HPLC Method (2022)

A.24.

Dissociation constant

OECD Test Guideline 112: Dissociation Constants in Water. (1981)

A.25.

Viscosity

OECD Test Guideline 114: Viscosity of Liquids (2012)

 

Flash point

Test methods according to table 2.6.3 of Annex I, Part 2 of Regulation (EC) No 1272/2008

 

Lower and upper explosion limit

EN 1839:2017 – Determination of the explosion limits and the limiting oxygen concentration (LOC) for flammable gases and vapours

 

Flammability

Test methods according to section 2.2.4.1. of Annex I, Part 2 of Regulation (EC) No 1272/2008

 

Test L.2: sustained combustibility test, Part III, section 32 of the UN RTDG Manual of Tests and Criteria

 

Test N.1: test method for readily combustible solids, Part III, sub-section 33.2.4 of the UN RTDG Manual of Tests and Criteria

 

Test N.5: test method for substances which in contact with water emit flammable gases, Part III, sub-section 33.5.4 of the UN RTDG Manual of Tests and Criteria

 

Self-ignition temperature (solids)

Test N.4: test method for self-heating substances, Part III, sub-section 33.4.6 of the UN RTDG Manual of Tests and Criteria

 

EN 15188:2020 – Determination of the spontanous ignition behaviour of dust accumulations

 

Auto-ignition temperature (liquids, gases)

ISO/IEC 80079-20-1:2017 – Explosive atmospheres – Part 20-1: Material characteristics for gas and vapour classification – Test methods and data

 

Decomposition temperature

Test Series H, part II, section 28, of the UN RTDG Manual of Tests and Criteria

 

Explosive properties

Test methods according to Test series 1-3, Part I, sections 11-13 of the UN RTDG Manual of Tests and Criteria

 

EU Test method A.14 Explosive Properties

A.14

Oxidising properties

Test method according to section 2.4.4. of Annex I, Part 2 of Regulation (EC) No 1272/2008

 

Test O.2: test for oxidizing liquids, Part III, sub-section 34.4.2 of the UN RTDG Manual of Tests and Criteria

 

Test O.1: Test for oxidizing solids, Part III, sub-section 34.4.1 of the UN RTDG Manual of Tests and Criteria

 

Test O.3 Gravimetric test for oxidizing solids, Part III, sub-section 34.4 3 of the UN RTDG Manual of Tests and Criteria

 

Pyrophoricity

Test N.3: test method for pyrophoric liquids, Part III, sub-section 33.3.1.5 of the UN RTDG Manual of Tests and Criteria

 

Test N.2: test method for pyrophoric solids, Part III, sub-section 33.3.1.4 of the UN RTDG Manual of Tests and Criteria

 

Granulometry/particle characteristics

EU test method A.22. Length Weighted Geometric Mean Diameter of Fibres

A.22.

ISO 13318 – Determination of Particle Size Distribution by Centrifugal Liquid Sedimentation Methods

 

ISO 21501 – Determination of Particle Size Distribution – Single Particle Light Interaction Methods

 

OECD Test Guideline 124: Determination of the Volume Specific Surface Area of Manufactured Nanomaterials (2022)

 

OECD Test Guideline 125: Particle Size and Particle Size Distribution of Nanomaterials (2022)

 

pH

OECD Test Guideline 122: Determination of pH, Acidity and Alkalinity (2013)

 

Properties of polymers

OECD Test Guideline 118: Determination of the Number-Average Molecular Weight and the Molecular Weight Distribution of Polymers using Gel Permeation Chromatography (1996)

A.18.

OECD Test Guideline 119: Determination of the Low Molecular Weight Content of a Polymer Using Gel Permeation Chromatography (1996)

A.19.

OECD Test Guideline 120: Solution/Extraction Behaviour of Polymers in Water (2000)

(A.20.)

TABLE 2: TEST METHODS FOR TOXICOLOGICAL PROPERTIES



Endpoint

Test method

Corresponding chapter, containing the full description of the test method, in Part B of this Annex (numbers in brackets indicate that a chapter, containing the full description of the test method, has been deleted from Part B; empty cell: no corresponding EU test method in Part B of this Annex)

Skin corrosion/irritation

In vitro:

OECD Test Guideline 430: In vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER) (2015)

B.40.

OECD Test Guideline 431: In vitro Skin Corrosion: Reconstructed Human Epidermis (RhE) Test Method (2019)

(B.40bis.)

OECD Test Guideline 435: In vitro Membrane Barrier Test Method for Skin Corrosion (2015)

B.65.

OECD Test Guideline 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2021)

(B.46.)

In vivo:

OECD Test Guideline 404: Acute Dermal Irritation/Corrosion (2015)

B.4.

Serious eye damage/eye irritation

In vitro:

OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020)

(B.47.)

OECD Test Guideline 438: Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2018)

(B.48.)

OECD Test Guideline 460: Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants (2017)

(B.61.)

OECD Test Guideline 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020)

(B.68.)

OECD Test Guideline 492: Reconstructed Human Cornea-Like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage (2019)

(B.69.)

OECD Test Guideline 492B: Reconstructed Human Cornea-like Epithelium (RHCE) Test Method for Eye Hazard Identification (2022)

 

OECD Test Guideline 494: Vitrigel-Eye Irritancy Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage (2021)

 

OECD Test Guideline 496: In vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2019)

 

OECD Test Guideline 467: Defined Approaches for Serious Eye Damage and Eye Irritation (2022)

 

In vivo:

OECD Test Guideline 405: Acute Eye Irritation/Corrosion (2021)

(B.5.)

Skin sensitisation

In vitro:

OECD Test Guideline 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) (2022)

(B.59.)

OECD Test Guideline 442D: In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation (2022)

(B.60.)

OECD Test Guideline 442E: In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays Addressing the Key Event on Activation of Dendritic Cells on the Adverse Outcome Pathway for Skin Sensitisation (2022)

(B.71.)

OECD Test Guideline 497: Defined Approaches on Skin Sensitisation (2021)

 

In vivo:

OECD Test Guideline 429: Skin Sensitisation – Local Lymph Node Assay (2010)

B.42.

OECD Test Guideline 442A: Skin Sensitisation – Local Lymph Node Assay: DA (2010)

B.50.

OECD Test Guideline 442B: Skin Sensitisation – Local Lymph Node Assay: BrdU-ELISA or –FCM (2018)

(B.51.)

OECD Test Guideline 406: Skin Sensitisation Guinea Pig

Maximisation Test and Buehler Test (2022)

(B.6.)

Mutagenicity

In vitro:

OECD Test Guideline 471: Bacterial Reverse Mutation Test (2020)

(B.13./14.)

OECD Test Guideline 476: In Vitro Mammalian Cell Gene Mutation Test Using the Hprt and xprt Genes (2016)

(B.17.)

OECD Test Guideline 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (2016)

B.67.

OECD Test Guideline 473: In vitro Mammalian Chromosome Aberration Test (2016)

B.10.

OECD Test Guideline 487. In vitro Mammalian Cell Micronucleus Test (2016)

B.49.

In vivo:

OECD Test Guideline 475: Mammalian Bone Marrow Chromosome Aberration Test (2016)

B.11.

OECD Test Guideline 474: Mammalian Erythrocyte Micronucleus Test (2016)

B.12.

OECD Test Guideline 483: Mammalian Spermatogonial Chromosome Aberration Test (2016)

B.23.

OECD Test Guideline 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays (2022)

(B.58.)

OECD Test Guideline 489: In Vivo Mammalian Alkaline Comet Assay (2016)

B.62.

OECD Test Guideline 470: Mammalian Erythrocyte Pig-a Gene mutation Assay (2022)

 

Acute toxicity

Oral:

OECD Test Guideline 420: Acute Oral Toxicity: Fixed Dose Procedure (2002)

B.1 bis.

OECD Test Guideline 423: Acute Oral Toxicity: Acute Toxic Class Method (2002)

B.1 tris.

OECD Test Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure (2022)

 

Dermal:

OECD Test Guideline 402: Acute Dermal Toxicity – Fixed Dose Procedure (2017)

(B.3.)

Inhalation:

OECD Test Guideline 403: Acute Inhalation Toxicity (2009)

B.2.

OECD Test Guideline 436: Acute Inhalation Toxicity – Acute Toxic Class Method (2009)

B.52.

OECD Test Guideline 433: Acute Inhalation Toxicity: Fixed Concentration Procedure (2018)

 

Repeated dose toxicity

OECD Test Guideline 407: Repeated Dose 28-Day Oral Toxicity Study in Rodents (2008)

B.7.

OECD Test Guideline 412: Subacute Inhalation Toxicity: 28-Day Study (2018)

(B.8.)

OECD Test Guideline 410: Repeated Dose Dermal Toxicity: 21/28-Day Study (1981)

B.9.

OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2016)

B.64.

OECD Test Guideline 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents (2018)

(B.26.)

OECD Test Guideline 409: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents (1998)

B.27.

OECD Test Guideline 413: Subchronic Inhalation Toxicity: 90-Day Study (2018)

(B.29.)

OECD Test Guideline 411: Subchronic Dermal Toxicity: 90-Day Study (1981)

B.28.

OECD Test Guideline 452: Chronic Toxicity Studies (2018)

(B.30.)

OECD Test Guideline 453: Combined Chronic Toxicity/Carcinogenicity Studies (2018)

(B.33.)

Reproductive/developmental toxicity

OECD Test Guideline 443: Extended One-Generation Reproduction Toxicity Study (2018)

(B.56.)

OECD Test Guideline 421: Reproduction/Developmental Toxicity Screening Test (2016)

B.63.

OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2016)

B.64.

OECD Test Guideline 414: Prenatal Developmental Toxicity Study (2018)

(B.31.)

Toxicokinetics

OECD Test Guideline 417: Toxicokinetics (2010)

B.36.

OECD Test Guideline 428: Skin Absorption: In Vitro Method (2004)

B.45.

OECD Test Guideline 427: Skin Absorption: In Vivo Method (2004)

B.44.

Carcinogenicity

OECD Test Guideline 451: Carcinogenicity Studies (2018)

(B.32.)

OECD Test Guideline 453: Combined Chronic Toxicity/Carcinogenicity Studies (2018)

(B.33.)

EU test method B.21. In Vitro Mammalian Cell Transformation Test

B.21.

(Developmental) Neurotoxicity

OECD Test Guideline 424: Neurotoxicity Study in Rodents (1997)

B.43.

OECD Test Guideline 426: Developmental Neurotoxicity Study (2007)

B.53.

OECD Test Guideline 418: Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (1995)

B.37.

OECD Test Guideline 419: Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (1995)

B.38.

Endocrine disrupting properties

In vitro

OECD Test Guideline 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonistsals (2021)

(B.66.)

OECD Test Guideline 456: H295R Steroidogenesis Assay (2022)

B.57.

OECD Test Guideline 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals (2020)

 

OECD Test Guideline 493: Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity (2015)

B.70.

In vivo

OECD Test Guideline 440: Uterotrophic Bioassay in Rodents A short-term screening test for oestrogenic properties (2007)

B.54.

OECD Test Guideline 441: Hershberger Bioassay in Rats, A Short-term Screening Assay for (Anti)Androgenic Properties (2009)

B.55.

Phototoxicity

OECD Test Guideline 432: In Vitro 3T3 NRU Phototoxicity Test (2019)

(B.41.)

OECD Test Guideline 495: Ros (Reactive Oxygen Species) Assay for Photoreactivity (2019)

 

OECD Test Guideline 498: In Vitro Phototoxicity Test Method Using the Reconstructed Human Epidermis (RhE) (2021)

 

TABLE 3: TEST METHODS FOR ECOTOXICOLOGICAL PROPERTIES



Endpoint

Test method

Corresponding chapter in Part C, containing the full description of the test method, of this Annex (numbers in brackets indicate that a chapter, containing the full description of the test method, has been deleted from Part C; empty cell: no corresponding EU test method in Part C of this Annex)

Aquatic toxicity

OECD Test Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test (2011)

C.3.

OECD Test Guideline 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) (2010)

C.11.

OECD Test Guideline 224: Determination of the Inhibition of the Activity of Anaerobic Bacteria (2007)

C.34.

OECD Test Guideline 244: Protozoan Activated Sludge Inhibition Test (2017)

 

OECD Test Guideline 221: Lemna sp. Growth Inhibition Test (2006)

C.26.

OECD Test Guideline 202: Daphnia sp. Acute Immobilisation Test (2004)

C.2.

OECD Test Guideline 211: Daphnia magna Reproduction Test (2012)

C.20.

OECD Test Guideline 203: Fish, Acute Toxicity Test (2019)

(C.1.)

OECD Test Guideline 210: Fish, Early-life Stage Toxicity Test (2013)

C.47.

OECD Test Guideline 215: Fish, Juvenile Growth Test (2000)

C.14.

OECD Test Guideline 236: Fish Embryo Acute Toxicity (FET) Test (2013)

C.49.

OECD Test Guideline 249: Fish Cell Line Acute Toxicity – the RTgill-W1 Cell Line Assay (2021)

 

OECD Test Guideline 242: Potamopyrgus antipodarum Reproduction Test (2016)

 

OECD Test Guideline 243: Lymnaea stagnalis Reproduction Test (2016)

 

Degradation

OECD Test Guideline 111: Hydrolysis as a Function of pH (2004)

C.7.

OECD Test Guideline 301: Ready Biodegradability (1992)

C.4.

OECD Test Guideline 302A: Inherent Biodegradability: Modified SCAS Test (1981)

C.12.

OECD Test Guideline 302B: Inherent Biodegradability: Zahn-Wellens/EMPA Test (1992)

(C.9).

OECD Test Guideline 302C: Inherent Biodegradability: Modified MITI Test (II) (2009)

 

OECD Test Guideline 303: Simulation Test – Aerobic Sewage Treatment — A: Activated Sludge Units; B: Biofilms (2001)

C.10.

OECD Test Guideline 304A: Inherent Biodegradability in Soil (1981)

 

OECD Test Guideline 306: Biodegradability in Seawater (1992)

C.42.

OECD Test Guideline 307: Aerobic and Anaerobic Transformation in Soil (2002)

C.23.

OECD Test Guideline 308: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems (2002)

C.24.

OECD Test Guideline 309: Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test (2004)

C.25.

OECD Test Guideline 310: Ready Biodegradability – CO2 in sealed vessels (Headspace Test) (2014)

C.29.

OECD Test Guideline 311: Anaerobic Biodegradability of Organic Compounds in Digested Sludge: by Measurement of Gas Production (2006)

C.43.

OECD Test Guideline 314: Simulation Tests to Assess the Biodegradability of Chemicals Discharged in Wastewater (2008)

 

OECD Test Guideline 316: Phototransformation of Chemicals in Water – Direct Photolysis (2008)

 

EU test method C.5. Degradation – Biochemical Oxygen Demand

C.5.

EU test method C.6. Degradation – Chemical Oxygen Demand

C.6.

Fate and behaviour in the environment

OECD Test Guideline 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure (2012)

C.13.

OECD Test Guideline 315: Bioaccumulation in Sediment-Dwelling Benthic Oligochaetes (2008)

C.46.

OECD Test Guideline 317: Bioaccumulation in Terrestrial Oligochaetes (2010)

C.30.

OECD Test Guideline 318: Dispersion Stability of Nanomaterials in Simulated Environmental Media (2017)

 

OECD Test Guideline 121: Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC) (2001)

C.19.

OECD Test Guideline 106: Adsorption – Desorption Using a Batch Equilibrium Method (2000)

C.18.

OECD Test Guideline 312: Leaching in Soil Columns (2004)

C.44.

OECD Test Guideline 313: Estimation of Emissions from Preservative – Treated Wood to the Environment (2007)

C.45.

OECD Test Guideline 319A: Determination of In Vitro Intrinsic Clearance Using Cryopreserved Rainbow Trout Hepatocytes (RT-HEP) (2018)

 

OECD Test Guideline 319B: Determination of In Vitro Intrinsic Clearance Using Rainbow Trout Liver S9 Sub-Cellular Fraction (RT-S9) (2018)

 

OECD Test Guideline 320: Anaerobic Transformation of Chemicals in Liquid Manure (2022)

 

Effects on terrestrial organisms

OECD Test Guideline 216: Soil Microorganisms: Nitrogen Transformation Test (2000)

C.21.

OECD Test Guideline 217: Soil Microorganisms: Carbon Transformation Test (2000)

C.22.

OECD Test Guideline 207: Earthworm, Acute Toxicity Tests (1984)

C.8.

OECD Test Guideline 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) (2016)

(C.33.)

OECD Test Guideline 220: Enchytraeid Reproduction Test (2016)

(C.32.)

OECD Test Guideline 226: Predatory Mite (Hypoaspis (Geolaelaps) aculeifer) Reproduction Test in Soil (2016)

(C.36.)

OECD Test Guideline 232: Collembolan Reproduction Test in Soil (2016)

(C.39.)

OECD Test Guideline 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test (2006)

C.31.

OECD Test Guideline 227: Terrestrial Plant Test: Vegetative Vigour Test (2006)

 

Effects on sediment organisms

OECD Test Guideline 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment (2004)

C.27.

OECD Test Guideline 219: Sediment-Water Chironomid Toxicity Using Spiked Water (2004)

C.28.

OECD Test Guideline 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment (2010)

C.40.

OECD Test Guideline 235: Chironomus sp., Acute Immobilisation Test (2011)

 

OECD Test Guideline 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (2007)

C.35.

OECD Test Guideline 238: Sediment-Free Myriophyllum Spicatum Toxicity Test (2014)

C.50.

OECD Test Guideline 239: Water-Sediment Myriophyllum Spicatum Toxicity Test (2014)

C.51.

Effects on birds

OECD Test Guideline 205: Avian Dietary Toxicity Test (1984)

 

OECD Test Guideline 206: Avian Reproduction Test (1984)

 

OECD Test Guideline 223: Avian Acute Oral Toxicity Test (2016)

 

Effects on insects

OECD Test Guideline 213: Honeybees, Acute Oral Toxicity Test (1998)

C.16.

OECD Test Guideline 214: Honeybees, Acute Contact Toxicity Test (1998)

C.17.

OECD Test Guideline 237: Honey Bee (Apis Mellifera) Larval Toxicity Test, Single Exposure (2013)

 

OECD Test Guideline 245: Honey Bee (Apis Mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding) (2017)

 

OECD Test Guideline 246: Bumblebee, Acute Contact Toxicity Test (2017)

 

OECD Test Guideline 247: Bumblebee, Acute Oral Toxicity Test (2017)

 

OECD Test Guideline 228: Determination of Developmental Toxicity to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) (2016)

 

Endocrine disrupting properties

OECD Test Guideline 230: 21-Day Fish Assay (2009)

C.37.

OECD Test Guideline 229: Fish Short Term Reproduction Assay (2012)

C.48.

OECD Test Guideline 231: Amphibian Metamorphosis Assay (2009)

C.38.

OECD Test Guideline 234: Fish Sexual Development Test (2011)

C.41.

OECD Test Guideline 240: Medaka Extended OneGeneration Reproduction Test (MEOGRT) (2015)

C.52.

OECD Test Guideline 241: The Larval Amphibian Growth and Development Assay (LAGDA) (2015)

C.53.

OECD Test Guideline 248: Xenopus Eleutheroembryonic Thyroid Assay (XETA) (2019)

 

OECD Test Guideline 250: EASZY assay – Detection of Endocrine Active Substances, Acting Through Estrogen Receptors, Using Transgenic tg(cyp19a1b:GFP) Zebrafish embrYos (2021)

 

OECD Test Guideline 251: Rapid Androgen Disruption Activity Reporter (RADAR) Assay (2022)

 

▼B




PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES

TABLE OF CONTENTS

A.1.

MELTING/FREEZING TEMPERATURE

A.2.

BOILING TEMPERATURE

A.3.

RELATIVE DENSITY

A.4.

VAPOUR PRESSURE

A.5.

SURFACE TENSION

A.6.

WATER SOLUBILITY

A.8.

PARTITION COEFFICIENT

A.9.

FLASH-POINT

A.10.

FLAMMABILITY (SOLIDS)

A.11.

FLAMMABILITY (GASES)

A.12.

FLAMMABILITY (CONTACT WITH WATER)

A.13.

PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS

A.14.

EXPLOSIVE PROPERTIES

A.15.

AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)

A.16.

RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS

A.17.

OXIDISING PROPERTIES (SOLIDS)

A.18.

NUMBER — AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS

A.19.

LOW MOLECULAR WEIGHT CONTENT OF POLYMERS

A.20.

SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER

A.21.

OXIDISING PROPERTIES (LIQUIDS)

A.22.

LENGTH WEIGHTED GEOMETRIC MEAN DIAMETER OF FIBRES

A.23.

PARTITION COEFFICIENT (1-OCTANOL/WATER): SLOW-STIRRING METHOD

A.24.

PARTITION COEFFICIENT (N-OCTANOL/WATER), HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC) METHOD

A.25.

DISSOCIATION CONSTANTS IN WATER (TITRATION METHOD — SPECTROPHOTOMETRIC METHOD — CONDUCTOMETRIC METHOD)

A.1.   MELTING/FREEZING TEMPERATURE

1.   METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1.   INTRODUCTION

The methods and devices described are to be applied for the determination of the melting temperature of substances, without any restriction with respect to their degree of purity.

The selection of the method is dependent on the nature of the substance to be tested. In consequence the limiting factor will be according to, whether or not the substance can be pulverised easily, with difficulty, or not at all.

For some substances, the determination of the freezing or solidification temperature is more appropriate and the standards for these determinations have also been included in this method.

Where, due to the particular properties of the substance, none of the above parameters can be conveniently measured, a pour point may be appropriate.

1.2.   DEFINITIONS AND UNITS

The melting temperature is defined as the temperature at which the phase transition from solid to liquid state occurs at atmospheric pressure and this temperature ideally corresponds to the freezing temperature.

As the phase transition of many substances takes place over a temperature range, it is often described as the melting range.

Conversion of units (K to oC)

t = T - 273,15

t

:

Celsius temperature, degree Celsius (oC)

T

:

thermodynamic temperature, kelvin (K)

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

Some calibration substances are listed in the references (4).

1.4.   PRINCIPLE OF THE TEST METHOD

The temperature (temperature range) of the phase transition from the solid to the liquid state or from the liquid to the solid state is determined. In practice while heating/cooling a sample of the test substance at atmospheric pressure the temperatures of the initial melting/freezing and the final stage of melting/freezing are determined. Five types of methods are described, namely capillary method, hot stage methods, freezing temperature determinations, methods of thermal analysis, and determination of the pour point (as developed for petroleum oils).

In certain cases, it may be convenient to measure the freezing temperature in place of the melting temperature.

1.4.1.   Capillary method

1.4.1.1.   Melting temperature devices with liquid bath

A small amount of the finely ground substance is placed in a capillary tube and packed tightly. The tube is heated, together with a thermometer, and the temperature rise is adjusted to less than about 1 K/min during the actual melting. The initial and final melting temperatures are determined.

1.4.1.2.   Melting temperature devices with metal block

As described under 1.4.1.1, except that the capillary tube and the thermometer are situated in a heated metal block, and can be observed through holes in the block.

1.4.1.3.   Photocell detection

The sample in the capillary tube is heated automatically in a metal cylinder. A beam of light is directed through the substance, by way of a hole in the cylinder, to a precisely calibrated photocell. The optical properties of most substances change from opaque to transparent when they are melting. The intensity of light reaching the photocell increases and sends a stop signal to the digital indicator reading out the temperature of a platinum resistance thermometer located in the heating chamber. This method is not suitable for some highly coloured substances.

1.4.2.   Hot stages

1.4.2.1.   Kofler hot bar

The Kofler hot bar uses two pieces of metal of different thermal conductivity, heated electrically, with the bar designed so that the temperature gradient is almost linear along its length. The temperature of the hot bar can range from 283 to 573 K with a special temperature-reading device including a runner with a pointer and tab designed for the specific bar. In order to determine a melting temperature, the substance is laid, in a thin layer, directly on the surface of the hot bar. In a few seconds a sharp dividing line between the fluid and solid phase develops. The temperature at the dividing line is read by adjusting the pointer to rest at the line.

1.4.2.2.   Melt microscope

Several microscope hot stages are in use for the determination of melting temperatures with very small quantities of material. In most of the hot stages the temperature is measured with a sensitive thermocouple but sometimes mercury thermometers are used. A typical microscope hot stage melting temperature apparatus has a heating chamber which contains a metal plate upon which the sample is placed on a slide. The centre of the metal plate contains a hole permitting the entrance of light from the illuminating mirror of the microscope. When in use, the chamber is closed by a glass plate to exclude air from the sample area.

The heating of the sample is regulated by a rheostat. For very precise measurements on optically anisotropic substances, polarised light may be used.

1.4.2.3.   Meniscus method

This method is specifically used for polyamides.

The temperature at which the displacement of a meniscus of silicone oil, enclosed between a hot stage and a cover-glass supported by the polyamide test specimen, is determined visually.

1.4.3.   Method to determine the freezing temperature

The sample is placed in a special test tube and placed in an apparatus for the determination of the freezing temperature. The sample is stirred gently and continuously during cooling and the temperature is measured at suitable intervals. As soon as the temperature remains constant for a few readings this temperature (corrected for thermometer error) is recorded as the freezing temperature.

Supercooling must be avoided by maintaining equilibrium between the solid and the liquid phases.

1.4.4.   Thermal analysis

1.4.4.1   Differential thermal analysis (DTA)

This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the temperature record.

1.4.4.2   Differential scanning calorimetry (DSC)

This technique records the difference in energy inputs into a substance and a reference material, as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the heat flow record.

1.4.5.   Pour point

This method was developed for use with petroleum oils and is suitable for use with oily substances with low melting temperatures.

After preliminary heating, the sample is cooled at a specific rate and examined at intervals of 3 K for flow characteristics. The lowest temperature at which movement of the substance is observed is recorded as the pour point.

1.5.   QUALITY CRITERIA

The applicability and accuracy of the different methods used for the determination of the melting temperature/melting range are listed in the following table:

TABLE: APPLICABILITY OF THE METHODS



A.  Capillary methods

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Melting temperature devices with liquid bath

yes

only to a few

273 to 573 K

± 0,3 K

JIS K 0064

Melting temperature with metal block

yes

only to a few

293 to > 573 K

± 0,5 K

ISO 1218 (E)

Photocell detection

yes

several with appliance devices

253 to 573 K

± 0,5 K

 

(1)   

Dependent on type of instrument and on degree of purity of the substance.



B.  Hot stages and freezing methods

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Kofler hot bar

yes

no

283 to > 573 K

± 1K

ANSI/ASTM D 3451-76

Melt microscope

yes

only to a few

273 to > 573 K

± 0,5 K

DIN 53736

Meniscus method

no

specifically for polyamides

293 to > 573 K

± 0,5 K

ISO 1218 (E)

Freezing temperature

yes

yes

223 to 573 K

± 0,5 K

e.g. BS 4695

(1)   

Dependent on type of instrument and on degree of purity of the substance



C.  Thermal analysis

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Differential thermal analysis

yes

yes

173 to 1 273 K

up to 600 K ± 0,5 K up to 1 273 K ± 2,0 K

ASTM E 537-76

Differential scanning calorimetry

yes

yes

173 to 1 273 K

up to 600 K ± 0,5 K up to 1 273 K ± 2,0 K

ASTM E 537-76

(1)   

Dependent on type of instrument and on degree of purity of the substance



D.  Pour point

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Pour point

for petroleum oils and oily substances

for petroleum oils and oily substances

223 to 323 K

± 0,3 K

ASTM D 97-66

(1)   

Dependent on type of instrument and on degree of purity of the substance

1.6.   DESCRIPTION OF THE METHODS

The procedures of nearly all the test methods have been described in international and national standards (see Appendix 1).

1.6.1.   Methods with capillary tube

When subjected to a slow temperature rise, finely pulverised substances usually show the stages of melting shown in figure 1.

Figure 1

image

During the determination of the melting temperature, the temperatures are recorded at the beginning of the melting and at the final stage.

1.6.1.1.   Melting temperature devices with liquid bath apparatus

Figure 2 shows a type of standardised melting temperature apparatus made of glass (JIS K 0064); all specifications are in millimeters.

Figure 2

image

Bath liquid:

A suitable liquid should be chosen. The choice of the liquid depends upon the melting temperature to be determined, e.g. liquid paraffin for melting temperatures no higher than 473 K, silicone oil for melting temperatures no higher than 573 K.

For melting temperatures above 523 K, a mixture consisting of three parts sulphuric acid and two parts potassium sulphate (in mass ratio) can be used. Suitable precautions should be taken if a mixture such as this is used.

Thermometer:

Only those thermometers should be used which fulfil the requirements of the following or equivalent standards:

ASTM E 1-71, DIN 12770, JIS K 8001.

Procedure:

The dry substance is finely pulverised in a mortar and is put into the capillary tube, fused at one end, so that the filling level is approximately 3 mm after being tightly packed. To obtain a uniform packed sample, the capillary tube should be dropped from a height of approximately 700 mm through a glass tube vertically onto a watch glass.

The filled capillary tube is placed in the bath so that the middle part of the mercury bulb of the thermometer touches the capillary tube at the part where the sample is located. Usually the capillary tube is introduced into the apparatus about 10 K below the melting temperature.

The bath liquid is heated so that the temperature rise is approximately 3 K/min. The liquid should be stirred. At about 10 K below the expected melting temperature the rate of temperature rise is adjusted to a maximum of 1 K/min.

Calculation:

The calculation of the melting temperature is as follows:

T = TD + 0,00016 (TD - TE) n

where:

T

=

corrected melting temperature in K

TD

=

temperature reading of thermometer D in K

TE

=

temperature reading of thermometer E in K

n

=

number of graduations of mercury thread on thermometer D at emergent stem.

1.6.1.2.   Melting temperature devices with metal block

Apparatus:

This consists of:

— 
a cylindrical metal block, the upper part of which is hollow and forms a chamber (see figure 3),
— 
a metal plug, with two or more holes, allowing tubes to be mounted into the metal block,
— 
a heating system, for the metal block, provided for example by an electrical resistance enclosed in the block,
— 
a rheostat for regulation of power input, if electric heating is used,
— 
four windows of heat-resistant glass on the lateral walls of the chamber, diametrically disposed at right-angles to each other. In front of one of these windows is mounted an eye-piece for observing the capillary tube. The other three windows are used for illuminating the inside of the enclosure by means of lamps,
— 
a capillary tube of heat-resistant glass closed at one end (see 1.6.1.1).

Thermometer:

See standards mentioned in 1.6.1.1. Thermoelectrical measuring devices with comparable accuracy are also applicable.

Figure 3

image

1.6.1.3.   Photocell detection

Apparatus and procedure:

The apparatus consists of a metal chamber with automated heating system. Three capillary are filled accordingly to 1.6.1.1 and placed in the oven.

Several linear increases of temperature are available for calibrating the apparatus and the suitable temperature rise is electrically adjusted at a pre-selected constant and linear rate. recorders show the actual oven temperature and the temperature of the substance in the capillary tubes.

1.6.2.   Hot stages

1.6.2.1.   Kofler hot bar

See Appendix.

1.6.2.2.   Melt microscope

See Appendix.

1.6.2.3.   Meniscus method (polyamides)

See Appendix.

The heating rate through the melting temperature should be less than 1 K/min.

1.6.3.   Methods for the determination of the freezing temperature

See Appendix.

1.6.4.   Thermal analysis

1.6.4.1.   Differential thermal analysis

See Appendix.

1.6.4.2.   Differential scanning calorimetry

See Appendix.

1.6.5.   Determination of the pour point

See Appendix.

2.   DATA

A thermometer correction is necessary in some cases.

3.   REPORTING

The test report shall, if possible, include the following information:

— 
method used,
— 
precise specification of the substance (identity and impurities) and preliminary purification step, if any,
— 
an estimate of the accuracy.

The mean of at least two measurements which are in the range of the estimated accuracy (see tables) is reported as the melting temperature.

If the difference between the temperature at the beginning and at the final stage of melting is within the limits of the accuracy of the method, the temperature at the final stage of melting is taken as the melting temperature; otherwise the two temperatures are reported.

If the substance decomposes or sublimes before the melting temperature is reached, the temperature at which the effect is observed shall be reported.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 102, Decision of the Council C(81) 30 final.

(2) IUPAC, B. Le Neindre, B. Vodar, eds. Experimental thermodynamics, Butterworths, London 1975, vol. II, p. 803-834.

(3) R. Weissberger ed.: Technique of organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter VII.

(4) IUPAC, Physicochemical measurements: Catalogue of reference materials from national laboratories, Pure and applied chemistry, 1976, vol. 48, p. 505-515.

Appendix

For additional technical details, the following standards may be consulted for example.

1.   Capillary methods

1.1.   Melting temperature devices with liquid bath



ASTM E 324-69

Standard test method for relative initial and final melting points and the melting range of organic chemicals

BS 4634

Method for the determination of melting point and/or melting range

DIN 53181

Bestimmung des Schmelzintervalles von Harzen nach Kapillarverfarehn

JIS K 00-64

Testing methods for melting point of chemical products

1.2.   Melting temperature devices with metal block



DIN 53736

Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen

ISO 1218 (E)

Plastics — polyamides — determination of ‘melting point’

2.   Hot stages

2.1.   Kofler hot bar



ANSI/ASTM D 3451-76

Standard recommended practices for testing polymeric powder coatings

2.2.   Melt microscope



DIN 53736

Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen

2.3.   Meniscus method (polyamides)



ISO 1218 (E)

Plastics — polyamides — determination of ‘melting point’

ANSI/ASTM D 2133-66

Standard specification for acetal resin injection moulding and extrusion materials

NF T 51-050

Résines de polyamides. Détermination du ‘point de fusion’ méthode du ménisque

3.   Methods for the determination of the freezing temperature



BS 4633

Method for the determination of crystallising point

BS 4695

Method for Determination of Melting Point of petroleum wax (Cooling Curve)

DIN 51421

Bestimmung des Gefrierpunktes von Flugkraftstoffen, Ottokraftstoffen und Motorenbenzolen

ISO 2207

Cires de pétrole: détermination de la température de figeage

DIN 53175

Bestimmung des Erstarrungspunktes von Fettsäuren

NF T 60-114

Point de fusion des paraffines

NF T 20-051

Méthode de détermination du point de cristallisation (point de congélation)

ISO 1392

Method for the determination of the freezing point

4.   Thermal analysis

4.1.   Differential thermal analysis



ASTM E 537-76

Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis

ASTM E 473-85

Standard definitions of terms relating to thermal analysis

ASTM E 472-86

Standard practice for reporting thermoanalytical data

DIN 51005

Thermische Analyse, Begriffe

4.2.   Differential scanning calorimetry



ASTM E 537-76

Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis

ASTM E 473-85

Standard definitions of terms relating to thermal analysis

ASTM E 472-86

Standard practice for reporting thermoanalytical data

DIN 51005

Thermische Analyse, Begriffe

5.   Determination of the pour point



NBN 52014

Echantillonnage et analyse des produits du pétrole: Point de trouble et point d'écoulement limite — Monsterneming en ontleding van aardolieproducten: Troebelingspunt en vloeipunt

ASTM D 97-66

Standard test method for pour point of petroleum oils

ISO 3016

Petroleum oils — Determination of pour point

A.2.   BOILING TEMPERATURE

1.   METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1.   INTRODUCTION

The methods and devices described here can be applied to liquid and low melting substances, provided that these do not undergo chemical reaction below the boiling temperature (for example: auto-oxidation, rearrangement, degradation, etc.). The methods can be applied to pure and to impure liquid substances.

Emphasis is put on the methods using photocell detection and thermal analysis, because these methods allow the determination of melting as well as boiling temperatures. Moreover, measurements can be performed automatically.

The ‘dynamic method’ has the advantage that it can also be applied to the determination of the vapour pressure and it is not necessary to correct the boiling temperature to the normal pressure (101,325 kPa) because the normal pressure can be adjusted during the measurement by a manostat.

Remarks:

The influence of impurities on the determination of the boiling temperature depends greatly upon the nature of the impurity. When there are volatile impurities in the sample, which could affect the results, the substance may be purified.

1.2.   DEFINITIONS AND UNITS

The normal boiling temperature is defined as the temperature at which the vapour pressure of a liquid is 101,325 kPa.

If the boiling temperature is not measured at normal atmospheric pressure, the temperature dependence of the vapour pressure can be described by the Clausius-Clapeyron equation:

image

where:

p

=

the vapour pressure of the substance in pascals

Δ Hv

=

its heat of vaporisation in J mol-1

R

=

the universal molar gas constant = 8,314  J mol-1 K-1

T

=

thermodynamic temperature in K

The boiling temperature is stated with regard to the ambient pressure during the measurement.

Conversions

Pressure (units: kPa)

100 kPa

=

1 bar = 0,1 MPa

(‘bar’ is still permissible but not recommended)

133 Pa

=

1 mm Hg = 1 Torr

(the units ‘mm Hg’ and ‘Torr’ are not permitted)

1 atm

=

standard atmosphere = 101 325 Pa

(the unit ‘atm’ is not permitted)

Temperature (units: K)

t = T - 273,15

t

:

Celsius temperature, degree Celsius (oC)

T

:

thermodynamic temperature, kelvin (K)

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

Some calibration substances can be found in the methods listed in the Appendix.

1.4.   PRINCIPLE OF THE TEST METHOD

Five methods for the determination of the boiling temperature (boiling range) are based on the measurement of the boiling temperature, two others are based on thermal analysis.

1.4.1.   Determination by use of the ebulliometer

Ebulliometers were originally developed for the determination of the molecular weight by boiling temperature elevation, but they are also suited for exact boiling temperature measurements. A very simple apparatus is described in ASTM D 1120-72 (see Appendix). The liquid is heated in this apparatus under equilibrium conditions at atmospheric pressure until it is boiling.

1.4.2.   Dynamic method

This method involves the measurement of the vapour recondensation temperature by means of an appropriate thermometer in the reflux while boiling. The pressure can be varied in this method.

1.4.3.   Distillation method for boiling temperature

This method involves distillation of the liquid and measurement of the vapour recondensation temperature and determination of the amount of distillate.

1.4.4.   Method according to Siwoloboff

A sample is heated in a sample tube, which is immersed in a liquid in a heat-bath. A fused capillary, containing an air bubble in the lower part, is dipped in the sample tube.

1.4.5.   Photocell detection

Following the principle according to Siwoloboff, automatic photo-electrical measurement is made using rising bubbles.

1.4.6.   Differential thermal analysis

This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the temperature record.

1.4.7.   Differential scanning calorimetry

This technique records the difference in energy inputs into a substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the heat flow record.

1.5.   QUALITY CRITERIA

The applicability and accuracy of the different methods used for the determination of the boiling temperature/boiling range are listed in table 1.



Table 1:

Comparison of the methods

Method of measurement

Estimated accuracy

Existing standard

Ebulliometer

± 1,4 K (up to 373 K) (1) (2)

± 2,5 K (up to 600 K) (1) (2)

ASTM D 1120-72 (1)

Dynamic method

± 0,5 K (up to 600 K) (2)

 

Distillation process (boiling range)

± 0,5 K (up to 600 K)

ISO/R 918, DIN 53171, BS 4591/71

According to Siwoloboff

± 2 K (up to 600 K) (2)

 

Photocell detection

± 0,3 K (up to 373 K) (2)

 

Differential thermal calorimetry

± 0,5 K (up to 600 K)

± 2,0 K (up to 1 273 K)

ASTM E 537-76

Differential scanning calorimetry

± 0,5 K (up to 600 K)

± 2,0 K (up to 1 273 K)

ASTM E 537-76

(1)   

This accuracy is only valid for the simple device as for example described in ASTM D 1120-72; it can be improved with more sophisticated ebulliometer devices.

(2)   

Only valid for pure substances. The use in other circumstances should be justified.

1.6.   DESCRIPTION OF THE METHODS

The procedures of some test methods have been described in international and national standards (see Appendix).

1.6.1.   Ebulliometer

See Appendix.

1.6.2.   Dynamic method

See test method A.4 for the determination of the vapour pressure.

The boiling temperature observed with an applied pressure of 101,325 kPa is recorded.

1.6.3.   Distillation process (boiling range)

See Appendix.

1.6.4.   Method according to Siwoloboff

The sample is heated in a melting temperature apparatus in a sample tube, with a diameter of approximately 5 mm (figure 1).

Figure 1 shows a type of standardised melting and boiling temperature apparatus (JIS K 0064) (made of glass, all specifications in millimetres).

Figure 1

image

A capillary tube (boiling capillary) which is fused about 1 cm above the lower end is placed in the sample tube. The level to which the test substance is added is such that the fused section of the capillary is below the surface of the liquid. The sample tube containing the boiling capillary is fastened either to the thermometer with a rubber band or is fixed with a support from the side (see figure 2).



Figure 2

Principle according to Siwoloboff

Figure 3

Modified principle

image

image

The bath liquid is chosen according to boiling temperature. At temperatures up to 573 K, silicone oil can be used. Liquid paraffin may only be used up to 473 K. The heating of the bath liquid should be adjusted to a temperature rise of 3 K/min at first. The bath liquid must be stirred. At about 10 K below the expected boiling temperature, the heating is reduced so that the rate of temperature rise is less than 1 K/min. Upon approach of the boiling temperature, bubbles begin to emerge rapidly from the boiling capillary.

The boiling temperature is that temperature when, on momentary cooling, the string of bubbles stops and fluid suddenly starts rising in the capillary. The corresponding thermometer reading is the boiling temperature of the substance.

In the modified principle (figure 3) the boiling temperature is determined in a melting temperature capillary. It is stretched to a fine point about 2 cm in length (a) and a small amount of the sample is sucked up. The open end of the fine capillary is closed by melting, so that a small air bubble is located at the end. While heating in the melting temperature apparatus (b), the air bubble expands. The boiling temperature corresponds to the temperature at which the substance plug reaches the level of the surface of the bath liquid (c).

1.6.5.   Photocell detection

The sample is heated in a capillary tube inside a heated metal block.

A light beam is directed, via suitable holes in the block, through the substance onto a precisely calibrated photocell.

During the increase of the sample temperature, single air bubbles emerge from the boiling capillary. When the boiling temperature is reached the number of bubbles increases greatly. This causes a change in the intensity of light, recorded by a photocell, and gives a stop signal to the indicator reading out the temperature of a platinum resistance thermometer located in the block.

This method is especially useful because it allows determinations below room temperature down to 253,15 K (– 20 oC) without any changes in the apparatus. The instrument merely has to be placed in a cooling bath.

1.6.6.   Thermal analysis

1.6.6.1.   Differential thermal analysis

See Appendix.

1.6.6.2.   Differential scanning calorimetry

See Appendix.

2.   DATA

At small deviations from the normal pressure (max. ± 5 kPa) the boiling temperatures are normalised to Tn by means of the following number-value equation by Sidney Young:

Tn = T + (fT × Δp)

where:

Δp

=

(101,325 - p) [note sign]

P

=

pressure measurement in kPa

fT

=

rate of change of boiling temperature with pressure in K/kPa

T

=

measured boiling temperature in K

Tn

=

boiling temperature corrected to normal pressure in K

The temperature-correction factors, fT, and equations for their approximation are included in the international and national standards mentioned above for many substances.

For example, the DIN 53171 method mentions the following rough corrections for solvents included in paints:



Table 2:

Temperature — corrections factors fT

Temperature T (K)

Correction factor fT (K/kPa)

323,15

0,26

348,15

0,28

373,15

0,31

398,15

0,33

423,15

0,35

448,15

0,37

473,15

0,39

498,15

0,41

523,15

0,4

548,15

0,45

573,15

0,47

3.   REPORTING

The test report shall, if possible, include the following information:

— 
method used,
— 
precise specification of the substance (identity and impurities) and preliminary purification step, if any,
— 
an estimate of the accuracy.

The mean of at least two measurements which are in the range of the estimated accuracy (see table 1) is reported as the boiling temperature.

The measured boiling temperatures and their mean shall be stated and the pressure(s) at which the measurements were made shall be reported in kPa. The pressure should preferably be close to normal atmospheric pressure.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 103, Decision of the Council C (81) 30 final.

(2) IUPAC, B. Le Neindre, B. Vodar, editions. Experimental thermodynamics, Butterworths, London, 1975, vol. II.

(3) R. Weissberger edition: Technique of organic chemistry, Physical methods of organic chemistry, Third Edition, Interscience Publications, New York, 1959, vol. I, Part I, Chapter VIII.

Appendix

For additional technical details, the following standards may be consulted for example.

1.   Ebulliometer

1.1. Melting temperature devices with liquid bath



ASTM D 1120-72

Standard test method for boiling point of engine anti-freezes

2.   Distillation process (boiling range)



ISO/R 918

Test Method for Distillation (Distillation Yield and Distillation Range)

BS 4349/68

Method for determination of distillation of petroleum products

BS 4591/71

Method for the determination of distillation characteristics

DIN 53171

Losungsmittel für Anstrichstoffe, Bestimmung des Siedeverlaufes

NF T 20-608

Distillation: détermination du rendement et de l'intervalle de distillation

3.   Differential thermal analysis and differential scanning calorimetry



ASTM E 537-76

Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis