COUNCIL 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)
Article 1
Article 2
Article 3
Article 4
ANNEX
PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES
A.1. MELTING/FREEZING TEMPERATURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Capillary method
1.4.1.1. Melting temperature devices with liquid bath
1.4.1.2. Melting temperature devices with metal block
1.4.1.3. Photocell detection
1.4.2. Hot stages
1.4.2.1. Kofler hot bar
1.4.2.2. Melt microscope
1.4.2.3. Meniscus method
1.4.3. Method to determine the freezing temperature
1.4.4. Thermal analysis
1.4.4.1 Differential thermal analysis (DTA)
1.4.4.2 Differential scanning calorimetry (DSC)
1.4.5. Pour point
1.5. QUALITY CRITERIA
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 |
|
B.
Hot stages and freezing methods
Method of measurement |
Substances which can be pulverised |
Substances which are not readily pulverised |
Temperature range |
Estimated accuracy(2) |
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 |
C.
Thermal analysis
Method of measurement |
Substances which can be pulverised |
Substances which are not readily pulverised |
Temperature range |
Estimated accuracy(3) |
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 |
D.
Pour point
Method of measurement |
Substances which can be pulverised |
Substances which are not readily pulverised |
Temperature range |
Estimated accuracy(4) |
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 |
1.6. DESCRIPTION OF THE METHODS
1.6.1. Methods with capillary tube
Figure 1
1.6.1.1. Melting temperature devices with liquid bath apparatus
Figure 2
Bath liquid:
Thermometer:
Procedure:
Calculation:
1.6.1.2. Melting temperature devices with metal block
Apparatus:
Thermometer:
Figure 3
1.6.1.3. Photocell detection
1.6.2. Hot stages
1.6.2.1. Kofler hot bar
1.6.2.2. Melt microscope
1.6.2.3. Meniscus method (polyamides)
1.6.3. Methods for the determination of the freezing temperature
1.6.4. Thermal analysis
1.6.4.1. Differential thermal analysis
1.6.4.2. Differential scanning calorimetry
1.6.5. Determination of the pour point
2. DATA
3. REPORTING
4. REFERENCES
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
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Determination by use of the ebulliometer
1.4.2. Dynamic method
1.4.3. Distillation method for boiling temperature
1.4.4. Method according to Siwoloboff
1.4.5. Photocell detection
1.4.6. Differential thermal analysis
1.4.7. Differential scanning calorimetry
1.5. QUALITY CRITERIA
Method of measurement |
Estimated accuracy |
Existing standard |
Ebulliometer |
±1,4 K (up to 373 K)(5) (6) ±2,5 K (up to 600 K)(5) (6) |
ASTM D 1120-72(5) |
Dynamic method |
±0,5 K (up to 600 K)(6) |
|
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)(6) |
|
Photocell detection |
±0,3 K (up to 373 K)(6) |
|
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 |
1.6. DESCRIPTION OF THE METHODS
1.6.1. Ebulliometer
1.6.2. Dynamic method
1.6.3. Distillation process (boiling range)
1.6.4. Method according to Siwoloboff
Figure 1
Figure 2 Principle according to Siwoloboff |
Figure 3 Modified principle |
[Bild bitte in Originalquelle ansehen] |
[Bild bitte in Originalquelle ansehen] |
1.6.5. Photocell detection
1.6.6. Thermal analysis
1.6.6.1. Differential thermal analysis
1.6.6.2. Differential scanning calorimetry
2. DATA
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
4. REFERENCES
Appendix
For additional technical details, the following standards may be consulted for example.
1. Ebulliometer
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 |
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 |
A.3. RELATIVE DENSITY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES (1) (3)
1.4. PRINCIPLE OF THE METHODS
1.4.1. Buoyancy methods
1.4.1.1. Hydrometer
(for liquid substances)
1.4.1.2. Hydrostatic balance
(for liquid and solid substances)
1.4.1.3. Immersed body method
(for liquid substances) (4)
1.4.2. Pycnometer methods
1.4.3. Air comparison pycnometer
(for solids)
1.4.4. Oscillating densitimeter
(5) (6) (7)
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHODS
2. DATA
3. REPORTING
Method of measurement |
Density |
Maximum possible dynamic viscosity |
Existing Standards |
|||
solid |
liquid |
|||||
|
|
yes |
5 Pa s |
ISO 387, ISO 649-2, NF T 20-050 |
||
|
|
|
|
|
||
|
yes |
|
|
ISO 1183 (A) |
||
|
|
yes |
5 Pa s |
ISO 901 and 758 |
||
|
|
yes |
20 Pa s |
DIN 53217 |
||
|
|
|
|
ISO 3507 |
||
|
yes |
|
|
ISO 1183(B), NF T 20-053 |
||
|
|
yes |
500 Pa s |
ISO 758 |
||
|
yes |
|
|
DIN 55990 Teil 3, DIN 53243 |
||
|
|
yes |
5 Pa s |
|
4. REFERENCES
Appendix
For additional technical details, the following standards may be consulted for example.
1. Buoyancy methods
1.1. Hydrometer
DIN 12790, ISO 387 |
Hydrometer; general instructions |
DIN 12791 |
Part I: Density hydrometers; construction, adjustment and use Part II: Density hydrometers; standardised sizes, designation Part III: Use and test |
ISO 649-2 |
Laboratory glassware: Density hydrometers for general purpose |
NF T 20-050 |
Chemical products for industrial use — Determination of density of liquids — Areometric method |
DIN 12793 |
Laboratory glassware: range find hydrometers |
1.2. Hydrostatic balance
For solid substances
ISO 1183 |
Method A: Methods for determining the density and relative density of plastics excluding cellular plastics |
NF T 20-049 |
Chemical products for industrial use — Determination of the density of solids other than powders and cellular products — Hydrostatic balance method |
ASTM-D-792 |
Specific gravity and density of plastics by displacement |
DIN 53479 |
Testing of plastics and elastomers; determination of density |
For liquid substances
ISO 901 |
ISO 758 |
DIN 51757 |
Testing of mineral oils and related materials; determination of density |
ASTM D 941-55, ASTM D 1296-67 and ASTM D 1481-62 |
|
ASTM D 1298 |
Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method |
BS 4714 |
Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method |
1.3. Immersed body method
DIN 53217 |
Testing of paints, varnishes and similar coating materials; determination of density; immersed body method |
2. Pycnometer methods
2.1. For liquid substances
ISO 3507 |
Pycnometers |
ISO 758 |
Liquid chemical products; determination of density at 20 oC |
DIN 12797 |
Gay-Lussac pycnometer (for non-volatile liquids which are not too viscous) |
DIN 12798 |
Lipkin pycnometer (for liquids with a kinematic viscosity of less than 100 10-6 m2 s-1 at 15 oC) |
DIN 12800 |
Sprengel pycnometer (for liquids as DIN 12798) |
DIN 12801 |
Reischauer pycnometer (for liquids with a kinematic viscosity of less than 100. 10-6 m2 s-1 at 20 oC, applicable in particular also to hydrocarbons and aqueous solutions as well as to liquids with higher vapour pressure, approximately 1 bar at 90 oC) |
DIN 12806 |
Hubbard pycnometer (for viscous liquids of all types which do not have too high a vapour pressure, in particular also for paints, varnishes and bitumen) |
DIN 12807 |
Bingham pycnometer (for liquids, as in DIN 12801) |
DIN 12808 |
Jaulmes pycnometer (in particular for ethanol — water mixture) |
DIN 12809 |
Pycnometer with ground-in thermometer and capillary side tube (for liquids which are not too viscous) |
DIN 53217 |
Testing of paints, varnishes and similar products; determination of density by pycnometer |
DIN 51757 |
Point 7: Testing of mineral oils and related materials; determination of density |
ASTM D 297 |
Section 15: Rubber products — chemical analysis |
ASTM D 2111 |
Method C: Halogenated organic compounds |
BS 4699 |
Method for determination of specific gravity and density of petroleum products (graduated bicapillary pycnometer method) |
BS 5903 |
Method for determination of relative density and density of petroleum products by the capillary — stoppered pycnometer method |
NF T 20-053 |
Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method |
2.2. For solid substances
ISO 1183 |
Method B: Methods for determining the density and relative density of plastics excluding cellular plastics |
NF T 20-053 |
Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method |
DIN 19683 |
Determination of the density of soils |
3. Air comparison pycnometer
DIN 55990 |
Part 3: Prüfung von Anstrichstoffen und ähnlichen Beschichtungsstoffen; Pulverlack; Bestimmung der Dichte |
DIN 53243 |
Anstrichstoffe; chlorhaltige Polymere; Prüfung |
A.4. VAPOUR PRESSURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1 Torr (≡ 1 mm Hg) |
= 1,333 × 102 Pa |
1 atmosphere |
= 1,013 × 105 Pa |
1 bar |
= 105 Pa |
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHODS
1.4.1. Dynamic method
1.4.2. Static method
1.4.3. Isoteniscope
1.4.4. Effusion method: Vapour pressure balance
1.4.5. Effusion method: By loss of weight or by trapping vaporisate
1.4.6. Gas saturation method
1.4.7. Spinning rotor
1.5. QUALITY CRITERIA
Measuring Method |
Substances |
Estimated Repeatability(7) |
Estimated Reproducibility(7) |
Recommended Range |
Existing Standard |
|||
solid |
liquid |
|||||||
|
Low melting |
yes |
Up to 25 % |
Up to 25 % |
103 Pa to 2 × 103 Pa |
— |
||
|
|
|
1 to 5 % |
1 to 5 % |
2 × 103 Pa to 105 Pa |
— |
||
|
yes |
yes |
5 to 10 % |
5 to 10 % |
10 Pa to 105 Pa(8) |
NFT 20-048 (5) |
||
|
yes |
yes |
5 to 10 % |
5 to 10 % |
102 Pa to 105 Pa |
ASTM-D 2879-86 |
||
|
yes |
yes |
5 to 20 % |
5 to 50 % |
10-3 Pa to 1 Pa |
NFT 20-047(6) |
||
|
yes |
yes |
10 to 30 % |
— |
10-3 Pa to 1 Pa |
— |
||
|
yes |
yes |
10 to 30 % |
Up to 50 % |
10-4 Pa to 1 Pa(8) |
— |
||
|
yes |
yes |
10 to 20 % |
— |
10-4 Pa to 0,5 Pa |
— |
1.6. DESCRIPTION OF THE METHODS
1.6.1. Dynamic measurement
1.6.1.1. Apparatus
1.6.1.2. Measurement procedure
1.6.2. Static measurement
1.6.2.1. Apparatus
1.6.2.2. Measurement procedure
1.6.3. Isoteniscope
1.6.4. Effusion method: vapour pressure balance
1.6.4.1. Apparatus
1.6.4.2. Measurement procedure
1.6.5. Effusion method — by loss of weight
1.6.5.1. Apparatus
1.6.5.2. Measurement procedure
ratio |
0,1 |
0,2 |
0,6 |
1,0 |
2,0 |
K |
0,952 |
0,909 |
0,771 |
0,672 |
0,514 |
1.6.6. Gas saturation method
1.6.6.1. Apparatus
Inert gas:
Flow control:
Traps to collect vapour:
Heat exchanger:
Saturator column:
1.6.6.2. Measurement procedure
Preparation of the saturator column:
Measurement:
1.6.6.3. Calculation of vapour pressure
1.6.7. Spinning rotor (8, 11, 13)
1.6.7.1. Apparatus
1.6.7.2. Measurement procedure
2. DATA
3. REPORTING
4. REFERENCES
Appendix 1
Estimation method
INTRODUCTION
ESTIMATION METHOD
CALCULATION PROCEDURE
REPORT
LITERATURE
Appendix 2
Figure 1
Apparatus for determining the vapour pressure curve according to the dynamic method
Figure 2a
Apparatus for determining the vapour pressure curve according to the static method (using a U-tube manometer)
Figure 2b
Apparatus for determining the vapour pressure curve according to the static method (using a pressure indicator)
Figure 3
Isoteniscope (see reference 7)
Figure 4
Apparatus for determining the vapour pressure curve according to the vapour pressure balance method
Figure 5
Example of apparatus for evaporation at low pressure by effusion methode, with an effusion cell volume of 8 cm
3
Figure 6a
An example of a flow system for the determination of vapour pressure by the gas saturation method
Figure 6b
An example of system for the determination of vapour pressure by the gas saturation method, with a capillary placed after the saturation chamber
Figure 7
Example of the experimental set-up for spinning rotor method
Figure 8
Example of spinning rotor measuring head
A.5. SURFACE TENSION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHODS
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHODS
1.6.1. Plate method
1.6.2. Stirrup method
1.6.3. Ring method
1.6.4. OECD harmonised ring method
1.6.4.1. Apparatus
1.6.4.1.1.
Mobile sample table
1.6.4.1.2.
Force measuring system
1.6.4.1.3.
Measuring body (ring)
Figure
Measuring body
1.6.4.1.4.
Measurement vessel
1.6.4.2. Preparation of the apparatus
1.6.4.2.1.
Cleaning
1.6.4.2.2.
Calibration of the apparatus
Mounting:
Zero point adjustment:
Calibrations:
[Bild bitte in Originalquelle ansehen] |
|
[Bild bitte in Originalquelle ansehen] |
|
1.6.4.3. Preparation of samples
1.6.5. Test conditions
1.6.6. Performance of test
2. DATA
r |
= 9,55 mm (average ring radius) |
r |
= 0,185 mm (ring wire radius) |
Experimental Value (mN/m) |
Corrected Value (mN/m) |
|
Weight calibration (see 1.6.4.2.2(a)) |
Water calibration (see 1.6.4.2.2(b)) |
|
20 |
16,9 |
18,1 |
22 |
18,7 |
20,1 |
24 |
20,6 |
22,1 |
26 |
22,4 |
24,1 |
28 |
24,3 |
26,1 |
30 |
26,2 |
28,1 |
32 |
28,1 |
30,1 |
34 |
29,9 |
32,1 |
36 |
31,8 |
34,1 |
38 |
33,7 |
36,1 |
40 |
35,6 |
38,2 |
42 |
37,6 |
40,3 |
44 |
39,5 |
42,3 |
46 |
41,4 |
44,4 |
48 |
43,4 |
46,5 |
50 |
45,3 |
48,6 |
52 |
47,3 |
50,7 |
54 |
49,3 |
52,8 |
56 |
51,2 |
54,9 |
58 |
53,2 |
57,0 |
60 |
55,2 |
59,1 |
62 |
57,2 |
61,3 |
64 |
59,2 |
63,4 |
66 |
61,2 |
65,5 |
68 |
63,2 |
67,7 |
70 |
65,2 |
69,9 |
72 |
67,2 |
72,0 |
74 |
69,2 |
— |
76 |
71,2 |
— |
78 |
73,2 |
— |
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
A.6. WATER SOLUBILITY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Column elution method
1.4.2. Flask method
1.5. QUALITY CRITERIA
1.5.1. Repeatability
1.5.2. Sensitivity
1.6. DESCRIPTION OF THE METHOD
1.6.1. Test conditions
1.6.2. Preliminary test
0,1 g soluble in ‘x’ ml of water |
0,1 |
0,5 |
1 |
2 |
10 |
100 |
> 100 |
Approximative solubility (grams per litre) |
> 1 000 |
1 000 to 200 |
200 to 100 |
100 to 50 |
50 to 10 |
10 to 1 |
< 1 |
1.6.3. Column elution method
1.6.3.1. Support material, solvent and eluent
1.6.3.2. Loading of the support
Test procedure:
1.6.3.3. Column elution method with recirculating pump
Apparatus
Measurement procedure
1.6.3.4. Column elution method with levelling vessel
Apparatus
(see figures 4 and 3)
Measurement procedure
1.6.4. Flask method
1.6.4.1. Apparatus
1.6.4.2. Measurement procedure
1.6.5. Analysis
2. DATA
2.1. COLUMN ELUTION METHOD
2.2. FLASK METHOD
3. REPORTING
3.1. COLUMN ELUTION METHOD
3.2. FLASK METHOD
4. REFERENCES
Appendix
Figure 1
Column elution method with recirculating pump
Figure 2
A typical microcolumn
Figure 3
A typical microcolumn
Figure 4
Column elution method with levelling vessel
A.8. PARTITION COEFFICIENT
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
Shake-flask method
HPLC method
1.4. PRINCIPLE OF THE METHOD
1.4.1. Shake-flask method
1.4.2. HPLC method
1.5. QUALITY CRITERIA
1.5.1. Repeatability
Shake-flask method
HPLC method
1.5.2. Sensitivity
Shake-flask method
HPLC method
1.5.3. Specificity
Shake-flask method
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preliminary estimate of the partition coefficient
1.6.2. Shake-flask method
1.6.2.1. Preparation
1.6.2.1.1.
Pre-saturation of the solvents
1.6.2.1.2.
Preparation for the test
1.6.2.1.3.
Test substance
1.6.2.2. Test conditions
1.6.2.3. Measurement procedure
1.6.2.3.1.
Establishment of the partition equilibrium
1.6.2.3.2.
Phase separation
1.6.2.4. Analysis
1.6.3. HPLC method
1.6.3.1. Preparation
Apparatus
Mobile phase
Solutes
Test conditions
1.6.3.2.
Measurement
Calculation of dead time t
o
Calibration graph
Determination of the capacity factor of the test substance
2. DATA
Shake-flask method
3. REPORTING
For shake-flask method:
For HPLC method:
4. REFERENCES
Appendix 1
Calculation/estimation methods
INTRODUCTION
ESTIMATION METHOD
Preliminary estimate of the partition coefficient
CALCULATION METHODS
Principle of the calculation methods
Quality criteria
Calculation procedures
Hansch π-method
Rekker method
Hansch-Leo method
Combined method
Remarks
Report
LITERATURE
Appendix 2
Recommended Reference Substances for the HLPC Method
No |
Reference Substance |
log Pow |
pKa |
1 |
2-Butanone |
0,3 |
|
2 |
4-Acetylpyridine |
0,5 |
|
3 |
Aniline |
0,9 |
|
4 |
Acetanilide |
1,0 |
|
5 |
Benzylalcohol |
1,1 |
|
6 |
p-Methoxyphenol |
1,3 |
pKa = 10,26 |
7 |
Phenoxy acetic acid |
1,4 |
pKa = 3,12 |
8 |
Phenol |
1,5 |
pKa = 9,92 |
9 |
2,4-Dinitrophenol |
1,5 |
pKa = 3,96 |
10 |
Benzonitrile |
1,6 |
|
11 |
Phenylacetonitrile |
1,6 |
|
12 |
4-Methylbenzyl alcohol |
1,6 |
|
13 |
Acetophenone |
1,7 |
|
14 |
2-Nitrophenol |
1,8 |
pKa = 7,17 |
15 |
3-Nitrobenzoic acid |
1,8 |
pKa = 3,47 |
16 |
4-Chloraniline |
1,8 |
pKa = 4,15 |
17 |
Nitrobenzene |
1,9 |
|
18 |
Cinnamic alcohol |
1,9 |
|
19 |
Benzoic acid |
1,9 |
pKa = 4,19 |
20 |
p-Cresol |
1,9 |
pKa = 10,17 |
21 |
Cinnamic acid |
2,1 |
pKa = 3,89 cis 4,44 trans |
22 |
Anisole |
2,1 |
|
23 |
Methylbenzoate |
2,1 |
|
24 |
Benzene |
2,1 |
|
25 |
3-Methylbenzoic acid |
2,4 |
pKa = 4,27 |
26 |
4-Chlorophenol |
2,4 |
pKa = 9,1 |
27 |
Trichloroethylene |
2,4 |
|
28 |
Atrazine |
2,6 |
|
29 |
Ethylbenzoate |
2,6 |
|
30 |
2,6-Dichlorobenzonitrile |
2,6 |
|
31 |
3-Chlorobenzoic acid |
2,7 |
pKa = 3,82 |
32 |
Toluene |
2,7 |
|
33 |
1-Naphthol |
2,7 |
pKa = 9,34 |
34 |
2,3-Dichloroaniline |
2,8 |
|
35 |
Chlorobenzene |
2,8 |
|
36 |
Allyl-phenylether |
2,9 |
|
37 |
Bromobenzene |
3,0 |
|
38 |
Ethylbenzene |
3,2 |
|
39 |
Benzophenone |
3,2 |
|
40 |
4-Phenylphenol |
3,2 |
pKa = 9,54 |
41 |
Thymol |
3,3 |
|
42 |
1,4-Dichlorobenzene |
3,4 |
|
43 |
Diphenylamine |
3,4 |
pKa = 0,79 |
44 |
Naphthalene |
3,6 |
|
45 |
Phenylbenzoate |
3,6 |
|
46 |
Isopropylbenzene |
3,7 |
|
47 |
2,4,6-Trichlorophenol |
3,7 |
pKa = 6 |
48 |
Biphenyl |
4,0 |
|
49 |
Benzylbenzoate |
4,0 |
|
50 |
2,4-Dinitro-6 sec. butyophenol |
4,1 |
|
51 |
1,2,4-Trichlorobenzene |
4,2 |
|
52 |
Dodecanoic acid |
4,2 |
|
53 |
Diphenylether |
4,2 |
|
54 |
n-Butylbenzene |
4,5 |
|
55 |
Phenanthrene |
4,5 |
|
56 |
Fluoranthene |
4,7 |
|
57 |
Dibenzyl |
4,8 |
|
58 |
2,6-Diphenylpyridine |
4,9 |
|
59 |
Triphenylamine |
5,7 |
|
60 |
DDT |
6,2 |
|
Other reference substances of low log Pow |
|||
1 |
Nicotinic acid |
-0,07 |
|
A.9. FLASH-POINT
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.5.1. Repeatability
1.5.2. Sensitivity
1.5.3. Specificity
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.3. Performance of the test
1.6.3.1. Equilibrium method
1.6.3.2. Non-equilibrium method
Abel apparatus:
Abel-Pensky apparatus:
Tag apparatus:
Pensky-Martens apparatus:
Remarks:
3. REPORTING
4. REFERENCES
A.10. FLAMMABILITY (SOLIDS)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF METHOD
1.6.1. Preliminary screening test
1.6.2. Burning rate test
1.6.2.1. Preparation
1.6.2.2. Test conditions
1.6.2.3. Performance of the test
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULT
4. REFERENCES
Appendix
Figure
Mould and accessories for the preparation of the pile
A.11. FLAMMABILITY (GASES)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Apparatus
1.6.2. Test conditions
1.6.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
A.12. FLAMMABILITY (CONTACT WITH WATER)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. PRINCIPLE OF THE METHOD
1.3.1. Step 1
1.3.2. Step 2
1.3.3. Step 3
1.3.4. Step 4
1.4. REFERENCE SUBSTANCES
1.5. QUALITY CR1TERIA
1.6. DESCRIPTION OF METHODS
1.6.1. Step 1
1.6.1.1. Test conditions
1.6.1.2. Performance of the test
1.6.2. Step 2
1.6.2.1. Apparatus
1.6.2.2. Test conditions
1.6.2.3. Performance of the test
1.6.3. Step 3
1.6.3.1. Test conditions
1.6.3.2. Performance of the test
1.6.4. Step 4
1.6.4.1. Apparatus
1.6.4.2. Test conditions
1.6.4.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
Appendix
Figure
Apparatus
A.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Performance of the test
(a) Powdery solids
(b) liquids
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION
3. REPORTING
4. REFERENCES
A.14. EXPLOSIVE PROPERTIES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.4.1. Safety-in-handling tests (3)
1.4.2. Thermal sensitivity
1.4.3. Mechanical sensitivity (shock)
1.4.4. Mechanical sensitivity (friction)
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF METHOD
1.6.1. Thermal sensitivity (effect of a flame)
1.6.1.1. Apparatus
1.6.1.2. Test conditions
1.6.1.3. Performance of the tests
1.6.1.4. Evaluation
1.6.2. Mechanical sensitivity (shock)
1.6.2.1. Apparatus (figure 4)
1.6.2.2. Test conditions
1.6.2.3. Performance of the tests
1.6.2.4. Evaluation
1.6.3. Mechanical sensitivity (friction)
1.6.3.1. Apparatus (figure 5)
1.6.3.2. Test conditions
1.6.3.3. Performance of the tests
1.6.3.4. Evaluation
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION AND EVALUATION OF RESULTS
4. REFERENCES
Appendix
Example of material specification for thermal sensitivity test (see DIN 1623)
Figure 1
Thermal sensitivity test apparatus
Figure 2
Thermal sensitivity test
Figure 3
Heating rate calibration for thermal sensitivity test
Figure 4
Shock test apparatus
Figure 4
Continued
Figure 5
Friction sensitivity apparatus
A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Apparatus
1.6.2. Test conditions
1.6.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Apparatus
1.6.1.1. Oven
1.6.1.2. Wire mesh cube
1.6.1.3. Thermocouples
1.6.1.4. Recorder
1.6.2. Test conditions
1.6.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
Figure 1
Pattern of 20 mm test cube
Figure 2
Typical temperature/time curve
A.17. OXIDISING PROPERTIES (SOLIDS)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preparation
1.6.1.1. Test substance
1.6.1.2. Combustible substance
1.6.1.3. Ignition source
1.6.2. Performance of the test
1.6.2.1. Preliminary test
1.6.2.2. Train test
2. DATA
3. REPORT
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULT
4. REFERENCES
Appendix
Figure
Mould and accessories for the preparations of the pile
A.18. NUMBER-AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
[Bild bitte in Originalquelle ansehen] |
[Bild bitte in Originalquelle ansehen] |
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
Mp < 2 000 |
Mw/Mn < 1,20 |
2 000 ≤ Mp ≤ 106 |
Mw/Mn < 1,05 |
Mp > 106 |
Mw/Mn < 1,20 |
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparation of the standard polystyrene solutions
1.6.2. Preparation of the sample solution
1.6.3. Apparatus
1.6.4. Injection and solvent delivery system
1.6.5. Column
1.6.6. Theoretical plates
[Bild bitte in Originalquelle ansehen] |
or |
[Bild bitte in Originalquelle ansehen] |
1.6.7. Separation efficiency
1.6.8. Solvents
1.6.9. Temperature control
1.6.10. Detector
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Instrumentation:
2.2.3. Calibration of the system:
2.2.4. Evaluation:
3. REFERENCES
Appendix
Examples of other methods for determination of number average molecular weight (Mn) for polymers
1. Use of colligative properties
2. End-group analysis
3. References
A.19. LOW MOLECULAR WEIGHT CONTENT OF POLYMERS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
[Bild bitte in Originalquelle ansehen] |
[Bild bitte in Originalquelle ansehen] |
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
Mp < 2 000 |
Mw/Mn < 1,20 |
2 000< Mp < 106 |
Mw/Mn < 1,05 |
Mp > 106 |
Mw/Mn < 1,20 |
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparation of the standard polystyrene solutions
1.6.2. Preparation of the sample solution
1.6.3. Correction for content of impurities and additives
1.6.4. Apparatus
1.6.5. Injection and solvent delivery system
1.6.6. Column
1.6.7. Theoretical plates
[Bild bitte in Originalquelle ansehen] |
or |
[Bild bitte in Originalquelle ansehen] |
1.6.8. Separation efficiency
1.6.9. Solvents
1.6.10. Temperature control
1.6.11. Detector
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Instrumentation:
2.2.3. Calibration of the system:
2.2.4. Information on the low molecular weight polymer content:
2.2.5. Evaluation:
3. REFERENCES
Appendix
Guidance for correcting low molecular content for the presence of insoluble polymer
A.20. SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER
1. METHOD
1.1. INTRODUCTION
1.2. REFERENCE SUBSTANCES
1.3. PRINCIPLE OF THE TEST METHOD
1.4. QUALITY CRITERIA
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Equipment
1.5.2. Sample preparation
1.5.3. Procedure
1.5.4. Analysis
1.5.4.1. Test conducted with one sample size
1.5.4.2. Test conducted with two different sample sizes
2. DATA
2.1. TEST CONDUCTED WITH ONE SAMPLE SIZE
2.2. TEST CONDUCTED WITH TWO DIFFERENT SAMPLE SIZES
3. REPORTING
3.1. TEST REPORT
3.1.1. Test substance:
3.1.2. Experimental conditions:
3.1.3. Results:
4. REFERENCES
A.21. OXIDISING PROPERTIES (LIQUIDS)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preparation
1.6.1.1. Combustible substance
1.6.1.2. Apparatus
1.6.1.2.1.
Pressure vessel
1.6.1.2.2.
Ignition system
1.6.2. Performance of the test
(15)
2. DATA
2.1. TREATMENT OF RESULTS
Substance(19) |
Mean pressure rise time for a 1:1 mixture with celulose (ms) |
Ammonium dichromate, saturated aqueous solution |
20 800 |
Calcium nitrate, saturated aqueous solution |
6 700 |
Ferric nitrate, saturated aqueous solution |
4 133 |
Lithium perchlorate, saturated aqueous solution |
1 686 |
Magnesium perchlorate, saturated aqueous solution |
777 |
Nickel nitrate, saturated aqueous solution |
6 250 |
Nitric acid, 65 % |
4 767(20) |
Perchloric acid, 50 % |
121(20) |
Perchloric acid, 55 % |
59 |
Potassium nitrate, 30 % aqueous solution |
26 690 |
Silver nitrate, saturated aqueous solution |
(21) |
Sodium chlorate, 40 % aqueous solution |
2 555(20) |
Sodium nitrate, 45 % aqueous solution |
4 133 |
Inert substance |
|
Water: cellulose |
(21) |
3. REPORT
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULTS(22)
4. REFERENCES
Figure 1
Pressure vessel
Figure 2
Support stand
Figure 3
Ignition system
PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS
GENERAL INTRODUCTION
A. CHARACTERISATION OF THE TEST SUBSTANCE
B. ANIMAL CARE
(i) Housing conditions
(ii) Feeding conditions
C. ALTERNATIVE TESTING
D. EVALUATION AND INTERPRETATION
E. LITERATURE REFERENCES
B.1 bis. ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
1.4.2. Housing and feeding conditions
1.4.3. Preparation of animals
1.4.4. Preparation of doses
1.5. PROCEDURE
1.5.1. Administration of doses
1.5.2. Sighting study
1.5.3. Main study
1.5.3.1. Numbers of animals and dose levels
1.5.3.2. Limit test
1.6. OBSERVATIONS
1.6.1. Body weight
1.6.2. Pathology
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
FLOW CHART FOR THE SIGHTING STUDY
Appendix 2
FLOW CHART FOR THE MAIN STUDY
Appendix 3
CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD
50
VALUES EXCEEDING 2 000 MG/KG WITHOUT THE NEED FOR TESTING.
TESTING AT DOSES ABOVE 2 000 MG/KG
Sighting study
Main study
Appendix 4
TEST METHOD B.1 bis
Guidance on classification according to the EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))
B.1 tris. ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST
1.4. DESCRIPTION OF THE METHOD
1.4.1. Selection of animal species
1.4.2. Housing and feeding conditions
1.4.3. Preparation of animals
1.4.4. Preparation of doses
1.5. PROCEDURE
1.5.1. Administration of doses
1.5.2. Number of animals and dose levels
1.5.3. Limit test
1.6. OBSERVATIONS
1.6.1. Body weight
1.6.2. Pathology
2. DATA
3. REPORTING
3.1. Test report
4. REFERENCES
Appendix 1
PROCEDURE TO BE FOLLOWED FOR EACH OF THE STARTING DOSES
GENERAL REMARKS
Appendix 1A
TEST PROCEDURE WITH A STARTING DOSE OF 5 MG/KG BODY WEIGHT
Appendix 1B
TEST PROCEDURE WITH A STARTING DOSE OF 50 MG/KG BODY WEIGHT
Appendix1C
TEST PROCEDURE WITH A STARTING DOSE OF 300 MG/KG BODY WEIGHT
Appendix 1D
TEST PROCEDURE WITH A STARTING DOSE OF 2 000 MG/KG BODY WEIGHT
Appendix 2
CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD
50
VALUES EXCEEDING 2 000 MG/KG WITHOUT THE NEED FOR TESTING
TESTING AT DOSES ABOVE 2 000 MG/KG
Appendix 3
TEST METHOD B.1 tris: Guidance on classififcation according to EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))
B.2. ACUTE TOXICITY (INHALATION)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Exposure concentrations
1.6.2.4. Limit test
1.6.2.5. Exposure time
1.6.2.6. Equipment
1.6.2.7. Observation period
1.6.3. Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.3. ACUTE TOXICITY (DERMAL)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Dose levels
1.6.2.4. Limit test
1.6.2.5. Observation period
1.6.3. Procedure
Assessment of toxicity in the other sex
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparation for the
in vivo
test
1.4.1.1. Selection of animal species
1.4.1.2. Preparation of the animals
1.4.1.3. Housing and feeding conditions
1.4.2. Test procedure
1.4.2.1. Application of the test substance
1.4.2.2. Dose level
1.4.2.3. Initial test (
in vivo
dermal irritation/corrosion test using one animal)
1.4.2.4. Confirmatory test (
in vivo
dermal irritation test with additional animals)
1.4.2.5. Observation period
1.4.2.6. Clinical observations and grading of skin reactions
2. DATA
2.1. PRESENTATION OF RESULTS
2.2. EVALUATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Table I
GRADING OF SKIN REACTIONS
No erythema … |
0 |
Very slight erythema (barely perceptible) … |
1 |
Well defined erythema … |
2 |
Moderate to severe erythema … |
3 |
Severe erythema (beef redness) to eschar formation preventing grading of erythema … |
4 |
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 mm) … |
3 |
Severe oedema (raised more than 1 mm and extending beyond area of exposure) … |
4 |
Appendix
A Sequential Testing Strategy for Dermal Irritation and Corrosion
GENERAL CONSIDERATIONS
DESCRIPTION OF THE EVALUATION AND TESTING STRATEGY
REFERENCES
Figure
TESTING AND EVALUATION STRATEGY FOR DERMAL IRRITATION/CORROSION
B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparation for the
in vivo
test
1.4.1.1. Selection of species
1.4.1.2. Preparation of animals
1.4.1.3. Housing and feeding conditions
1.4.2. Test procedure
1.4.2.1. Application of the test substance
1.4.2.2. Irrigation
1.4.2.3. Dose level
1.4.2.3.1.
Testing of liquids
1.4.2.3.2.
Testing of solids
1.4.2.3.3.
Testing of aerosols
1.4.2.4. Initial test (
in vivo
eye irritation/corrosion test using one animal)
1.4.2.5. Local anaesthetics
1.4.2.6. Confirmatory test (
in vivo
eye irritation test with additional animals)
1.4.2.7. Observation period
1.4.2.7.1.
Clinical observations and grading of eye reactions
2. DATA
2.2. EVALUATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULTS
4. REFERENCES
Table I
GRADING OF OCULAR LESIONS
Cornea
No ulceration or opacity … |
0 |
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible … |
1 |
Easily discernible translucent area; details of iris slightly obscured … |
2 |
Nacrous area; no details of iris visible; size of pupil barely discernible … |
3 |
Opaque cornea; iris not discernible through the opacity … |
4 |
Iris
Normal … |
0 |
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect) … |
1 |
Hemorrhage, gross destruction, or no reaction to light … |
2 |
Conjunctivae
Normal … |
0 |
Some blood vessels hyperaemic (injected) … |
1 |
Diffuse, crimson colour; individual vessels not easily discernible … |
2 |
Diffuse beefy red … |
3 |
Chemosis
Normal … |
0 |
Some swelling above normal … |
1 |
Obvious swelling, with partial eversion of lids … |
2 |
Swelling, with lids about half closed … |
3 |
Swelling, with lids more than half closed … |
4 |
Appendix
A Sequential Testing Strategy for Eye Irritation and Corrosion
GENERAL CONSIDERATIONS
DESCRIPTION OF THE STEPWISE TESTING STRATEGY
REFERENCES
Figure
TESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION
B.6. SKIN SENSITISATION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
CAS numbers |
EINECS numbers |
EINECS names |
Common names |
101-86-0 |
202-983-3 |
α-hexylcinnamaldehyde |
α-hexylcinnamaldehyde |
149-30-4 |
205-736-8 |
Benzothiazole-2-thiol (mercaptobenzothiazole) |
kaptax |
94-09-7 |
202-303-5 |
Benzocaine |
norcaine |
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHODS
1.5.1.
Guinea-Pig Maximisation Test (GPMT)
1.5.1.1.
Preparations
1.5.1.2.
Test conditions
1.5.1.2.1. Test animals
1.5.1.2.2. Number and sex
1.5.1.2.3. Dose levels
1.5.1.3.
Procedure
1.5.1.3.1. Induction
1.5.1.3.2. Challenge
1.5.1.3.3. Observation and Grading: treated and control groups
1.5.2.
Buehler test
1.5.2.1.
Preparations
1.5.2.2.
Test conditions
1.5.2.2.1. Test animals
1.5.2.2.2. Number and sex
1.5.2.2.3. Dose levels
1.5.2.3.
Procedure
1.5.2.3.1. Induction
1.5.2.3.2. Challenge
1.5.2.3.3. Observation and grading
2. DATA (GPMT and Buehler test)
3. REPORTING (GPMT and Buehler test)
Test report (GMPT and Buehler test)
4. REFERENCES
Appendix
TABLE
Magnusson/Kligman grading scale for the evaluation of challenge patch test reactions
B.7. REPEATED DOSE (28 DAYS) TOXICITY (ORAL)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.2. Test conditions
1.4.2.1. Test animals
1.4.2.2. Number and sex
1.4.2.3. Dose levels
1.4.2.4. Limit test
1.4.2.5. Observation period
1.4.3. Procedure
1.4.3.1. General observations
1.4.3.2. Body weight and food/water consumption
1.4.3.3. Haematology
1.4.3.4. Clinical biochemistry
1.4.3.5. Gross necropsy
1.4.3.6. Histopathological examination
2. DATA
3. REPORTING
TEST REPORT
4. REFERENCES
B.8. REPEATED DOSE (28 DAYS) TOXICITY (INHALATION)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Exposure concentration
1.6.2.4. Exposure time
1.6.2.5. Equipment
1.6.2.6. Observation period
1.6.3. Procedure
1.6.3.1. Gross necropsy
1.6.3.2. Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Dose levels
1.6.2.4. Limit test
1.6.2.5. Observation period
1.6.3. Procedure
1.6.4. Gross necropsy
1.6.5. Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.10. MUTAGENICITY —
IN VITRO
MAMMALIAN CHROMOSOME ABERRATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
1.4.1.2. Media and culture conditions
1.4.1.3. Preparation of cultures
1.4.1.4. Metabolic activation
1.4.1.5. Test substance/Preparation
1.4.2. Test conditions
1.4.2.1. Solvent/vehicle
1.4.2.2. Exposure concentrations
1.4.2.3. Negative and positive controls
Metabolic Activation condition |
Substance |
CAS No |
EINECS No |
Absence of exogenous metabolic Activation |
Methyl methanesulphonate |
66-27-3 |
200-625-0 |
Ethyl methanesulphonate |
62-50-0 |
200-536-7 |
|
Ethyl nitrosourea |
759-73-9 |
212-072-2 |
|
Mitomycin C |
50-07-7 |
200-008-6 |
|
4-Nitroquinoline-N-oxide |
56-57-5 |
200-281-1 |
|
Presence of exogenous metabolic Activation |
Benzo[a]pyrene |
50-32-8 |
200-028-5 |
Cyclophosphamide Cyclophosphamide monohydrate |
50-18-0 6055-19-2 |
200-015-4 |
1.4.3. Procedure
1.4.3.1. Treatment with the test substance
1.4.3.3. Culture harvest time
1.4.3.4. Chromosome preparation
1.4.3.5. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.11. MUTAGENICITY —
IN VIVO
MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Preparation of doses
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
Substance |
CAS No |
EINECS No |
Ethyl methanesulphonate |
62-50-0 |
200-536-7 |
Ethyl nitrosourea |
759-73-9 |
212-072-2 |
Mitomycin C |
50-07-7 |
200-008-6 |
Cyclophosphamide Cyclophosphamide monohydrate |
50-18-0 6055-19-2 |
200-015-4 |
Triethylenemelamine |
51-18-3 |
200-083-5 |
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Chromosome preparation
1.5.7. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.12. MUTAGENICITY —
IN VIVO
MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Preparation of doses
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
Substance |
CAS No |
EINECS No |
Ethyl methanesulphonate |
62-50-0 |
200-536-7 |
N-ethyl-N-nitrosourea |
759-73-9 |
212-072-2 |
Mitomycin C |
50-07-7 |
200-008-6 |
Cyclophosphamide Cyclophosphamide monohydrate |
50-18-0 6055-19-2 |
200-015-4 |
Triethylenemelamine |
51-18-3 |
200-083-5 |
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Bone marrow/blood preparation
1.5.7. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.13/14. MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. INITIAL CONSIDERATIONS
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Preparations
1.5.1.1. Bacteria
1.5.1.2. Medium
1.5.1.3. Metabolic activation
1.5.1.4. Test substance/Preparation
1.5.2. Test conditions
1.5.2.1. Test strains (see 1.5.1.1)
1.5.2.2. Exposure concentration
1.5.2.3. Negative and positive controls
CA numbers |
EINECS numbers |
Names |
781-43-1 |
212-308-4 |
9,10-dimethylanthracene |
57-97-6 |
200-359-5 |
7,12-dimethylbenz[a]anthracene |
50-32-8 |
200-028-5 |
benzo[a]pyrene |
613-13-8 |
210-330-9 |
2-aminoanthracene |
50-18-0 |
|
cyclophosphamide |
6055-19-2 |
200-015-4 |
cyclophosphamide monohydrate |
CA numbers |
EINECS numbers |
Names |
573-58-0 |
209-358-4 |
Congo Red |
CAS numbers |
EINECS numbers |
Names |
Strain |
26628-22-8 |
247-852-1 |
Sodium azide |
TA 1535 and TA 100 |
607-57-8 |
210-138-5 |
2-nitrofluorene |
TA 98 |
90-45-9 |
201-995-6 |
9-aminoacridine |
TA 1537, TA 97 and TA 97a |
17070-45-0 |
241-129-4 |
ICR 191 |
TA 1537, TA 97 and TA 97a |
80-15-9 |
201-254-7 |
Cumene hydroperoxide |
TA 102 |
50-07-7 |
200-008-6 |
Mitomycin C |
WP2 uvrA and TA102 |
70-25-7 |
200-730-1 |
N-ethyl-N-nitro-N-nitrosoguanidine |
WP2, WP2uvrA and WP2uvrA(pKM101) |
56-57-5 |
200-281-1 |
4-nitroquinoline-1-oxide |
WP2, WP2uvrA and WP2uvrA(pKM101) |
3688-53-7 |
|
Furylfuramide (AF2) |
plasmid-containing strains |
1.5.3. Procedure
1.5.4. Incubation
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.15. MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY GENE MUTATION —
SACCHAROMYCES CEREVISIAE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Metabolic activation
Test conditions
Tester strains
Media
Use of negative and positive controls
Exposure concentration
Incubation conditions
Spontaneous mutation frequencies
Number of replicates
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.16. MITOTIC RECOMBINATION —
SACCHAROMYCES CEREVISIAE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Metabolic activation
Test conditions
Tester strains
Media
Use of negative and positive controls
Exposure concentrations
Incubation conditions
Spontaneous mitotic recombination frequencies
Number of replicates
Procedures
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.17. MUTAGENICITY —
IN VITRO
MAMMALIAN CELL GENE MUTATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
1.4.1.2. Media and culture conditions
1.4.1.3. Preparation of cultures
1.4.1.4. Metabolic activation
1.4.1.5. Test substance/Preparation
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Exposure concentrations
1.4.2.3. Controls
Metabolic activation condition |
Locus |
Substance |
CAS No |
EINECS No |
Absence of exogenous metabolic activation |
HPRT |
Ethyl methanesulphonate |
62-50-0 |
200-536-7 |
Ethyl nitrosourea |
759-73-9 |
212-072-2 |
||
TK (small and large colonies) |
Methyl methanesulphonate |
66-27-3 |
200-625-0 |
|
XPRT |
Ethyl methanesulphonate |
62-50-0 |
200-536-7 |
|
Ethyl nitrosourea |
759-73-9 |
212-072-2 |
||
Presence of exogenous metabolic activation |
HPRT |
3-Methylcholanthrene |
56-49-5 |
200-276-4 |
N-Nitrosodimethylamine |
62-75-9 |
200-549-8 |
||
7,12-Dimethylbenzanthracene |
57-97-6 |
200-359-5 |
||
TK (small and large colonies) |
Cyclophosphamide |
50-18-0 |
200-015-4 |
|
Cyclophosphamide monohydrate |
6055-19-2 |
|
||
Benzo[a]pyrene |
50-32-8 |
200-028-5 |
||
3-Methylcholanthrene |
56-49-5 |
200-276-5 |
||
XPRT |
N-Nitrosodimethylamine (for high levels of S-9) |
62-75-9 |
200-549-8 |
|
Benzo[a]pyrene |
50-32-8 |
200-028-5 |
1.4.3. Procedure
1.4.3.1. Treatment with the test substance
1.4.3.2. Measurement of survival, viability and mutant frequency
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.18. DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS — MAMMALIAN CELLS
IN VITRO
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Test conditions
Number of cultures
Use of negative and positive controls
Exposure concentrations
Cells
Metabolic activation
Procedure
Preparation of cultures
Treatment of the cultures with the test substance
Primary rat hepatocytes
Established cell lines and lymphocytes
Analysis
Autoradiographic determinations
LSC determinations
2. DATA
2.1. AUTORADIOGRAPHIC DETERMINATIONS
2.2. LSC DETERMINATIONS
3. REPORTING
3.1. TEST REPORT
3.2. VALUATION AND INTERPRETATION
4. REFERENCES
B.19. SISTER CHROMATID EXCHANGE ASSAY
IN VITRO
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Test conditions
Number of cultures
Use of negative and positive controls
Exposure concentrations
1.6.3.
Procedure
Preparation of cultures
Treatment
Harvesting of cells
Chromosome preparation and staining
Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.20. SEX-LINKED RECESSIVE LETHAL TEST IN
DROSOPHILA MELANOGASTER
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLES OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Stocks
Test substance
Number of animals
Route of administration
Use of negative and positive controls
Exposure levels
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.21.
IN VITRO
MAMMALIAN CELL TRANSFORMATION TESTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Cells
Medium
Test substance
Metabolic activation
Test conditions
Use of negative and positive controls
Exposure concentrations
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.22. RODENT DOMINANT LETHAL TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Test conditions
Route of administration
Experimental animals
Number and sex
Use of negative and positive controls
Dose levels
Limit test
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.23. MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Preparation of doses
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
Substance |
CAS No |
Einecs No |
Cyclophosphamide Cyclophosphamide monohydrate |
50-18-0 6055-19-2 |
200-015-4 |
Cyclohexylamine |
108-91-8 |
203-629-0 |
Mitomycin C |
50-07-7 |
200-008-6 |
Monomeric acrylamide |
79-06-1 |
201-173-7 |
Triethylenemelamine |
51-18-3 |
200-083-5 |
1.5. PROCEDURE
1.5.1. Number of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Chromosome preparation
1.5.7. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.24. MOUSE SPOT TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Experimental animals
Number and sex
Use of negative and positive controls
Route of administration
Dose levels
Procedure
Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.25. MOUSE HERITABLE TRANSLOCATION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Route of administration
Experimental animals
Number of animals
Use of negative and positive controls
Dose levels
Procedure
Treatment and mating
Testing for translocation heterozygosity
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.26. SUB-CHRONIC ORAL TOXICITY TESTREPEATED DOSE 90 – DAY ORAL TOXICITY STUDY IN RODENTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations of animals
1.4.2. Preparations of doses
1.4.3. Test conditions
1.4.3.1. Experimental animals
1.4.3.2. Number and sex
1.4.3.3. Dose levels
1.4.3.4. Limit test
1.5. PROCEDURE
1.5.1. Administration of doses
1.5.2. Observations
1.5.2.1. Body weight and food/water consumption
1.5.2.2. Haematology and clinical biochemistry
1.5.2.3. Gross necropsy
1.5.2.4. Histopathology
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
B.27. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90-DAY ORAL TOXICITY STUDY IN NON-RODENTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
1.4.2. Preparation of animals
1.4.3. Preparations of doses
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Dosage
1.5.3. Limit test
1.5.4. Administration of doses
1.5.5. Observations
1.5.5.1. Body weight and food/water consumption
1.5.5.2. Haematology and clinical biochemistry
1.5.5.3. Gross necropsy
1.5.5.4. Histopathology
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
B.28. SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY REPEATED DERMAL DOSE STUDY USING RODENT SPECIES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2.
Test conditions
1.6.2.1.
Experimental animals
1.6.2.2.
Number and sex
1.6.2.3.
Dose levels
1.6.3.
Limit test
1.6.4.
Observation period
1.6.5.
Procedure
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.29. SUB-CHRONIC INHALATION TOXICITY STUDY 90-DAY REPEATED INHALATION DOSE STUDY USING RODENT SPECIES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Test conditions
1.6.2.1.
Experimental animals
1.6.2.2.
Number and sex
1.6.2.3.
Exposure concentrations
1.6.2.4.
Exposure time
1.6.2.5.
Equipment
1.6.2.6.
Observation period
1.6.3.
Procedure
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.30. CHRONIC TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Test conditions
1.6.2.1.
Experimental animals
1.6.2.2.
Number and sex
1.6.2.3.
Dose levels and frequency of exposure
1.6.2.4.
Controls
1.6.2.5.
Route of administration
1.6.2.6.
Oral studies
1.6.2.7.
Inhalation studies
1.6.2.8.
Exposure chambers
1.6.2.9.
Duration of study
1.6.3.
Procedure
Observations
Haematological examination
Urinalysis
Clinical chemistry
Gross necropsy
Histopathology
2. DATA
3. REPORTING
TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.31. PRENATAL DEVELOPMENTAL TOXICITY STUDY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Selection of animal species
1.5.2. Housing and feeding conditions
1.5.3. Preparation of the animals
1.6. PROCEDURE
1.6.1. Number and sex of animals
1.6.2. Preparation of doses
1.6.3. Dosage
1.6.4. Limit test
1.6.5. Administration of doses
1.6.6. Observations of the dams
1.6.7. Body weight and food consumption
1.6.8. Post-mortem examination
1.6.9. Examination of uterine contents
1.6.10. Examination of foetuses
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION OF RESULTS
2.3. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.32. CARCINOGENICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Experimental animals
1.6.2.
Number and sex
1.6.3.
Dose levels and frequency of exposure
1.6.4.
Controls
1.6.5.
Route of administration
1.6.5.1. Oral studies
1.6.5.2. Dermal studies
1.6.5.3. Inhalation studies
1.6.6.
Exposure chambers
1.6.7.
Duration of study
1.6.8.
Procedure
1.6.8.1.
Observations
1.6.8.2.
Clinical examinations
Haematology
Gross necropsy
Histopathology
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.33. COMBINED CHRONIC TOXICITY/CARCINOGENICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Experimental animals
1.6.2.
Number and sex
1.6.3.
Dose levels and frequency of exposure
1.6.4.
Controls
1.6.5.
Route of administration
1.6.5.1. Oral tests
1.6.5.2. Dermal tests
1.6.5.3. Inhalation tests
1.6.6.
Exposure chambers
1.6.7.
Duration of test
1.6.8.
Procedure
1.6.8.1.
Observations
1.6.8.2.
Clinical examinations
Haematology
Urinalysis
Clinical chemistry
Gross necropsy
Histopathology
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.34. ONE-GENERATION REPRODUCTION TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Experimental animals
Selection of species
Number and sex
1.6.3.
Test conditions
1.6.3.1.
Dose levels
1.6.3.2.
Limit test
1.6.3.3.
Performance of the test
Experimental schedules
Mating procedure
Litter sizes
1.6.4.
Observations
1.6.5.
Pathology
1.6.5.1.
Necropsy
1.6.5.2.
Histopathology
2. DATA
3. REPORTING
TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.35. TWO-GENERATION REPRODUCTION TOXICITY STUDY
1. METHOD
1.1. INTRODUCTION
1.2. PRINCIPLE OF THE TEST METHOD
1.3. DESCRIPTION OF THE TEST METHOD
1.3.1. Selection of animal species
1.3.2. Housing and feeding conditions
1.3.3. Preparation of animals
1.4. PROCEDURE
1.4.1. Number and sex of animals
1.4.2. Preparation of doses
1.4.3. Dosage
1.4.4. Limit test
1.4.5. Administration of doses
1.4.6. Experimental schedules
1.4.7. Mating procedure
1.4.7.1. Parental (P) mating
1.4.7.2. F1 mating
1.4.7.3. Second mating
1.4.7.4. Litter size
1.5. OBSERVATIONS
1.5.1. Clinical observations
1.5.2. Body weight and food/water consumption of parent animals
1.5.3. Oestrus cycle
1.5.4. Sperm parameters
1.5.5. Offspring
1.5.6. Gross necropsy
1.5.7. Organ weights
1.5.8. Histopathology
1.5.8.1. Parental animals
1.5.8.2. Weanlings
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION OF RESULTS
2.3. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.36. TOXICOKINETICS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.3. Dose levels
1.6.4. Route of administration
1.6.5. Observation period
1.6.6. Procedure
Absorption
Distribution
Excretion
Metabolism
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.37. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES FOLLOWING ACUTE EXPOSURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Preparations
1.5.2. Test conditions
1.5.2.1. Test animals
1.5.2.2. Number and sex
1.5.2.3. Dose levels
1.5.2.4. Limit test
1.5.3. Observation period
1.5.4. Procedure
General observation
Body weight
Biochemistry
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
TEST REPORT
4. REFERENCES
B.38. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES 28-DAY REPEATED DOSE STUDY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.2. Test conditions
1.4.2.1. Test animals
1.4.2.2. Number and sex
1.4.2.3. Dose levels
1.4.2.4. Limit test
1.4.2.5. Observation period
1.4.3. Procedure
General observations
Body weight
Biochemistry
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
TEST REPORT
4. REFERENCES
B.39. UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Test substance/Preparation
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
Sampling Times |
Substance |
CAS No |
EINECS No |
Early sampling times (2-4 hours) |
N-Nitrosodimethylamine |
62-75-9 |
200-249-8 |
Late sampling times (12-16 hours) |
N-2-Fluorenylacetamide (2-AAF) |
53-96-3 |
200-188-6 |
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Preparation of liver cells
1.5.7. Determination of UDS
1.5.8. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
positive |
(i) |
NNG values above a pre-set threshold which is justified on the basis of laboratory historical data; or |
|
(ii) |
NNG values significantly greater than concurrent control; |
negative |
(i) |
NNG values within/below historical control threshold; or |
|
(ii) |
NNG values not significantly greater than concurrent control. |
3. REPORTING
TEST REPORT
4. REFERENCES
B.40.
IN VITRO
SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST (TER)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
Name |
EINECS No |
CAS No |
|
1,2-Diaminopropane |
201-155-9 |
78-90-0 |
Severely corrosive |
Acrylic Acid |
201-177-9 |
79-10-7 |
Severely Corrosive |
2-tert. Butylphenol |
201-807-2 |
88-18-6 |
Corrosive |
Potassium hydroxide (10 %) |
215-181-3 |
1310-58-3 |
Corrosive |
Sulfuric acid (10 %) |
231-639-5 |
7664-93-9 |
Corrosive |
Octanoic acid (caprylic acid) |
204-677-5 |
124-07-02 |
Corrosive |
4-Amino-1,2,4-triazole |
209-533-5 |
584-13-4 |
Not corrosive |
Eugenol |
202-589-1 |
97-53-0 |
Not corrosive |
Phenethyl bromide |
203-130-8 |
103-63-9 |
Not corrosive |
Tetrachloroethylene |
204-825-9 |
27-18-4 |
Not corrosive |
Isostearic acid |
250-178-0 |
30399-84-9 |
Not corrosive |
4-(Methylthio)-benzaldehyde |
222-365-7 |
3446-89-7 |
Not corrosive |
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Animals
1.5.2. Preparation of the skin discs
1.5.3. Application of the test and control substances
1.5.4. TER measurements
1.5.5. Dye binding methods
1.5.5.1. Sulforhodamine B dye application and removal
1.5.5.2. Calculation of dye content
2. DATA
2.1. INTERPRETATION OF RESULTS
Control |
Substance |
Resistance range (kΩ) |
Positive |
10M Hydrochloric acid |
0,5-1,0 |
Negative |
Distilled water |
10-25 |
Control |
Substance |
Dye content range (μg/disc) |
Positive |
10M Hydrochloric acid |
40-100 |
Negative |
Distilled water |
15-35 |
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Figure 1
Apparatus for the rat skin TER assay
Figure 2
Dimensions of the polytetrafluoroethylene (PFTE) and receptor tubes and electrodes used
B.40 BIS.
IN VITRO
SKIN CORROSION: HUMAN SKIN MODEL TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
Name |
EINECS No |
CAS No |
|
1,2-Diaminopropane |
201-155-9 |
78-90-0 |
Severely corrosive |
Acrylic Acid |
201-177-9 |
79-10-7 |
Severely corrosive |
2-tert. Butylphenol |
201-807-2 |
88-18-6 |
Corrosive |
Potassium hydroxide (10 %) |
215-181-3 |
1310-58-3 |
Corrosive |
Sulfuric acid (10 %) |
231-639-5 |
7664-93-9 |
Corrosive |
Octanoic acid (caprylic acid) |
204-677-5 |
124-07-02 |
Corrosive |
4-Amino-1,2,4-triazole |
209-533-5 |
584-13-4 |
Not corrosive |
Eugenol |
202-589-1 |
97-53-0 |
Not corrosive |
Phenethyl bromide |
203-130-8 |
103-63-9 |
Not corrosive |
Tetrachloroethylene |
204-825-9 |
27-18-4 |
Not Corrosive |
Isostearic acid |
250-178-0 |
30399-84-9 |
Not corrosive |
4-(Methylthio)-benzaldehyde |
222-365-7 |
3446-89-7 |
Not corrosive |
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Procedure
1.4.1.1. Human skin models
1.4.1.1.1.
General model conditions:
1.4.1.1.2.
Functional model conditions:
1.4.1.2. Application of the test and control substances
1.4.1.3. Cell viability measurements
2. DATA
2.1. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.41.
IN VITRO
3T3 NRU PHOTOTOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
1.4.1.2. Media and culture conditions
1.4.1.3. Preparation of cultures
1.4.1.4. Preparation of test substance
1.4.1.5. Irradiation conditions
1.4.1.5.1.
Light source
1.4.1.5.2.
Dosimetry
[Bild bitte in Originalquelle ansehen] |
(1 J = 1 Wsec) |
1.4.2. Test conditions
1.4.2.1. Test substance concentrations
1.4.2.2. Controls
1.4.2.2.1.
Radiation sensitivity of the cells, establishing of historical data:
1.4.2.2.2.
Radiation sensitivity, check of current test:
1.4.2.2.3.
Viability of solvent controls:
1.4.2.2.4.
Positive control:
1.4.3. Test procedure (6)(7)(8)(16)(17):
1.4.3.1. 1st day:
1.4.3.2. 2nd day:
1.4.3.3. 3rd day:
1.4.3.3.1.
Microscopic evaluation
1.4.3.3.2.
Neutral Red uptake test
2. DATA
2.1. QUALITY AND QUANTITY OF DATA
2.2. EVALUATION OF RESULTS
2.3. INTERPRETATION OF RESULTS
Chemical name |
EINECS No |
CAS No |
PIF |
MPE |
Absorption peak |
Solvent(9) |
Amiodarone HCL |
243-293-2 |
[19774-82-4] |
> 3,25 |
0,2-0,54 |
242 nm 300 nm (shoulder) |
ethanol |
Choloropromazine HCL |
200-701-3 |
[69-09-0] |
> 14,4 |
0,33-0,63 |
309 nm |
ethanol |
Norfloxacin |
274-614-4 |
[70458-96-7] |
> 71,6 |
0,34-0,90 |
316 nm |
acetonitrile |
Anthracene |
204-371-1 |
[120-12-7] |
> 18,5 |
0,19-0,81 |
356 nm |
acetonitrile |
Protoporphyrin IX, Disodium |
256-815-9 |
[50865-01-5] |
> 45,3 |
0,54-0,74 |
402 nm |
ethanol |
L-Histidine |
|
[7006-35-1] |
no PIF |
0,05-0,10 |
211 nm |
water |
Hexacholorophene |
200-733-8 |
[70-30-4] |
1,1-1,7 |
0,00-0,05 |
299 nm 317 nm (shoulder) |
ethanol |
Sodium lauryl sulphate |
205-788-1 |
[151-21-3] |
1,0-1,9 |
0,00-0,05 |
no absorption |
water |
2.4. INTERPRETATION OF DATA
3. REPORTING
TEST REPORT
4. REFERENCES
Appendix 1
Role of the 3T3 NRU PT in a sequential approach to the phototoxicity testing of chemicals
Appendix 2
Figure 1
Spectral power distribution of a filtered solar simulator
Figure 2
Irradiation sensivity of Balb/c 3T3 cells (as measured in the UVA range)
B.42. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY
1. METHOD
1.1. INTRODUCTION
1.2. PRINCIPLE OF THE TEST METHOD
1.3. DESCRIPTION OF THE TEST METHOD
1.3.1. Preparations
1.3.1.1. Housing and feeding conditions
1.3.1.2. Preparation of animals
1.3.2. Test conditions
1.3.2.1. Experimental animals
1.3.2.2. Reliability check
1.3.2.3. Number of animals, dose levels and vehicle selection.
1.3.3. Test procedure
1.3.3.1. Experimental schedule
1.3.3.2. Preparation of cell suspensions
1.3.3.3. Determination of cell proliferation (incorporated radioactivity)
1.3.3.4. Observations
1.3.3.4.1.
Clinical observations
1.3.3.4.2.
Body weights
1.3.4. Calculation of results
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.43. NEUROTOXICITY STUDY IN RODENTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
1.4.2. Housing and feeding conditions
1.4.3. Preparation of animals
1.4.4. Route of administration and preparation of doses
1.5. PROCEDURES
1.5.1. Number and sex animals
1.5.2. Treatment and control group
1.5.3. Reliability check
1.5.4. Dose selection
1.5.5. Limit test
1.5.6. Administration of doses
1.6. OBSERVATION
1.6.1. Frequency of observations and tests
1.6.1.1. Observations of general health condition and mortality/morbidity
1.6.1.2. Detailed clinical observations
1.6.1.3. Functional tests
1.6.2. Body weight and food/water consumption
1.6.3. Ophthalmology
1.6.4. Haematology and clinical biochemistry
1.6.5. Histopathology
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
|
NEUROTOXICITY STUDY CONDUCTED AS: |
|||
Separate study |
Combined study with the 28-day study |
Combined study with the 90-day study |
Combined study with the chronic toxicity study |
|
Total number of animals per group |
10 males and 10 females |
10 males and 10 females |
15 males and 15 females |
25 males and 25 females |
Number of animals selected for functional testing including detailed clinical observations |
10 males and 10 females |
10 males and 10 females |
10 males and 10 females |
10 males and 10 females |
Number of animals selected per perfusion in situ and neurohistopathology |
5 males and 5 females |
5 males and 5 females |
5 males and 5 females |
5 males and 5 females |
Number of animals selected for repeated dose/subchronic/chronic toxicity observations, haematology, clinical biochemistry, histopathology, etc. as indicate in the respective Guidelines |
|
5 males and 5 females |
10 males(10) and 10 females(10) |
20 males(10) and 20 females(10) |
Supplemental observations, as appropriate |
5 males and 5 females |
|
|
|
Type of observations |
Study duration |
||||||||||||||||||||||||||
Acute |
28-day |
90-day |
Chronic |
||||||||||||||||||||||||
In all animals |
General health condition |
daily |
daily |
daily |
daily |
||||||||||||||||||||||
Mortality/morbidity |
Twice daily |
Twice daily |
Twice daily |
Twice daily |
|||||||||||||||||||||||
In animals selected for functional observations |
Detailed clinical observations |
|
|
|
|
||||||||||||||||||||||
Functional tests |
|
|
|
|
B.44. SKIN ABSORPTION:
IN VIVO
METHOD
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE METHOD
1.4.1. Selection of animal species
1.4.2. Number and sex of animals
1.4.3. Housing and feeding conditions
1.4.4. Preparation of animals
1.4.5. Test substance
1.4.6. Test preparation
1.4.7. Application to the skin
1.4.8. Duration of exposure and sampling
1.4.9. Terminal procedures
1.4.10. Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Figure 1
An example of a design of a typical device used to define and protect dermal application site during
in vivo
percutaneous absorption studies
B.45. SKIN ABSORPTION:
IN VITRO
METHOD
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Diffusion cell
1.4.2. Receptor fluid
1.4.3. Skin preparations
1.4.4. Skin preparation integrity
1.4.5. Test substance
1.4.6. Test preparation
1.4.7. Test substances concentrations and formulations
1.4.8. Application to the skin
1.4.9. Temperature
1.4.10. Duration of exposure and sampling
1.4.11. Terminal procedures
1.4.12. Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Figure 1
An example of a typical design of a static diffusion cell for
in vitro
percutaneous absorption studies
PART C: METHODS FOR THE DETERMINATION OF ECOTOXICITY
C.1. ACUTE TOXICITY FOR FISH
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Reagents
1.6.1.1. Solutions of test substances
1.6.1.2. Holding and dilution water
1.6.2. Apparatus
1.6.3. Test fish
1.6.3.1. Holding
1.6.3.2. Mortality
1.6.4. Adaptation
1.6.5. Test procedure
Limit test
2. DATA AND EVALUATION
3. REPORTING
4. REFERENCES
Appendix 1
Reconstituted water
Example of a suitable dilution water
Stock solutions
CaCI2. 2H2O (calcium chloride dihydrate): Dissolve in, and make up to 1 litre with water. |
11,76 g |
MgSO4. 7H2O (magnesium sulphate heptahydrate): Dissolve in, and make up to 1litre with water. |
4,93 g |
NaHCO3 (sodium hydrogen carbonate): Dissolve in, and make up to 1 litre with water. |
2,59 g |
KCI (potassium chloride): Dissolve in, and make up to 11itre with water. |
0,23 g |
Reconstituted dilution water
Appendix 2
Fish species recommended for testing
Recommended species |
Recommended range of test temperature (oC) |
Recommended total length of test animal (cm) |
Brachydanio rerio (Teleostei, Cyprinidae) (Hamilton-Buchanan) Zebrab-fish |
20 to 24 |
3,0 ± 0,5 |
Pimephales promelas (Teleostei, Cyprinidae) (Rafinesque) Fathead minnow |
20 to 24 |
5,0 ± 2,5 |
Cyprinus carpio (Teleostei, Cyprinidae) (Linneaus 1758) Common carp |
20 to 24 |
6,0 ± 2,0 |
Oryzias latipes (Teleostei, Poeciliidae) Cyprinodontidae (Tomminck and Schlege 1850) Red killifish |
20 to 24 |
3,0 ± 1,0 |
Poecilia reticulata (Teleostei, Poeciliidae) (Peters 1859) Guppy |
20 to 24 |
3,0 ± 1,0 |
Lepomis macrochirus (Teleostei, Centrarchidae) (Rafinesque Linneaus 1758) Bluegill |
20 to 24 |
5,0 ± 2,0 |
Onchorhynchus mykiss (Teleostei, Salmonidae) (Walbaum 1988) Rainbow trout |
12 to 17 |
6,0 ± 2,0 |
Leuciscus idus (Teleostei, Cyprinidae) (Linneaus 1758) Golden Orfe |
20 to 24 |
6,0 ± 2,0 |
Collection
Appendix 3
Example of concentration: percentage mortality
C.2.
DAPHNIA
SP. ACUTE IMMOBILISATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. REFERENCE SUBSTANCES
1.6. QUALITY CRITERIA
1.7. DESCRIPTION OF THE TEST METHOD
1.7.1. Apparatus
1.7.2. Test organism
1.7.3. Holding and dilution water
1.7.4. Test solutions
1.8. PROCEDURE
1.8.1. Conditions of exposure
1.8.1.1. Test groups and controls
1.8.1.2. Test concentrations
1.8.1.3. Incubation conditions
1.8.1.4. Duration
1.8.2. Observations
1.8.3. Analytical measurements
1.9. LIMIT TEST
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
Substance |
Concentration |
Particulate matter |
< 20 mg/l |
Total organic carbon |
< 2 mg/l |
Unionised ammonia |
< 1 μg/l |
Residual chlorine |
< 10 μg/l |
Total organophosphorus pesticides |
< 50 ng/l |
Total organochorine pesticides plus polychlorinated biphenyls |
< 50 ng/l |
Total organic chlorine |
< 25 ng/l |
Appendix 2
EXAMPLES OF SUITABLE RECONSTITUTED TEST WATER
ISO Test water (1)
Stock solutions (single substance) |
To prepare the reconstituted water, add the following volumes of stock solutions to 1 litre water(2) |
|
Substance |
Amount added to 1 litre water(2) |
|
Calcium chloride CaCl2, 2H2O |
11,76 g |
25 ml |
Magnesium sulfate MgSO4, 7H2O |
4,93 g |
25 ml |
Sodium bicarbonate NaHCO3 |
2,59 g |
25 ml |
Potassium chloride KCl |
0,23 g |
25 ml |
Elendt M7 and M4 medium
Acclimation to Elendt M4 and M7 medium
Preparation
Trace element
Stock solution(s) I (single substance) |
Amount added to water (mg/l) |
Concentration (related to medium M4) |
To prepare the combined stock solution II, add the following amount of stock solution I to water (ml/l) |
|
M4 |
M7 |
|||
H3 BO3 |
57 190 |
20 000-fold |
1,0 |
0,25 |
MnCl2.4H2O |
7 210 |
20 000-fold |
1,0 |
0,25 |
LiCl |
6 120 |
20 000-fold |
1,0 |
0,25 |
RbCl |
1 420 |
20 000-fold |
1,0 |
0,25 |
SrCl2.6H2O |
3 040 |
20 000-fold |
1,0 |
0,25 |
NaBr |
320 |
20 000-fold |
1,0 |
0,25 |
Na2 MoO4.2H2O |
1 230 |
20 000-fold |
1,0 |
0,25 |
CuCl2.2H2O |
335 |
20 000-fold |
1,0 |
0,25 |
ZnCl2 |
260 |
20 000-fold |
1,0 |
1,0 |
CoCl2.6H2O |
200 |
20 000-fold |
1,0 |
1,0 |
KI |
65 |
20 000-fold |
1,0 |
1,0 |
Na2 SeO3 |
43,8 |
20 000-fold |
1,0 |
1,0 |
NH4 VO3 |
11,5 |
20 000-fold |
1,0 |
1,0 |
Na2 EDTA.2H2O |
5 000 |
2 000-fold |
— |
— |
FeSO4.7H2O |
1 991 |
2 000-fold |
— |
— |
Both Na 2 EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. |
||||
This gives: |
||||
2 l Fe-EDTA solution |
|
1 000-fold |
20,0 |
5,0 |
M4 and M7 media
|
Amount added to water (mg/l) |
Concentration (related to medium M4) |
Amount of stock solution II added to prepare medium (ml/l) |
|
M4 |
M7 |
|||
Stock solution II (combined trace elements) |
|
20-fold |
50 |
50 |
Macro nutrient stock solutions (single substance) |
|
|
|
|
CaCl2 · 2H20 |
293 800 |
1 000-fold |
1,0 |
1,0 |
MgSO4 · 7H2O |
246 600 |
2 000-fold |
0,5 |
0,5 |
KCl |
58 000 |
10 000-fold |
0,1 |
0,1 |
NaHCO3 |
64 800 |
1 000-fold |
1,0 |
1,0 |
Na2 SiO3 · 9H2O |
50 000 |
5 000-fold |
0,2 |
0,2 |
NaNO3 |
2 740 |
10 000-fold |
0,1 |
0,1 |
KH2 PO4 |
1 430 |
10 000-fold |
0,1 |
0,1 |
K2HPO4 |
1 840 |
10 000-fold |
0,1 |
0,1 |
Combined Vitamin stock |
— |
10 000-fold |
0,1 |
0,1 |
The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below: |
||||
Thiamine hydrochloride |
750 |
10 000-fold |
|
|
Cyanocobalamine (B12) |
10 |
10 000-fold |
|
|
Biotine |
7,5 |
10 000-fold |
|
|
C.3. ALGAL INHIBITION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST PROCEDURE
1.6.1. Reagents
1.6.1.1. Solutions of test substances
1.6.1.2. Test medium
Nutrient |
Concentration in the stock solution |
Final concentration in the test solution |
Stock solution 1: macro-nutrients |
||
NH4Cl |
1,5 g/l |
15 mg/l |
MgCl2.6H2O |
1,2 g/l |
12mg/l |
CaCl2.2H2O |
1,8 g/l |
18 mg/l |
MgSO4.7H2O |
1,5 g/l |
15 mg/l |
KH2PO4 |
0,16 g/l |
1,6 mg/l |
Stock solution 2: Fe-EDTA |
||
FeCl3.6H2O |
80 mg/l |
0,08 mg/l |
Na2EDTA.2H2O |
100 mg/l |
0,1 mg/l |
Stock solution 3: trace elements |
||
H3BO3 |
185 mg/l |
0,185 mg/l |
MnCl2.4H2O |
415 mg/l |
0,415 mg/l |
ZnCl2 |
3 mg/l |
3 × 10-3 mg/l |
CoCl2.6H2O |
1,5 mg/l |
1,5 × 10-3 mg/l |
CuCl2.2H2O |
0,01 mg/l |
10-5 mg/l |
Na2MoO4.2H2O |
7 mg/l |
7 × 10-3mg/l |
Stock solution 4: NaHCO3 |
||
NaHCO3 |
50 g/l |
50 mg/l |
1.6.2. Apparatus
1.6.3. Test organisms
1.6.4. Test procedure
Initial cell density
Concentrations of test substance
Replicates and controls
Performance of the test
Testing volatile substances
Limit test
2. DATA AND EVALUATION
2.1. COMPARISON OF AREAS UNDER THE GROWTH CURVES
2.2. COMPARISON OF GROWTH RATES
2.3. CALCULATION OF THE NOEC
3. REPORTING
4. REFERENCES
Appendix 1
Example of a procedure for the culturing of algae
General observations
Procedures for obtaining algal cultures
Preparation of nutrient solutions (media):
Stock culture:
Pre-culture:
Appendix 2
|
Means (mg/l) |
Range (mg/l) |
ErC50 (0-72 h) |
0,84 |
0,60 to 1,03 |
EbC50 (0-72 h) |
0,53 |
0,20 to 0,75 |
C.4. DETERMINATION OF ‘READY’ BIODEGRADABILITY
PART I. GENERAL CONSIDERATIONS
I.1. INTRODUCTION
I.2. SELECTION OF THE APPROPRIATE METHOD
Test |
Analytical Method |
Suitability for substances which are: |
||
poorly soluble |
volatile |
adsorbing |
||
DOC Die-Away |
Dissolved organic carbon |
– |
– |
+/– |
Mod. OECD Die-Away |
Dissolved organic carbon |
– |
– |
+/– |
CO2 Evolution |
Respirometry: CO2 evolution |
+ |
– |
+ |
Manometric Respirometry |
Manometric respirometry: oxygen consumption |
+ |
+/– |
+ |
Closed Bottle |
Respirometry: dissolved oxygen |
+/– |
+ |
+ |
MITI |
Respirometry: oxygen consumption |
+ |
+/– |
+ |
I.3. REFERENCE SUBSTANCES
I.4. PRINCIPLE OF THE TEST METHODS
I.5. QUALITY CRITERIA
I.5.1. Reproducibility
I.5.2. Validity of the test
I.6. GENERAL PROCEDURES AND PREPARATIONS
Test |
DOC Die-Away |
CO2 Evolution |
Manometric Respirometry |
Modified OECD Screeing |
Closed Bottle |
MITI (I) |
|||||||||
Concentration of Test Substance |
|
|
|
|
|
|
|||||||||
as mg/l |
|
|
100 |
|
2-10 |
100 |
|||||||||
mg DOC/1 |
10-40 |
10-20 |
|
10-40 |
|
|
|||||||||
mg ThOD/1 |
|
|
50-100 |
|
5-10 |
|
|||||||||
Concentration of Inoculum (in cells/l, approximatively) |
≤ 30 mg/l SS or ≤ 100 ml effluent/l (107-108) |
0,5 ml secondary effluent/1 (105) |
≤ 5 ml of effluent/l (104-106) |
30 mg/l SS (107-108) |
|||||||||||
Concentration of elements in mineral medium (in mg/l): |
|
|
|
|
|
|
|||||||||
P |
116 |
11,6 |
29 |
||||||||||||
N |
1,3 |
0,13 |
1,3 |
||||||||||||
Na |
86 |
8,6 |
17,2 |
||||||||||||
K |
122 |
12,2 |
36,5 |
||||||||||||
Mg |
2,2 |
2,2 |
6,6 |
||||||||||||
Ca |
9,9 |
9,9 |
29,7 |
||||||||||||
Fe |
0,05-0,1 |
0,05-0,1 |
0,15 |
||||||||||||
pH |
7,4±0,2 |
preferably 7,0 |
|||||||||||||
Temperature |
22 ± 2 oC |
25 ± 1 oC |
|||||||||||||
|
|
|
I.6.1. Dilution water
I.6.2. Stock solutions of mineral components
Stock solutions:
(a) |
Monopotassium dihydrogen orthophosphate, KH2PO4 |
8,50 g |
Dipotassium monohydrogen orthophosphate, K2HPO4 |
21,75 g |
|
Disodium monohydrogen orthophosphate dihydrate Na2HPO4. 2 H2O |
33,40 g |
|
Ammonium chloride, NH4Cl |
0,50 g |
|
Dissolve in water and make up to 1 litre The pH of the solution should be 7,4. |
|
|
(b) |
Calcium chloride, anhydrous, CaCl2 |
27,50g |
or Calcium chloride dihydrate, CaCl2, 2 H2O |
36,40 g |
|
Dissolve in water and make up to 1 litre |
|
|
(c) |
Magnesium sulphate heptahydrate, MgSO4. 7 H2O |
22,50 g |
Dissolve in water and make up to 1 litre. |
|
|
(d) |
Iron (III) chloride hexahydrate, FeC13. 6H2O |
0,25 g |
Dissolve in water and make up to 1 litre. |
|
I.6.3. Stock solutions of chemicals
I.6.4. Inocula
I.6.4.1. Inoculum from activated sludges
I.6.4.2. Other sources of inoculum
I.6.5. Pre-conditioning of inocula
I.6.6. Abiotic controls
I.6.7. Number of flasks
I.7. DATA AND EVALUATION
I.7.1. Degradation measured by means of DOC determination
I.7.2. Degradation measured by means of specific analysis
1.7.3. Abiotic degradation
I.8. REPORTING
PART II. DOC DIE-AWAY TEST
(Method C.4-A)
II.1. PRINCIPLE OF THE METHOD
II.2. DESCRIPTION OF THE METHOD
II.2.1. Apparatus
II.2.2. Preparation of mineral medium
II.2.3. Preparation and pre-conditioning of inoculum
II.2.4. Preparation of flasks
II.2.5. Number of flasks in typical run
II.2.6. Performance of the test
II.3. DATA AND REPORTING
II.3.1. Treatment of results
II.3.2. Validity of results
II.3.3. Reporting
II.4. DATA SHEET
DOC DIE-AWAY TEST
3. TEST SUBSTANCE
4. INOCULUM
5. CARBON DETERMINATIONS
|
Flask nr |
|
DOC after n-days (mg/l) |
||||
0 |
n1 |
n2 |
n3 |
nx |
|||
Test chemical plus inoculum |
1 |
a1 |
|
|
|
|
|
a2 |
|
|
|
|
|
||
a, mean Ca(t) |
|
|
|
|
|
||
2 |
b1 |
|
|
|
|
|
|
b2 |
|
|
|
|
|
||
b, men Cb(t) |
|
|
|
|
|
||
Blank inoculum without test chemical |
3 |
C1 |
|
|
|
|
|
C2 |
|
|
|
|
|
||
C, mean Cc(t) |
|
|
|
|
|
||
4 |
d1 |
|
|
|
|
|
|
d2 |
|
|
|
|
|
||
d, mean Cd(t) |
|
|
|
|
|
||
[Bild bitte in Originalquelle ansehen] |
|
|
|
|
|
6. EVALUATION OF RAW DATA
Flask nr |
|
% degradation afer n days |
||||
0 |
n1 |
n2 |
n3 |
nx |
||
1 |
[Bild bitte in Originalquelle ansehen] |
0 |
|
|
|
|
2 |
[Bild bitte in Originalquelle ansehen] |
0 |
|
|
|
|
Mean(3) |
[Bild bitte in Originalquelle ansehen] |
0 |
|
|
|
|
7. ABIOTIC CONTROL
(optional)
|
Time (days) |
|
0 |
t |
|
DOC conc. (mg/l) in sterile control |
Cs(o) |
Cs(t) |
8. SPECIFIC CHEMICAL ANALYSIS
(optional)
|
residual amount of test chemical at end of test (mg/l) |
% primary degradation |
Sterile control |
Sb |
|
Inoculated test medium |
Sa |
[Bild bitte in Originalquelle ansehen] |
PART III. MODIFIED OECD SCREENING TEST
(Method C.4-B)
III.1. PRINCIPLE OF THE METHOD
III.2. DESCRIPTION OF THE METHOD
III.2.1.
Apparatus
III.2.2. Preparation of mineral medium
Manganese sulfate tetrahydrate, MnSO4. 4H2O |
39,9 mg |
Boric acid, H3BO3 |
57,2 mg |
Zinc sulfate heptahydrate, ZnSO4. 7H2O |
42,8 mg |
Ammonium heptamolybdate (NH4)6 Mo7O24 |
34,7 mg |
Fe-chelate (FeCl3 ethylenediamine-tetra-acetic acid) |
100,0 mg |
Dissolve in, and make up to 1 000 ml with dilution water |
|
Vitamin solution: |
|
Yeast extract |
15,0 mg |
III.2.3. Preparation and pre-conditioning of inoculum
III.2.4. Preparation of flasks
III.2.5. Number of flasks in typical run
III.2.6. Performance of the test
III.3. DATA AND REPORTING
III.3.1. Treatment of results
III.3.2.
Validity of results
III.3.3.
Reporting
III.4. DATA SHEET
MODIFIED OECD SCREENING TEST
3. TEST SUBSTANCE
4. INOCULUM
5.
CARBON DETERMINATIONS
|
Flask nr |
|
DOC after n-days (mg/l) |
||||
0 |
n1 |
n2 |
n3 |
nx |
|||
Test chemical plus inoculum |
1 |
a1 |
|
|
|
|
|
a2 |
|
|
|
|
|
||
a, mean Ca(t) |
|
|
|
|
|
||
2 |
b1 |
|
|
|
|
|
|
b2 |
|
|
|
|
|
||
b, men Cb(t) |
|
|
|
|
|
||
Blank inoculum without test chemical |
3 |
C1 |
|
|
|
|
|
C2 |
|
|
|
|
|
||
C, mean Cc(t) |
|
|
|
|
|
||
4 |
d1 |
|
|
|
|
|
|
d2 |
|
|
|
|
|
||
d, mean Cd(t) |
|
|
|
|
|
||
[Bild bitte in Originalquelle ansehen] |
|
|
|
|
|
6. EVALUATION OF RAW DATA
Flask nr |
|
% degradation afer n days |
||||
0 |
n1 |
n2 |
n3 |
nx |
||
1 |
[Bild bitte in Originalquelle ansehen] |
0 |
|
|
|
|
2 |
[Bild bitte in Originalquelle ansehen] |
0 |
|
|
|
|
Mean(4) |
[Bild bitte in Originalquelle ansehen] |
0 |
|
|
|
|
7. ABIOTIC CONTROL
(optional)
|
Time (days) |
|
0 |
t |
|
DOC conc. (mg/l) in sterile control |
Cs(o) |
Cs(t) |
8. SPECIFIC CHEMICAL ANALYSIS
(optional)
|
residual amount of test chemical at end of test (mg/l) |
% primary degradation |
Sterile control |
Sb |
|
Inoculated test medium |
Sa |
[Bild bitte in Originalquelle ansehen] |
PART IV.
CO
2
EVOLUTION TEST (Method C.4-C)
IV.1. PRINCIPLE OF THE METHOD
IV.2. DESCRIPTION OF THE METHOD
IV.2.1. Apparatus
IV.2.2. Preparation of mineral medium
IV.2.3. Preparation and pre-conditioning of inoculum
IV.2.4. Preparation of flasks
IV.2.5. Number of flasks in a typical run
IV.2.6. Performance of the test
IV.3. DATA AND REPORTING
IV.3.1. Treatment of results
IV.3.2. Validity of results
IV.3.3. Reporting
IV.4. DATA SHEET
CARBON DIOXIDE EVOLUTION TEST
3. TEST SUBSTANCE
4. INOCULUM
Time (day) |
CO2 formed Test (mg) |
CO2 formed blank (mg) |
CO2 formed cumulative (mg) (test minus blank mean) |
ThCO2 cumulative [Bild bitte in Originalquelle ansehen] |
|||||
1 2 |
mean |
3 4 |
mean |
1 |
2 |
1 |
2 |
mean |
|
0 |
|
|
|
|
|
|
|
|
|
n1 |
|
|
|
|
|
|
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|
n2 |
|
|
|
|
|
|
|
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|
n3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
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|
28 |
|
|
|
|
|
|
|
|
|
6. CARBON ANALYSIS
(optional)
Time (day) |
Blank mg/l |
Test chemical mg/l |
0 |
Cb(o) |
Co |
28(5) |
Cb(t) |
Ct |
7. ABIOTIC DEGRADATION
(optional)
PART V. MANOMETRIC RESPIROMETRY TEST
(Method C.4-D)
V.1. PRINCIPLE OF THE METHOD
V.2. DESCRIPTION OF THE METHOD
V.2.1. Apparatus
V.2.2. Preparation of mineral medium
V.2.3. Preparation and pre-conditioning of inoculum
V.2.4. Preparation of flasks
V.2.5. Number of flasks in a typical run
V.2.6. Performance of the test
V.3. DATA AND REPORTING
V.3.1. Treatment of results
V.3.2. Validity of results
V.3.3. Reporting
V.4.
DATA SHEET
MANOMETRIC RESPIROMETRY TEST
3. TEST SUBSTANCE
4. INOCULUM
5. OXYGEN UPTAKE: BIODEGRADABILITY
|
Time (Days) |
||||||||||||
0 |
|
7 |
|
14 |
|
|
21 |
|
|
28 |
|
||
O2 upt. (mg) test chemical |
1 |
|
|
|
|
|
|
|
|
|
|
|
|
2 |
|
|
|
|
|
|
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|
|
|
|
|
a, mean |
|
|
|
|
|
|
|
|
|
|
|
|
|
O2 upt. (mg) blank |
3 |
|
|
|
|
|
|
|
|
|
|
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|
4 |
|
|
|
|
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|
|
|
|
|
b, mean |
|
|
|
|
|
|
|
|
|
|
|
|
|
Corrected BOD (mg) |
(a1 - bm) |
|
|
|
|
|
|
|
|
|
|
|
|
(a2 - bm) |
|
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|
|
BOD per mg test chemical |
[Bild bitte in Originalquelle ansehen] |
|
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|
[Bild bitte in Originalquelle ansehen] |
|
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|
|
% degradation [Bild bitte in Originalquelle ansehen] |
D1 (a1) |
|
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|
|
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|
|
|
|
|
D2 (a2) |
|
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|
Mean(6) |
|
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|
|
V = volume of medium in test flask |
6. CORRECTION FOR NITRIFICATION
(see Annex V)
Day |
0 |
28 |
Difference |
||
|
|
|
(N) |
||
|
— |
— |
|
||
|
|
|
(N) |
||
|
— |
— |
|
||
|
— |
— |
|
7. CARBON ANALYSIS
(optional)
Time (day) |
Blank mg/litre |
Test chemical mg/litre |
0 |
(Cblo) |
(Co) |
28(7) |
(Cblt) |
(Ct) |
8. SPECIFIC CHEMICAL
(optional)
9. ABIOTIC DEGRADATION
(optional)
PART VI. CLOSED BOTTLE TEST
(Method C.4-E)
VI.1 PRINCIPLE OF THE TEST METHOD
VI.2 DESCRIPTION OF THE METHOD
VI.2.1. Apparatus
VI.2.2. Preparation of mineral medium
VI.2.3. Preparation of the inoculum
VI.2.4. Preparation of flasks
VI.2.5. Number of flasks in a typical run
VI.2.6.
Performance of the test
VI.3. DATA AND REPORTING
VI.3.1. Treatment of results
VI.3.2. Validity of results
VI.3.3. Reporting
VI.4. DATA SHEET
CLOSED BOTTLE TEST
3. TEST SUBSTANCE
4. INOCULUM
5. DO DETERMINATION
Time of incubation (d) |
DO (mg/l) |
|||||
0 |
n1 |
n2 |
|
|||
Blank (without chemical) |
1 |
C1 |
|
|
|
|
2 |
C2 |
|
|
|
|
|
Mean |
[Bild bitte in Originalquelle ansehen] |
|
|
|
|
|
Test chemical |
1 |
a1 |
|
|
|
|
|
2 |
a2 |
|
|
|
|
Mean |
[Bild bitte in Originalquelle ansehen] |
|
|
|
|
6. CORRECTION FOR NITRIFICATION
(see Annex V)
Time of incubation (d) |
0 |
n1 |
n2 |
n3 |
||
|
|
|
|
|
||
|
— |
|
|
|
||
|
— |
|
|
|
||
|
|
|
|
|
||
|
— |
|
|
|
||
|
— |
|
|
|
||
|
— |
|
|
|
7. DO DEPLETION: % DEGRADATION
|
Depletion after n days (mg/litre) |
|||
n1 |
n2 |
n3 |
|
|
FLASK 1: (mto - mtx) - (mbo - mbx) |
|
|
|
|
FLASK 2: (mto - mtx) - (mbo - mbx) |
|
|
|
|
FLASK 1: [Bild bitte in Originalquelle ansehen] |
|
|
|
|
FLASK 2: [Bild bitte in Originalquelle ansehen] |
|
|
|
|
% D mean(8) = [Bild bitte in Originalquelle ansehen] |
|
|
|
|
8. BLANK DO DEPLETIONS
PART VII. M.I.T.I. TEST
(Method C.4-F)
VII.1. PRINCIPLE OF THE METHOD
VII.2. DESCRIPTION OF THE METHOD
VII.2.1. Apparatus
VII.2.2. Preparation of mineral medium
(a) |
Monopotassium dihydrogen ortho phosphate, KH2PO4 |
8,50 g |
Dipotassium monohydrogen ortho phosphate, K2HPO4 |
21,75 g |
|
Disodium monohydrogen ortho phosphate dodecahydrate Na2HPO4 12 H2O |
44,60 g |
|
Ammonium chloride, NH4Cl |
1,70 g |
|
Dissolve in water and make up to 1 litre |
|
|
The pH value of the solution should be 7,2 |
|
|
(b) |
Magnesium sulphate heptahydrate, MgSO4 7 H2O |
22,50 g |
Dissolve in water and make up to 1 litre |
|
|
(c) |
Calcium chloride anhydrous, CaCl2 |
27,50 g |
Dissolve in water and make up to 1 litre |
|
|
(d) |
Iron (III) chloride hexahydrate, FeCl3 6 H2O |
0,25 g |
Dissolve in water and make up to 1 litre |
|
VII.2.3. Preparation of inoculum
VII.2.4. Preparation of flasks
VII.2.5. Performance of the test
VII.3. DATA AND REPORTING
VII.3.1. Treatment of results
VII.3.2. Validity of results
VII.3.3. Reporting
VII.4. DATA SHEET
MITI (I) TEST
3. TEST SUBSTANCE
4. INOCULUM
|
|
||||
|
|
||||
|
|
||||
|
|
||||
|
|
5. OXYGEN UPTAKE: BIODEGRADABILITY
|
Time (Days) |
||||||
0 |
7 |
14 |
21 |
28 |
|||
O2 upt. (mg) test chemical |
a1 |
|
|
|
|
|
|
a2 |
|
|
|
|
|
||
a3 |
|
|
|
|
|
||
O2 upt. (mg) blank |
b |
|
|
|
|
|
|
Corrected BOD (mg) |
(a1 - b1) (a1 - b1) (a1 - b1) |
|
|
|
|
|
|
BOD per mg test chemical |
[Bild bitte in Originalquelle ansehen] |
Flask 1 |
|
|
|
|
|
Flask 2 |
|
|
|
|
|
||
Flask 3 |
|
|
|
|
|
||
% degradation [Bild bitte in Originalquelle ansehen] |
|
1 |
|
|
|
|
|
2 |
|
|
|
|
|
||
3 |
|
|
|
|
|
||
Mean(9) |
|
|
|
|
|
6. CARBON ANALYSIS
(optional)
Flask |
DOC |
% DOC removed |
Mean |
|||
Measured |
Corrected |
|||||
Water + test substance |
a |
|
|
|
— |
— |
Sludge + test substance |
b1 |
|
b1 - c |
|
|
|
Sludge + test substance |
b2 |
|
b2 - c |
|
|
|
Sludge + test substance |
b3 |
|
b3 - c |
|
|
|
Control blank |
c |
|
— |
|
— |
— |
7. SPECIFIC CHEMICAL ANALYTICAL DATA
|
Residual amount of test chemical at end of test |
% degradation |
blank test with water |
Sb |
|
inoculated medium |
Sa1 |
|
Sa2 |
|
|
Sa3 |
|
8. REMARKS
Appendix 1
ABBREVIATIONS AND DEFINITIONS
Primary biodegradation:
Ultimate biodegradation (aerobic):
Readily biodegradable:
Inherently biodegradable:
Treatability:
Lag time
Degradation time
10-day window
Appendix 2
CALCULATION AND DETERMINATION OF SUITABLE SUMMARY PARAMETERS
1. Carbon content
2. Theoretical oxygen demand (ThOD)
3. Chemical Oxygen Demand (COD)
4. Dissolved organic carbon (DOC)
Remarks:
BIBLIOGRAPHY
Appendix 3
EVALUATION OF THE BIODEGRADABILITY OF POORLY SOLUBLE SUBSTANCES
BIBLIOGRAPHY
Appendix 4
EVALUATION OF THE BIODEGRADABILITY OF CHEMICALS SUSPECTED TO BE TOXIC TO THE INOCULUM
BIBLIOGRAPHY
Appendix 5
CORRECTION FOR OXYGEN UPTAKE FOR INTERFERENCE BY NITRIFICATION
2 NH4Cl + 3 O2 = 2 HNO2 + 2 HCl + 2 H2O |
(1) |
2 HNO2 + O2 = 2 HNO3 |
(2) |
Overall: |
|
2 NH4Cl + 4 O2 = 2 HNO3 + 2 HCl + 2 H2O |
(3) |
O2 consumed in nitrate formation = 4,57 × increase in nitrate concentration |
(4) |
and |
|
O2 consumed in nitrite formation = 3,43 × increase in nitrite concentration |
(5) |
and |
|
O2 lost in nitrite disappearance = -3,43 × decrease in nitrate concentration |
(6) |
So that |
|
O2 uptake due to nitrification = ±3,43 × change in nitrite conc. +4,57 × increase in nitrate conc. |
(7) |
and thus |
|
O2 uptake due to C oxidation = total observed uptake uptake due to nitrification |
(8). |
C.5 DEGRADATION — BIOCHEMICAL OXYGEN DEMAND
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
2. DATA AND EVALUATION
3. REPORTING
4.
REFERENCES
C.6. DEGRADATION — CHEMICAL OXYGEN DEMAND
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
2. DATA AND EVALUATION
3. REPORTING
4. REFERENCES
C.7. DEGRADATION — ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF PH
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. APPLICABILITY OF THE METHOD
1.4. PRINCIPLE OF THE TEST METHOD
1.5. INFORMATION ON THE TEST SUBSTANCE
1.6. REFERENCE SUBSTANCES
1.7. QUALITY CRITERIA
1.7.1. Recovery
1.7.2. Repeatability and sensitivity of analytical method
1.7.3. Confidence intervals for hydrolysis kinetic data
1.8. DESCRIPTION OF THE TEST METHOD
1.8.1. Equipment and apparatus
1.8.2. Test substance application
1.8.3. Buffer solutions
1.8.4. Test conditions
1.8.4.1. Test temperature
1.8.4.2. Light and oxygen
1.8.4.3. Test duration
1.8.5. Performance of the test
1.8.5.1. Preliminary test (Tier 1)
1.8.5.2. Hydrolysis of unstable substances (Tier 2)
1.8.5.3. Identification of hydrolysis products (Tier 3)
1.8.5.4. Optional tests
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
Tiered hydrolysis test scheme
Appendix 2
Definitions and units
RX + HOH → ROH + HX |
[1] |
rate = k [H2O] [RX] |
second order reaction |
or |
|
rate = k [RX] |
first order reaction |
kobs = k [H2O] |
[2] |
[Bild bitte in Originalquelle ansehen] ln[Bild bitte in Originalquelle ansehen] |
[3] |
[Bild bitte in Originalquelle ansehen] |
[4] |
Appendix 3
Buffer Systems
A. CLARK AND LUBS:
Buffer mixtures of CLARK and LUBS
(14)
Composition |
pH |
0,2 N HCl and 0,2 N KCl at 20 oC |
|
47,5 ml. HCl + 25 ml. KCl dil. to 100 ml |
1,0 |
32,25 ml. HCl + 25 ml. KCl dil. to 100 ml |
1,2 |
20,75 ml. HCl + 25 ml. KCl dil. to 100 ml |
1,4 |
13,15 ml. HCl + 25 ml. KCl dil. to 100 ml |
1,6 |
8,3 ml. HCl + 25 ml. KCl dil. to 100 ml |
1,8 |
5,3 ml. HCl + 25 ml. KCl dil. to 100 ml |
2,0 |
3,35 ml. HCl + 25 ml. KCl dil. to 100 ml |
2,2 |
0,1 M potassium biphthalate +0,1 N HCl at 20 oC |
|
46,70 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
2,2 |
39,60 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
2,4 |
32,95 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
2,6 |
26,42 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
2,8 |
20,32 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
3,0 |
14,70 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
3,2 |
9,90 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
3,4 |
5,97 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
3,6 |
2,63 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml |
3,8 |
0,1 M potassium biphthalate +0,1 N NaOH at 20 oC |
|
0,40 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
4,0 |
3,70 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
4,2 |
7,50 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
4,4 |
12,15 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
4,6 |
17,70 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
4,8 |
23,85 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
5,0 |
29,95 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
5,2 |
35,45 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
5,4 |
39,85 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
5,6 |
43,00 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
5,8 |
45,45 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml |
6,0 |
Buffer mixtures of CLARK and LUBS (Continued)
0,1 M monopotassium phosphate +0,1 N NaOH at 20 oC |
|
5,70 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
6,0 |
8,60 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
6,2 |
12,60 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
6,4 |
17,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
6,6 |
23,45 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
6,8 |
29,63 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
7,0 |
35,00 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
7,2 |
39,50 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
7,4 |
42,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
7,6 |
45,20 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
7,8 |
46,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml |
8,0 |
0,1 M H3B03 in 0,1 M KCl +0,1 N NaOH at 20 oC |
|
2,61 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
7,8 |
3,97 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
8,0 |
5,90 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
8,2 |
8,50 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
8,4 |
12,00 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
8,6 |
16,30 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
8,8 |
21,30 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
9,0 |
26,70 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
9,2 |
32,00 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
9,4 |
36,85 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
9,6 |
40,80 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
9,8 |
43,90 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml |
10,0 |
B. KOLTHOFF AND VLEESCHHOUWER:
Citrate buffers of KOLTHOFF and VLEESCHHOUWER
Composition |
pH |
0,1 M monopotassium citrate and 0,1 N HCl at 18 oC (15) |
|
49,7 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
2,2 |
43,4 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
2,4 |
36,8 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
2,6 |
30,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
2,8 |
23,6 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
3,0 |
17,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
3,2 |
10,7 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
3,4 |
4,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml |
3,6 |
0,1 M monopotassium citrate and 0,1 N NaOH at 18 oC (15) |
|
2,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
3,8 |
9,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
4,0 |
16,3 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
4,2 |
23,7 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
4,4 |
31,5 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
4,6 |
39,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
4,8 |
46,7 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
5,0 |
54,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
5,2 |
61,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
5,4 |
68,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
5,6 |
74,4 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
5,8 |
81,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml |
6,0 |
C. SÖRENSEN:
Borate mixtures of SÖRENSEN
Composition |
Sörensen 18 oC |
Walbum, pH at |
|||
ml. Borax |
ml. HCl/NaOH |
10 oC |
40 oC |
70 oC |
|
0,05 M borax +0,1 N HCl |
|||||
5,25 |
4,75 |
7,62 |
7,64 |
7,55 |
7,47 |
5,50 |
4,50 |
7,94 |
7,98 |
7,86 |
7,76 |
5,75 |
4,25 |
8,14 |
8,17 |
8,06 |
7,95 |
6,00 |
4,00 |
8,29 |
8,32 |
8,19 |
8,08 |
6,50 |
3,50 |
8,51 |
8,54 |
8,40 |
8,28 |
7,00 |
3,00 |
8,08 |
8,72 |
8,56 |
8,40 |
7,50 |
2,50 |
8,80 |
8,84 |
8,67 |
8,50 |
8,00 |
2,00 |
8,91 |
8,96 |
8,77 |
8,59 |
8,50 |
1,50 |
9,01 |
9,06 |
8,86 |
8,67 |
9,00 |
1,00 |
9,09 |
9,14 |
8,94 |
8,74 |
9,50 |
0,50 |
9,17 |
9,22 |
9,01 |
8,80 |
10,00 |
0,00 |
9,24 |
9,30 |
9,08 |
8,86 |
0,05 M borax +0,1 N NaOH |
|||||
10,0 |
0,0 |
9,24 |
9,30 |
9,08 |
8,86 |
9,0 |
1,0 |
9,36 |
9,42 |
9,18 |
8,94 |
8,0 |
2,0 |
9,50 |
9,57 |
9,30 |
9,02 |
7,0 |
3,0 |
9,68 |
9,76 |
9,44 |
9,12 |
6,0 |
4,0 |
9,97 |
10,06 |
9,67 |
9,28 |
Phosphate mixtures of SÖRENSEN
Composition |
pH |
0,0667 M Monopotassium phosphate + 0,0667 M Disodium phosphate at 20 oC |
|
99,2 ml. KH2PO4 + 0,8 ml Na2HPO4 |
5,0 |
98,4 ml. KH2PO4 + 1,6 ml Na2HPO4 |
5,2 |
97,3 ml. KH2PO4 + 2,7 ml Na2HPO4 |
5,4 |
95,5 ml. KH2PO4 + 4,5 ml Na2HPO4 |
5,6 |
92,8 ml. KH2PO4 + 7,2 ml Na2HPO4 |
5,8 |
88,9 ml. KH2PO4 + 11,1 ml Na2HPO4 |
6,0 |
83,0 ml. KH2PO4 + 17,0 ml Na2HPO4 |
6,2 |
75,4 ml. KH2PO4 + 24,6 ml Na2HPO4 |
6,4 |
65,3 ml. KH2PO4 + 34,7 ml Na2HPO4 |
6,6 |
53,4 ml. KH2PO4 + 46,6 ml Na2HPO4 |
6,8 |
41,3 ml. KH2PO4 + 58,7 ml Na2HPO4 |
7,0 |
29,6 ml. KH2PO4 + 70,4 ml Na2HPO4 |
7,2 |
19,7 ml. KH2PO4 + 80,3 ml Na2HPO4 |
7,4 |
12,8 ml. KH2PO4 + 87,2 ml Na2HPO4 |
7,6 |
7,4 ml. KH2PO4 + 92,6 ml Na2HPO4 |
7,8 |
3,7 ml. KH2PO4 + 96,3 ml Na2HPO4 |
8,0 |
C.8. TOXICITY FOR EARTHWORMS
ARTIFICIAL SOIL TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNIT
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE TEST
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Materials
1.6.1.1.
Test substrate
1.6.1.2.
Test containers
1.6.2.
Test conditions
1.6.3.
Test procedure
Test concentrations
Range finding test
Definitive test
Mixture of the basic test substrate and the test substance
1.6.4.
Test organisms
2. DATA
2.1. TREATMENT AND EVALUATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix
Breeding and keeping of the worms before testing
Keeping and breeding conditions
C.9. BIODEGRADATION
ZAHN-WELLENS TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST PROCEDURE
1.6.1.
Preparations
1.6.1.1.
Reagents
Sulphuric acid, analytical reagent (A.R.): |
50 g/l |
Sodium hydroxide solution A.R.: |
40 g/l |
Mineral nutrient solution: dissolve in one litre deionised water: |
|
ammonium chloride, NH4Cl, A.R.: |
38,5 g |
sodium dihydrogenphosphate, NaH2PO4.2H2O, A.R.: |
33,4 g |
potassium dihydrogenphosphate, KH2PO4, A.R.: |
8,5 g |
di-potassium mono-hydrogenphosphate, K2HPO4, A.R.: |
21,75 g |
1.6.1.2.
Apparatus
1.6.1.3.
Preparation of the inoculum
1.6.1.4.
Preparation of the test solutions
1.6.2.
Performance of the test
Functional control of activated sludge
Adaptation
Analytical means
2. DATA AND EVALUATION
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
Appendix
EVALUATION EXAMPLE
Organic compound: |
4-Ethoxybenzoic acid |
Theoretical test concentration: |
600 mg/1 |
Theoretical DOC: |
390 mg/l |
Inoculum |
Sewage treatment plant of… |
Concentration: |
1 gram dry material/litre |
Adaptation status: |
not adapted |
Analysis: |
DOC-determination |
Amount of sample: |
3 ml |
Control substance: |
Diethyleneglycol |
Toxicity of compound: |
No toxic effects below 1 000 mg/l Test used: fermentation tubes test |
Test time |
Control substance |
Test substance |
|||||
Blank DOC(16) mg/l |
DOC(16) mg/l |
DOC net mg/l |
Degradation % |
DOC(16) mg/l |
DOC net mg/l |
Degradation % |
|
0 |
— |
— |
300,0 |
— |
— |
390,0 |
— |
3 hours |
4,0 |
298,0 |
294,0 |
2 |
371,6 |
367,6 |
6 |
1 day |
6,1 |
288,3 |
282,2 |
6 |
373,3 |
367,2 |
6 |
2 days |
5,0 |
281,2 |
276,2 |
8 |
360,0 |
355,0 |
9 |
5 days |
6,3 |
270,5 |
264,2 |
12 |
193,8 |
187,5 |
52 |
6 days |
7,4 |
253,3 |
245,9 |
18 |
143,9 |
136,5 |
65 |
7 days |
11,3 |
212,5 |
201,2 |
33 |
104,5 |
93,2 |
76 |
8 days |
7,8 |
142,5 |
134,7 |
55 |
58,9 |
51,1 |
87 |
9 days |
7,0 |
35,0 |
28,0 |
91 |
18,1 |
11,1 |
97 |
10 days |
18,0 |
37,0 |
19,0 |
94 |
20,0 |
2,0 |
99 |
Figure 1
Examples of biodegradation curves
Figure 2
Examples of sludge adaptation
C.10. BIODEGRADATION
ACTIVATED SLUDGE SIMULATION TESTS
1. METHOD
1.1. INTRODUCTION
1.1.1.
General remarks
1.1.2.
Determination of ultimate biodegradability (DOC/COD analysis)
1.1.3.
Determination of primary biodegradability (specific analysis)
1.2. DEFINITIONS AND UNITS
1.2.1.
DOC/COD analysis
[Bild bitte in Originalquelle ansehen] |
[1(a)] |
1.2.2.
Specific analysis
[Bild bitte in Originalquelle ansehen] |
[1(b)] |
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHODS
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparation
1.6.1.1.
Apparatus
1.6.1.2.
Filtration
1.6.1.3.
Sewage
Peptone: |
160 mg, |
Meat extract: |
10 mg, |
Urea: |
30 mg, |
NaCl: |
7 mg, |
CaCl2 .2H2O: |
4 mg, |
MgSO4 .7H2O: |
2 mg, |
K2HPO4: |
28 mg. |
1.6.1.4.
Stock solution of test material
1.6.1.5.
Inoculum
1.6.2.
Procedure
1.6.2.1.
Running-in period: Sludge formation/stabilisation of the units
1.6.2.2.
Test procedure
1.6.2.3.
Analysis
2. DATA AND EVALUATION
2.1. COUPLED UNITS MODE
[Bild bitte in Originalquelle ansehen] |
[2] |
[Bild bitte in Originalquelle ansehen] |
[3] |
[Bild bitte in Originalquelle ansehen] |
[4] |
[Bild bitte in Originalquelle ansehen] |
[5] |
2.2. NON-COUPLED UNITS MODE
2.2.1.
Using COD/DOC determinations
[Bild bitte in Originalquelle ansehen] |
[6] |
[Bild bitte in Originalquelle ansehen] |
[7] |
2.2.2.
Using specific analysis
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
Appendix 1
Figure 1
Figure 2
Appendix 2
Figure 1
Equipment used for assessing biodegradability
Figure 2
Details of three-litre porous-pot aeration vessel
Appendix 3
Operating conditions for the Activated Sludge Simulation Test
Check in each group
Apparatus
OECD confirmatory |
|
Porous pot |
|
Mode of operation
Single unit |
|
Coupled units |
|
Non-coupled units |
|
Transinoculation
None |
|
Activated sludge |
|
Supernatant |
|
Mean retention time
Three hours |
|
Six hours |
|
Base nutrient
Domestic sewage |
|
Synthetic sewage |
|
Inoculum
Secondary effluent |
|
Composite |
|
Activated sludge |
|
Test material addition
Form the start |
|
Stepwise increase |
|
After sludge has formed |
|
Analysis
Specific |
|
COD |
|
DOC |
|
C.11. BIODEGRADATION
ACTIVATED SLUDGE RESPIRATION INHIBITION TEST
1. METHOD
1.1. INTRODUCTION
Recommendation
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Reagents
1.6.1.1.
Solutions of the test substance
1.6.1.2.
Solution of control substance
1.6.1.3.
Synthetic sewage
1.6.2.
Apparatus
1.6.3.
Preparation of the inoculum
1.6.4.
Performance of the test
Duration/contact time: |
30 minutes and/or three hours, during which aeration takes place |
Water: |
Drinking water (dechlorinated if necessary) |
Air supply: |
Clean, oil-free air. Air flow 0,5 to 1 litre/minute |
Measuring apparatus: |
Flat bottom flask such as a BOD-flask |
Oxygen meter: |
Suitable oxygen electrode, with a recorder |
Nutrient solution: |
Synthetic sewage (see above) |
Test substance: |
The test solution is freshly prepared at the start of the test |
Reference substance: |
e.g. 3,5-dichlorophenol (at least three concentrations) |
Controls: |
Inoculated sample without test substance |
Temperature: |
20 ± 2 oC |
2. DATA AND EVALUATION
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF DATA
4. REFERENCES
C.12. BIODEGRADATION
MODIFIED SCAS TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
[Bild bitte in Originalquelle ansehen] |
[1] |
[Bild bitte in Originalquelle ansehen] |
[2 (a)] |
[Bild bitte in Originalquelle ansehen] |
[2 (b)] |
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST PROCEDURE
1.6.1.
Preparations
1.6.2.
Test conditions
1.6.3.
Performance of the test
2. DATA AND EVALUATION
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
Appendix 1
SCAS test: example of results
Substance |
CT (mg/l) |
Ct - Cc (mg/l) |
Percentage biodegradition, Dda |
Test duration (days) |
4-acetyl aminobenzene sulphonate |
17,2 |
2,0 |
85 |
40 |
Tetra propylene benzene sulphonate |
17,3 |
8,4 |
51,4 |
40 |
4-nitrophenol |
16,9 |
0,8 |
95,3 |
40 |
Diethylene glycol |
16,5 |
0,2 |
98,8 |
40 |
Aniline |
16,9 |
1,7 |
95,9 |
40 |
Cyclopentane tetra carboxylate |
17,9 |
3,2 |
81,1 |
120 |
Appendix 2
Example of test apparatus
Figure 1
C.13 BIOCONCENTRATION: FLOW-THROUGH FISH TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. REFERENCE COMPOUNDS
1.7. DESCRIPTION OF THE TEST METHOD
1.7.1.
Apparatus
1.7.2.
Water
1.7.3.
Test Solutions
1.7.4.
Selection of species
1.7.5.
Holding of fish
1.8. PERFORMANCE OF THE TEST
1.8.1.
Preliminary test
1.8.2.
Conditions of exposure
1.8.2.1.
Duration of uptake phase
1.8.2.2.
Duration of the depuration phase
1.8.2.3.
Numbers of test fish
1.8.2.4.
Loading
1.8.2.5.
Feeding
1.8.2.6.
Light and temperature
1.8.2.7.
Test concentrations
1.8.2.8.
Controls
1.8.3.
Frequency of water quality measurements
1.8.4.
Sampling and analysis of fish and water
1.8.4.1.
Fish and water sampling schedule
1.8.4.2.
Sampling and Sample Preparation
1.8.4.3.
Quality of Analytical method
1.8.4.4.
Analysis of Fish Sample
2. DATA
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST SUBSTANCE:
3.2. TEST SPECIES
3.3. TEST CONDITIONS:
3.4. RESULTS:
4. REFERENCES
Appendix 1
Chemical characteristics of an acceptable dilution water
|
Substance |
Limit concentration |
1 |
Particulate Matter |
5 mg/l |
2 |
Total Organic Carbon |
2 mg/l |
3 |
Un-ionised ammonia |
1 μg/l |
4 |
Residual chlorine |
10 μg/l |
5 |
Total organophosphorous pesticides |
50 ng/l |
6 |
Total organochlorine pesticides plus polychlorinated biphenyls |
50 ng/l |
7 |
Total organic chlorine |
25 ng/l |
8 |
Aluminium |
1μg/l |
9 |
Arsenic |
1μg/l |
10 |
Chromium |
1μg/l |
11 |
Cobalt |
1μg/l |
12 |
Copper |
1μg/l |
13 |
Iron |
1μg/l |
14 |
Lead |
1μg/l |
15 |
Nickel |
1μg/l |
16 |
Zinc |
1μg/l |
17 |
Cadmium |
100 ng/l |
18 |
Mercury |
100 ng/l |
19 |
Silver |
100 ng/l |
Appendix 2
Fish species recommended for testing
|
Recommended species |
Recommended range of test temperature (oC) |
Recommended total length of test animal (cm) |
1 |
Danio rerio (17) (Teleostei, Cyprinidae) (Hamilton-Buchanan) Zebra — fish |
20-25 |
3,0±0,5 |
2 |
Pimephales promelas (Teleostei, Cyprinidae) (Rafinesque) Fathead minnow |
20-25 |
5,0±2,0 |
3 |
Cyprinus carpio (Teleostei, Cyprinidae) (Linnaeus) Common Carp |
20-25 |
5,0±3,0 |
4 |
Oryzias latipes (Teleostei, Poeciliidae) (Temminck and Schlegel) Ricefish |
20-25 |
4,0±1,0 |
5 |
Poecilia reticulata (Teleostei, Poeciliidae) (Peters) Guppy |
20-25 |
3,0±1,0 |
6 |
Lepomis macrochirus (Teleostei, Centrarchidae) (Rafinesque) Bluegill |
20-25 |
5,0±2,0 |
7 |
Oncorhynchus mykiss (Teleostei, Salmonidae) (Walbaum) Rainbow trout |
13-17 |
8,0±4,0 |
8 |
Gasterosteus aculeatus (Teleostei, Gasterosteidae) (Linnaeus) Three-spined stickleback |
18-20 |
3,0±1,0 |
Spot |
Leiostomus xanthurus |
Sheepshead minnow |
Cyprinodon variegatus |
Silverside |
Menidia beryllina |
Shiner perch |
Cymatogaster aggregata |
English sole |
Parophrys vetulus |
Staghorn sculpin |
Leptocottus armatus |
Three-spined stikleback |
Gasterosteus aculeatus |
Sea bass |
Dicentracus labrax |
Bleak |
Alburnus alburnus |
Collection
Appendix 3
Prediction of the duration of the uptake and depuration phases
1. Prediction of the duration of the uptake phase
log10k2 = 0,414 log10(Pow) + 1,47 (r2 = 0,95) |
[equation 1] |
log10(Pow) = 0,862 log10(s) + 0,710 (r2 = 0,994) |
[equation 2] |
[Bild bitte in Originalquelle ansehen] |
[equation 3] |
[Bild bitte in Originalquelle ansehen] or[Bild bitte in Originalquelle ansehen] |
[equation 4] |
[Bild bitte in Originalquelle ansehen] or[Bild bitte in Originalquelle ansehen] |
[equation 5] |
[Bild bitte in Originalquelle ansehen] |
[equation 6] |
log10k2 = -0,414.(4) + 1,47 |
k2 = 0,652 days-1 |
2. Prediction of the duration of the depuration phase
Literature
(of Appendix 3)
Appendix 4
Theoretical example of sampling schedule for bioconcentration tests of substances with log P
ow
= 4
Fish sampling |
Sample time schedule |
No of water samples |
No of fish per sample |
||||
Minimal required frequency (days) |
Additional sampling |
||||||
Uptake phase |
-1 0 |
|
2(18) 2 |
add 45-80 fish |
|||
1st |
0,3 |
0,4 |
2 (2) |
4 (4) |
|||
2nd |
0,6 |
0,9 |
2 (2) |
4 (4) |
|||
3rd |
1,2 |
1,7 |
2 (2) |
4 (4) |
|||
4th |
2,4 |
3,3 |
2 (2) |
4 (4) |
|||
5th |
4,7 |
|
2 |
6 |
|||
Depuration phase |
|
|
|
Transfer fish to water free of test chemical |
|||
6th |
5,0 |
5,3 |
|
4 (4) |
|||
7th |
5,9 |
7,0 |
|
4 (4) |
|||
8th |
9,3 |
11,2 |
|
4 (4) |
|||
9th |
14,0 |
17,5 |
|
6 (4) |
|||
Values in brackets are numbers of samples (water, fish) to be taken if additional sampling is carried out.
|
Appendix 5
Model discrimination
Graphical method for determination of depuration (loss) rate constant k
2
Graphical method for determination of uptake rate constant k
1
[Bild bitte in Originalquelle ansehen] |
[equation 1] |
Computer method for calculation of uptake and depuration (loss) rate constants
[Bild bitte in Originalquelle ansehen] 0 < t < tc |
[equation 2] |
[Bild bitte in Originalquelle ansehen] t > tc |
[equation 3] |
C.14. FISH JUVENILE GROWTH TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Water
1.6.3. Test solutions
1.6.4. Selection of species
1.6.5. Holding of fish
1.7. TEST DESIGN
1.7.1. Design for analysis by regression
1.7.2. Design for estimation of a NOEC/LOEC using Analysis of Variance (ANOVA)
1.8. PROCEDURE
1.8.1. Selection and weighing of test fish
1.8.2. Conditions of exposure
1.8.2.1. Duration
1.8.2.2. Loading rates and stocking densities
1.8.2.3. Feeding
1.8.2.4. Light and temperature
1.8.3. Test concentrations
1.8.4. Controls
1.8.5. Frequency of analytical determinations and measurements
1.8.6. Observations
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
2.1.1. Analysis of results by regression (concentration-response modelling)
2.1.2. Analysis of results for the estimation of the LOEC
2.2. INTERPRETATION OF RESULTS
2.3. TEST REPORT
2.3.1. Test substance:
2.3.2. Test species:
2.3.3. Test conditions:
2.3.4. Results:
3.
REFERENCES
Appendix 1
FISH SPECIES RECOMMENDED FOR TESTING AND SUITABLE TEST CONDITIONS
Species |
Recommended test temperature range (oC) |
Photoperiod (hours) |
Recommended range for initial fish weight (g) |
Required measurement precision |
Loading rate (g/l) |
Stocking density (per litre) |
Food |
Test duration (days) |
Recommended species: |
|
|
|
|
|
|
|
|
Oncorhynchus mykiss rainbow trout |
12,5-16,0 |
12-16 |
1-5 |
to nearest 100 mg |
1,2-2,0 |
4 |
Dry propietary salmonid fry food |
≥ 28 |
Other well documented species: |
|
|
|
|
|
|
|
|
Danio rerio zebrafish |
21-25 |
12-16 |
0,050-0,100 |
to nearest 1 mg |
0,2-1,0 |
5-10 |
Live food (Brachionus Artemia) |
≥ 28 |
Oryzias latipes ricefish (Medaka) |
21-25 |
12-16 |
0,050-0,100 |
to nearest 1 mg |
0,2-1,0 |
5-20 |
Live food (Brachionus Artemia) |
≥ 28 |
Appendix 2
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
Substance |
Concentrations |
Particulate matter |
< 20 mg/l |
Total organic carbon |
< 2 mg/l |
Unionised ammonia |
< 1 μg/l |
Residual chlorine |
< 10 μg/l |
Total organophosphorus pesticides |
< 50 ng/l |
Total organochlorine pesticides plus polychlorinated biphenyls |
< 50 ng/l |
Total organic chlorine |
< 25 ng/l |
Appendix 3
Logarithmic series of concentrations suitable for toxicity test (9)
Column (Number of concentrations between 100 and 10, or between 10 and 1)(20) |
||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
32 |
46 |
56 |
63 |
68 |
72 |
75 |
10 |
22 |
32 |
40 |
46 |
52 |
56 |
3,2 |
10 |
18 |
25 |
32 |
37 |
42 |
1,0 |
4,6 |
10 |
16 |
22 |
27 |
32 |
|
2,2 |
5,6 |
10 |
15 |
19 |
24 |
|
1,0 |
3,2 |
6,3 |
10 |
14 |
18 |
|
|
1,8 |
4,0 |
6,8 |
10 |
13 |
|
|
1,0 |
2,5 |
4,6 |
7,2 |
10 |
|
|
|
1,6 |
3,2 |
5,2 |
7,5 |
|
|
|
1,0 |
2,2 |
3,7 |
5,6 |
|
|
|
|
1,5 |
2,7 |
4,2 |
|
|
|
|
1,0 |
1,9 |
3,2 |
|
|
|
|
|
1,4 |
2,4 |
|
|
|
|
|
1,0 |
1,8 |
|
|
|
|
|
|
1,3 |
|
|
|
|
|
|
1,0 |
C.15. FISH, SHORT-TERM TOXICITY TEST ON
EMBRYO
AND SAC-FRY STAGES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Test chambers
1.6.2. Selection of fish species
1.6.3. Holding of the brood fish
1.6.4. Handling of embryos and larvae
1.6.5. Water
1.6.6. Test solutions
1.7. PROCEDURE
1.7.1. Conditions of exposure
1.7.1.1. Duration
1.7.1.2. Loading
1.7.1.3. Light and temperature
1.7.2. Test concentrations
1.7.3. Controls
1.7.4. Frequency of analytical determinations and measurements
1.7.5. Observations
1.7.5.1. Stage of embryonic development
1.7.5.2. Hatching and survival
1.7.5.3. Abnormal appearance
1.7.5.4. Abnormal behaviour
1.7.5.5. Length
1.7.5.6. Weight
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
2.3. THE TEST REPORT
2.3.1. Test substance:
2.3.2. Test species:
2.3.3. Test conditions:
2.3.4. Results:
3. REFERENCES
FRESHWATER |
Oncorhynchus mykiss Rainbow trout (9)(16) |
Danio rerio Zebrafish (7)(17)(18) |
Cyprinus caprio Common carp (8)(19) |
Oryzias latipes Japanese ricefish/Medaka (20)(21) |
Pimephales promelas Fathead minnow (8)(22) |
FRESHWATER |
SALTWATER |
Carassius auratus Goldfish (8) |
Menidia peninsulae Tidewater silverside (23)(24)(25) |
Lepomis macrochirus Bluegill (8) |
Clupea harengus Herring (24)(25) |
Gadus morhua Cod (24)(25) |
|
Cyprinodon variegatus Sheepshead minnow (23)(24)(25) |
Appendix 1
GUIDANCE ON PERFORMANCE OF A TOXICITY TEST ON EMBRYOS AND SAC-FRY OF ZEBRAFISH
(BRACHYDANIO RERIO)
INTRODUCTION
CONDITIONS OF PARENTAL FISH, REPRODUCTION AND EARLY-LIFE STAGES
CALCULATIONS AND STATISTICS
LC
50
AND EC
50
DETERMINATIONS
ESTIMATION OF LOEC AND NOEC
REFERENCES
Appendix 2
TEST CONDITIONS, DURATION AND SURVIVAL CRITERIA FOR RECOMMENDED SPECIES
Species |
Temp (0C) |
Salinity (0/00) |
Photo-period (hrs) |
Duration of stages (days) |
Typical duration of test |
Survival of control, (minimum %) |
||
Embryo |
Sac-fry |
Hatching success |
Post-hatch |
|||||
FRESHWATER |
||||||||
Brachydanio rerio Zebrafish |
25 ± 1 |
— |
12-16 |
3-5 |
8-10 |
As soon as possible after fertilisation (early gastrula stage) to 5 days post-hatch (8-10 days) |
80 |
90 |
Oncorhynchus mykiss Rainbow trout |
10 ± 1(22) 12 ± 1(23) |
— |
0(24) |
30-35 |
25-30 |
As soon as possible after fertilisation (early gastrula stage) to 20 days post-hatch (50-55 days) |
66 |
70 |
Cyprinus carpio Common carp |
21-25 |
— |
12-16 |
5 |
> 4 |
As soon as possible after fertilisation (early gastrula stage) to 4 days post-hatch (8-9 days) |
80 |
75 |
Oryzias latipes Japanese ricefish/Medaka |
24 ± 1(22) 23 ± 1(23) |
— |
12-16 |
8-11 |
4-8 |
As soon as possible after fertilisation (early gastrula stage) to 5 days post-hatch (13-16 days) |
80 |
80 |
Pimephales promelas Fathead minnow |
25 ± 2 |
— |
16 |
4-5 |
5 |
As soon as possible after fertilisation (early gastrula stage) to 4 days post-hatch (8-9 days) |
60 |
70 |
Appendix 3
Test conditions, duration and survival criteria for other well documented species
Species |
Temp ( oC) |
Salinity (0/00) |
Photo-period (hrs) |
Duration of stages (days) |
Typical duration of embryo and sac-fry test |
Survival of control (minimum %) |
||
|
|
|
|
Embryo |
Sac-fry test |
Hatching success |
Post-hatch |
|
FRESHWATER |
||||||||
Carassius auratus Goldfish |
24 ± 1 |
— |
— |
3-4 |
> 4 |
As soon as possible after fertilisation (early gastrula stage) to 4 days post-hatch (7 days) |
— |
80 |
Leopomis macrochirus Blugill sunfish |
21 ± 1 |
— |
16 |
3 |
> 4 |
As soon as possible after fertilisation (early gastrula stage) to 4 days post-hatch (7 days) |
— |
75 |
SALTWATER |
||||||||
Menidia peninsulae Tidewater silverside |
22-25 |
15-22 |
12 |
1,5 |
10 |
As soon as possible after fertilisation (early gastrula stage) to 5 days post-hatch (6-7 days) |
80 |
60 |
Clupea harengus Herring |
10 ± 1 |
8-15 |
12 |
20-25 |
3-5 |
As soon as possible after fertilisation (early gastrula stage) to 3 days post-hatch (23-27 days) |
60 |
80 |
Gadus morhua Cod |
5 ± 1 |
5-30 |
12 |
14-16 |
3-5 |
As soon as possible after fertilisation (early gastrula stage) to 3 days post-hatch (18 days) |
60 |
80 |
Cyprinodon variegatus Sheepshead minnow |
25 ± 1 |
15-30 |
12 |
— |
— |
As soon as possible after fertilisation (early gastrula stage) to 4/7 days post-hatch (28 days) |
> 75 |
80 |
Appendix 4
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
Substance |
Concentrations |
Particulate matter |
< 20 mg/l |
Total organic carbon |
< 2 mg/l |
Unionised ammonia |
< 1 μg/l |
Residual chlorine |
< 10 μg/l |
Total organophosphorus pesticides |
< 50 ng/l |
Total organochlorine pesticides plus polychlorinated biphenyls |
< 50 ng/l |
Total organic chlorine |
< 25 ng/l |
C.16. HONEYBEES — ACUTE ORAL TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. VALIDITY OF THE TEST
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Collection of bees
1.5.2. Housing and feeding conditions
1.5.3. Preparation of bees
1.5.4. Preparation of doses
1.6. PROCEDURE
1.6.1. Test and control groups
1.6.2. Toxic standard
1.6.3. Exposure
1.6.3.1. Administration of doses
1.6.3.2. Duration
1.6.4. Observations
1.6.5. Limit test
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
C.17. HONEYBEES — ACUTE CONTACT TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. VALIDITY OF THE TEST
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Collection of bees
1.5.2. Housing and feeding conditions
1.5.3. Preparation of bees
1.5.4. Preparation of doses
1.6. PROCEDURE
1.6.1. Test and control groups
1.6.2. Toxic standard
1.6.3. Exposure
1.6.3.1. Administration of doses
1.6.3.2. Duration
1.6.4. Observations
1.6.5. Limit test
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
C.18. ADSORPTION/DESORPTION USING A BATCH EQUILIBRIUM METHOD
1. METHOD
1.1. INTRODUCTION
1.2. SCOPE
1.3. DEFINITIONS AND UNITS
Symbol |
Definition |
Units |
[Bild bitte in Originalquelle ansehen] |
adsorption percentage at the time ti |
% |
Aeq |
adsorption percentage at adsorption equilibrium |
% |
[Bild bitte in Originalquelle ansehen] |
mass of the test substance adsorbed on the soil at the time ti |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the test substance adsorbed on the soil during the time interval Δti |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the test substance adsorbed on the soil at adsorption equilibrium |
μg |
m0 |
mass of the test substance in the test tube, at the beginning of the adsorption test |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the test substance measured in an aliquot ( [Bild bitte in Originalquelle ansehen] ) at the time point ti |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the substance in the solution at adsorption equilibrium |
μg |
msoil |
quantity of the soil phase, expressed in dry mass of soil |
g |
Cst |
mass concentration of the stock solution of the substance |
μg cm-3 |
C0 |
initial mass concentration of the test solution in contact with the soil |
μg cm-3 |
[Bild bitte in Originalquelle ansehen] |
mass concentration of the substance in the aqueous phase at the time ti that the analysis is performed |
μg cm-3 |
[Bild bitte in Originalquelle ansehen] |
content of the substance adsorbed on soil at adsorption equilibrium an equilibrium |
μg g-1 |
[Bild bitte in Originalquelle ansehen] |
mass concentration of the substance in the aqueous phase at adsorption equilibrium |
μg cm-3 |
V0 |
initial volume of the aqueous phase in contact with the soil during the adsorption test |
cm3 |
[Bild bitte in Originalquelle ansehen] |
volume of the aliquot in which the test substance is measured |
cm3 |
Kd |
distribution coefficient for adsorption |
cm3 g-1 |
Koc |
organic carbon normalised adsorption coefficient |
cm3 g-1 |
Kom |
organic matter normalised distribution coefficient |
cm3 g-1 |
[Bild bitte in Originalquelle ansehen] |
Freundlich adsorption coefficient |
μg 1-1/n (cm3) 1/n g-1 |
1/n |
Freundlich exponent |
|
[Bild bitte in Originalquelle ansehen] |
desorption percentage at a point time ti |
% |
[Bild bitte in Originalquelle ansehen] |
desorption percentage corresponding to a time interval Δti |
% |
Kdes |
apparent desorption coefficient |
cm3 g-1 |
[Bild bitte in Originalquelle ansehen] |
Freundlich desorption coefficient |
μg 1-1/n (cm3) 1/n g-1 |
[Bild bitte in Originalquelle ansehen] |
mass of the test substance desorbed from soil at the time ti |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the test substance desorbed from soil during the time Δti |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the substance determined analytically in the aqueous phase at desorption equilibrium |
μg |
[Bild bitte in Originalquelle ansehen] |
total mass of the test substance desorbed at desorption equilibrium |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the substance remaining adsorbed on the soil after the time interval Δti |
μg |
[Bild bitte in Originalquelle ansehen] |
mass of the substance left over from the adsorption equilibrium due to incomplete volume replacement |
μg |
[Bild bitte in Originalquelle ansehen] |
content of the test substance remaining adsorbed on the soil at desorption equilibrium |
μg g-1 |
[Bild bitte in Originalquelle ansehen] |
mass concentration of the test substance in the aqueous phase at desorption equilibrium |
μg cm-3 |
VT |
total volume of the aqueous phase in contact with the soil during the desorption kinetics experiment performed with the serial method |
cm3 |
VR |
volume of the supernatant removed from the tube after the attainment of adsorption equilibrium and replaced by the same volume of a 0,01 M CaCl2 solution |
cm3 |
[Bild bitte in Originalquelle ansehen] |
volume of the aliquot sampled for analytical purpose from the time (i), during the desorption kinetics experiment performed with the serial method |
cm3 |
[Bild bitte in Originalquelle ansehen] |
volume of the solution taken from the tube (i) for the measurement of the test substance, in desorption kinetics experiment (parallel method) |
cm3 |
[Bild bitte in Originalquelle ansehen] |
volume of the solution taken from the tube for the measurement of the test substance, at desorption equilibrium |
cm3 |
MB |
mass balance |
% |
mE |
total mass of the test substance extracted from soil and walls of the test vessel in two steps |
μg |
Vrec |
volume of the supernatant recovered after the adsorption equilibrium |
cm3 |
Pow |
octanol/water partition coefficient |
|
pKa |
dissociation constant |
|
Sw |
water solubility |
g l-1 |
1.4. PRINCIPLE OF THE TEST METHOD
1.5. INFORMATION ON THE TEST SUBSTANCE
1.6. APPLICABILITY OF THE TEST
1.7. DESCRIPTION OF THE METHOD
1.7.1. Apparatus and chemical reagents
1.7.2. Characterisation and selection of soils
Soil Type |
pH range (in 0,01 M CaCl2) |
Organic carbon content (%) |
Clay content (%) |
Soil texture(25) |
1 |
4,5 - 5,5 |
1,0 - 2,0 |
65-80 |
clay |
2 |
> 7,5 |
3,5 - 5,0 |
20-40 |
clay loam |
3 |
5,5 - 7,0 |
1,5 - 3,0 |
15-25 |
silt loam |
4 |
4,0 - 5,5 |
3,0 - 4,0 |
15-30 |
loam |
5 |
< 4,0 - 6,0(26) |
< 0,5 - 1,5(26) (27) |
< 10-15(26) |
loamy sand |
6 |
> 7,0 |
< 0,5 - 1,0(26) (27) |
40-65 |
clay loam/clay |
7 |
< 4,5 |
> 10 |
< 10 |
sand/loamy sand |
1.7.3. Collection and storage of soil samples
1.7.3.1. Collection
1.7.3.2. Storage
1.7.3.3. Handling and preparation of soil samples for the test
1.7.4. Preparation of the test substance for application to soil
1.8. PREREQUISITES FOR PERFORMING THE ADSORPTION/DESORPTION TEST
1.8.1. The analytical method
1.8.2. The selection of optimal soil/solution ratios
[Bild bitte in Originalquelle ansehen] |
(1) |
[Bild bitte in Originalquelle ansehen] |
(2) |
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
1.9.2. Tier 1 — Preliminary study
1.9.2.1. Selection of optimal soil/solution ratios
1.9.2.2. Determination of adsorption equilibration time and of the amount of test substance adsorbed at equilibrium
1.9.2.3. Adsorption on the surface of the test vessel and stability of the test substance
1.9.3. Tier 2 — Adsorption kinetics at one concentration of the test substance
1.9.4. Tier 3 — Adsorption isotherms and desorption kinetics/desorption isotherms
1.9.4.1.
Adsorption isotherms
1.9.4.2.
Desorption kinetics
1.9.4.3.
Desorption isotherms
2. DATA AND REPORTING
2.1. ADSORPTION
[Bild bitte in Originalquelle ansehen] |
(3) |
[Bild bitte in Originalquelle ansehen] (cm3 g-1) |
(4) |
[Bild bitte in Originalquelle ansehen] (cm3 g-1) |
(5) |
[Bild bitte in Originalquelle ansehen] (cm3 g-1) |
(6) |
2.1.1. Adsorption isotherms
[Bild bitte in Originalquelle ansehen] (μg g-1) |
(7) |
[Bild bitte in Originalquelle ansehen] (μg g-1) |
(8) |
[Bild bitte in Originalquelle ansehen] |
(9) |
2.1.2. Mass balance
[Bild bitte in Originalquelle ansehen] |
(10) |
2.2. DESORPTION
[Bild bitte in Originalquelle ansehen] |
(11) |
[Bild bitte in Originalquelle ansehen] (cm3 g-1) |
(12) |
2.2.1. Desorption isotherms
[Bild bitte in Originalquelle ansehen] (µg g-1) |
(13) |
[Bild bitte in Originalquelle ansehen] (μg) |
(14) |
[Bild bitte in Originalquelle ansehen] |
(15) |
[Bild bitte in Originalquelle ansehen] (μg g-1) |
(16) |
[Bild bitte in Originalquelle ansehen] |
(17) |
2.2.2. Test report
3. REFERENCES
Appendix 1
Testing scheme
Appendix 2
INFLUENCE OF ACCURACY OF ANALYTICAL METHOD AND CONCENTRATION CHANGE ON ACCURACY OF ADSORPTION RESULTS
Amount of soil |
msoil |
= 10 g |
Volume of solution |
V0 |
= 100 cm3 |
|
FOR-L_2008142EN.01044401.notes.0187.xml.jpg (μg) |
FOR-L_2008142EN.01044401.notes.0188.xml.jpg (μg cm-3) |
R |
FOR-L_2008142EN.01044401.notes.0189.xml.jpg (μg) |
FOR-L_2008142EN.01044401.notes.0190.xml.jpg (μg g-1) |
R‡ |
Kd* |
R‡ |
|
FOR A = 9 % |
|||||||
m0 = 110 μg or C0 = 1,100 μg/cm3 |
100 |
1,000 |
true value |
10 |
1,00 |
true value |
1 |
|
101 |
1,010 |
1 % |
9 |
0,90 |
10 % |
0,891 |
10,9 % |
|
105 |
1,050 |
5 % |
5 |
0,50 |
50 % |
0,476 |
52,4 % |
|
109 |
1,090 |
9 % |
1 |
0,10 |
90 % |
0,092 |
90,8 % |
|
|
FOR A = 55 % |
|||||||
m0= 110 μg or C0 = 1,100 μg/cm3 |
50,0 |
0,500 |
true value |
60,0 |
6,00 |
true value |
12,00 |
|
50,5 |
0,505 |
1 % |
59,5 |
5,95 |
0,8 % |
11,78 |
1,8 % |
|
52,5 |
0,525 |
5 % |
57,5 |
5,75 |
4,0 % |
10,95 |
8,8 % |
|
55,0 |
0,550 |
10 % |
55,0 |
5,50 |
8,3 % |
10,00 |
16,7 % |
|
|
FOR A = 99 % |
|||||||
m0 = 110 μg or C0 = 1,100 μg/cm3 |
1,100 |
0,011 |
true value |
108,9 |
10,89 |
true value |
990 |
|
1,111 |
0,01111 |
1 % |
108,889 |
10,8889 |
0,01 % |
980 |
1,0 % |
|
1,155 |
0,01155 |
5 % |
108,845 |
10,8845 |
0,05 % |
942 |
4,8 % |
|
1,21 |
0,0121 |
10 % |
108,790 |
10,8790 |
0,10 % |
899 |
9,2 % |
Appendix 3
ESTIMATION TECHNIQUES FOR K
D
[Bild bitte in Originalquelle ansehen] |
[Bild bitte in Originalquelle ansehen] |
Substances |
Correlations |
Authors |
Substituted ureas |
log Kom = 0,69 + 0,52 log Pow |
Briggs (1981) (39) |
Aromatic chlorinated |
log Koc = -0,779 + 0,904 log Pow |
Chiou et al. (1983) (65) |
Various pesticides |
log Kom = 4,4 + 0,72 log Pow |
Gerstl and Mingelgrin (1984) (66) |
Aromatic hydrocarbons |
log Koc = -2,53 + 1,15 log Pow |
Vowles and Mantoura (1987) (67) |
Compounds |
Correlations |
Authors |
Various pesticides |
log Kom = 3,8 - 0,561 log Sw |
Gerstl and Mingelgrin (1984) (66) |
Aliphatic, aromatic chlorinated substances |
log Kom = (4,040 +/- 0,038) - (0,557 +/- 0,012) log Sw |
Chiou et al. (1979) (70) |
α-naphtol |
logKoc = 4,273 - 0,686 log Sw |
Hasset et al. (1981) (71) |
Cyclic, aliphatic aromatic substances |
logKoc = -1,405 - 0,921 log Sw-0,00953 (mp-25) |
Karickhoff (1981) (72) |
Various compounds |
log Kom = 2,75 - 0,45 log Sw |
Moreale van Blade (1982) (73) |
Appendix 4
CALCULATIONS FOR DEFINING THE CENTRIFUGATION CONDITIONS
[Bild bitte in Originalquelle ansehen] |
(1) |
[Bild bitte in Originalquelle ansehen] |
(2) |
Fig. 1a.
Fig. 1b.
Appendix 5
CALCULATION OF ADSORPTION A (%) AND DESORPTION D (%)
ADSORPTION A (A%)
a) Parallel method
[Bild bitte in Originalquelle ansehen] (%) |
(1) |
m0 = C0 · V0 (μg) |
(2) |
[Bild bitte in Originalquelle ansehen] (μg) |
(3) |
b) Serial method
[Bild bitte in Originalquelle ansehen] |
(4) |
[Bild bitte in Originalquelle ansehen] |
(5) |
[Bild bitte in Originalquelle ansehen] |
(6) |
[Bild bitte in Originalquelle ansehen] |
(7) |
[Bild bitte in Originalquelle ansehen] |
(8) |
[Bild bitte in Originalquelle ansehen] |
(9) |
[Bild bitte in Originalquelle ansehen] |
(10) |
[Bild bitte in Originalquelle ansehen] |
(11) |
[Bild bitte in Originalquelle ansehen] |
(12) |
DESORPTION D (A %)
(a) Parallel method
[Bild bitte in Originalquelle ansehen] |
(13) |
[Bild bitte in Originalquelle ansehen] |
(14) |
[Bild bitte in Originalquelle ansehen] |
(15) |
[Bild bitte in Originalquelle ansehen] |
(16) |
[Bild bitte in Originalquelle ansehen] |
(17) |
(b) Serial method
[Bild bitte in Originalquelle ansehen] |
(18) |
[Bild bitte in Originalquelle ansehen] |
(19) |
[Bild bitte in Originalquelle ansehen] |
(20) |
[Bild bitte in Originalquelle ansehen] and[Bild bitte in Originalquelle ansehen] |
(21) |
[Bild bitte in Originalquelle ansehen] and [Bild bitte in Originalquelle ansehen] |
(22) |
[Bild bitte in Originalquelle ansehen] and [Bild bitte in Originalquelle ansehen] |
(23) |
[Bild bitte in Originalquelle ansehen] |
(24) |
[Bild bitte in Originalquelle ansehen] |
(25) |
[Bild bitte in Originalquelle ansehen] |
(26) |
[Bild bitte in Originalquelle ansehen] |
(27) |
Appendix 6
ADSORPTION-DESORPTION IN SOILS: DATA REPORTING SHEETS
Suitability of the analytical method
Weighed soil |
g |
|
Soil: dry mass |
g |
|
Volume CaCl2 sol. |
cm3 |
|
Nominal conc. final sol. |
μg cm-3 |
|
Analytical conc. final sol. |
μg cm-3 |
|
Analytical methodology followed: |
Indirect |
|
Parallel |
|
Serial |
|
|
Direct |
|
|
|
|
|
Adsorption test: test samples
|
Symbol |
Units |
Equilibration time |
Equilibration time |
Equilibration time |
Equilibration time |
||||
Tube No |
|
|
|
|
|
|
|
|
|
|
Weighed soil |
— |
g |
|
|
|
|
|
|
|
|
Soil: dry mass |
msoil |
g |
|
|
|
|
|
|
|
|
Water volume in weighed soil (calculated) |
VWS |
cm3 |
|
|
|
|
|
|
|
|
Volume 0,01 M CaCl2 sol. to equilibrate the soil |
|
cm3 |
|
|
|
|
|
|
|
|
Volume of stock solution |
|
cm3 |
|
|
|
|
|
|
|
|
Total volume of aq. phase in contact with soil |
V0 |
cm3 |
|
|
|
|
|
|
|
|
Initial concentration Test solution |
C0 |
μg cm-3 |
|
|
|
|
|
|
|
|
Mass test subst. at the beginning of the test |
m 0 |
μg |
|
|
|
|
|
|
|
|
After agitation and centrifugation |
||||||||||
INDIRECT METHOD |
||||||||||
Parallel method |
||||||||||
Concentration test subst. aq. phase Blank correction included |
[Bild bitte in Originalquelle ansehen] |
μg cm-3 |
|
|
|
|
|
|
|
|
Serial method |
||||||||||
Measured mass test subst. in the aliquot Va A |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
|
|
|
DIRECT METHOD |
||||||||||
Mass test substance adsorbed on soil |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
|
|
|
Calculation of adsorption |
||||||||||
Adsorption |
[Bild bitte in Originalquelle ansehen] |
% |
|
|
|
|
|
|
|
|
|
[Bild bitte in Originalquelle ansehen] |
% |
|
|
|
|
|
|
|
|
Means |
|
|
|
|
|
|
||||
Adsorption coefficient |
Kd |
cm3 g-1 |
|
|
|
|
|
|
|
|
Means |
|
|
|
|
|
|
||||
Adsorption coefficient |
Koc |
cm3 g-1 |
|
|
|
|
|
|
|
|
Means |
|
|
|
|
|
|
Adsorption test: blanks and control
|
Symbol |
Units |
Blank |
Blank |
Control |
|||
Tube No |
|
|
|
|
|
|
|
|
Weighed soils |
|
g |
|
|
|
|
0 |
0 |
Water amount in weighed soil (calculated) |
|
cm3 |
|
|
|
|
— |
— |
Volume of 0,01 M CaCl2 solution added |
|
cm3 |
|
|
|
|
|
|
Volume of the stock solution of the test substance added |
|
cm3 |
0 |
0 |
|
|
|
|
Total volume of aq. phase (calculated) |
|
cm3 |
|
|
|
|
— |
— |
Initial concentration of the test substance in aqueous phase |
|
μg cm-3 |
|
|
|
|
|
|
After agitation and centrifugation |
||||||||
Concentration in aqueous phase |
|
μg cm-3 |
|
|
|
|
|
|
Mass balance
|
Symbol |
Units |
|
|
|
|
Tube No |
|
|
|
|
|
|
Weighed soil |
— |
g |
|
|
|
|
Soil: dry mass |
msoil |
g |
|
|
|
|
Water volume in weighed soil (calculated) |
VWS |
ml |
|
|
|
|
Volume 0,01 M CaCl2 sol. to equilibrate the soil |
|
ml |
|
|
|
|
Volume of stock solution |
|
cm3 |
|
|
|
|
Total volume of aq. phase in contact with soil |
V0 |
cm3 |
|
|
|
|
Initial concentration test solution |
C0 |
μg cm-3 |
|
|
|
|
Equilibration time |
— |
h |
|
|
|
|
After agitation and centrifugation |
||||||
Concentr. test subst. aq. phase at adsorption equilibrium blank correction included |
[Bild bitte in Originalquelle ansehen] |
μg cm-3 |
|
|
|
|
Equalibration time |
teq |
h |
|
|
|
|
1st dilution with solvent |
||||||
Removed volume aq. phase |
Vrec |
cm3 |
|
|
|
|
Added volume of solvent |
ΔV |
cm3 |
|
|
|
|
1st extraction with solvent |
||||||
Signal analysed in solvent |
SE1 |
var. |
|
|
|
|
Conc. test subst. in solvent |
CE1 |
μg cm-3 |
|
|
|
|
Mass of substance extracted from soil and vessel walls |
mE1 |
μg |
|
|
|
|
2nd dilution with solvent |
||||||
Removed volume of solvent |
ΔVs |
cm3 |
|
|
|
|
Added volume of solvent |
ΔV' |
cm3 |
|
|
|
|
2nd extraction with solvent |
||||||
Signal analysed in solvent phase |
SE2 |
var. |
|
|
|
|
Conc. test subst. in solvent |
CE2 |
μg cm-3 |
|
|
|
|
Mass of substance extracted from soil and vessel walls |
mE2 |
μg |
|
|
|
|
Total mass test subst. extracted in two steps |
mE |
μg |
|
|
|
|
Mass balance |
MB |
% |
|
|
|
|
Adsorption isotherms
|
Symbol |
Units |
|
|
|
|
|
|
|
|
Tube No |
|
|
|
|
|
|
|
|
|
|
Weighed soil |
— |
g |
|
|
|
|
|
|
|
|
Soil: dry mass |
E |
g |
|
|
|
|
|
|
|
|
Water volume in weighed soil (calculated) |
VWS |
cm3 |
|
|
|
|
|
|
|
|
Volume 0,01 M CaCl2 sol. to equilibrate the soil |
|
cm3 |
|
|
|
|
|
|
|
|
Volume of stock solution added |
|
cm3 |
|
|
|
|
|
|
|
|
Total volume of aq. phase in contact with soil (calculated) |
V0 |
cm3 |
|
|
|
|
|
|
|
|
Concentration solution |
C0 |
μg cm-3 |
|
|
|
|
|
|
|
|
Equilibration time |
— |
h |
|
|
|
|
|
|
|
|
After agitation and centrifugation |
||||||||||
Concentration subst. aq. phase, blank correction included |
[Bild bitte in Originalquelle ansehen] |
μg cm-3 |
|
|
|
|
|
|
|
|
Temperature |
|
oC |
|
|
|
|
|
|
|
|
Adsorb. mass per unit soil |
[Bild bitte in Originalquelle ansehen] |
μg g-1 |
|
|
|
|
|
|
|
|
Analytical methodology followed: |
Indirect |
|
Parallel |
|
Serial |
|
Desorption test
|
Symbol |
Units |
Time interval |
Time interval |
Time interval |
Time interval |
|
Tube No coming from adsorption step |
|
|
|
|
|
|
|
Mass of substance adsorbed on soil at adsorption equilibrium |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
Removed volume aq. phase, replaced by 0,01 M CaCl2 |
VR |
cm3 |
|
|
|
|
|
Total volume of aq. phase in contact with soil |
PM |
V0 |
cm3 |
|
|
|
|
SM |
VT |
cm3 |
|
|
|
|
|
Mass test subst. left over the adsorption equilibrium due to incomplete volume replacement |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
Desorption kinetics |
|||||||
Measured mass of substance desorbed from soil at time ti |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
Volume of the solution taken from the tube (i) for the measurement of the test substance |
PM |
Vr i |
cm3 |
|
|
|
|
SM |
va D |
cm3 |
|
|
|
|
|
Mass of substance desorbed from soil at time ti (calculated) |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
Mass of substance desorbed from soil during time interval Δti (calculated) |
[Bild bitte in Originalquelle ansehen] |
μg |
|
|
|
|
|
Desorption percentage |
|||||||
Desorption at time ti |
Dti |
% |
|
|
|
|
|
Desorption at time interval Δti |
[Bild bitte in Originalquelle ansehen] |
% |
|
|
|
|
|
Apparent desorption coefficient |
Kdes |
|
|
|
|
|
C.19. ESTIMATION OF THE ADSORPTION COEFFICIENT (
K
OC
) ON SOIL AND ON SEWAGE SLUDGE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
[Bild bitte in Originalquelle ansehen] |
(1) |
[Bild bitte in Originalquelle ansehen] |
(2) |
[Bild bitte in Originalquelle ansehen] |
(3) |
[Bild bitte in Originalquelle ansehen] |
(4) |
[Bild bitte in Originalquelle ansehen] |
(5) |
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
— O — Si |
— CH2 — CH2 — CH2 |
— CN |
silica |
non-polar spacer |
polar moiety |
1.5. APPLICABILITY OF THE TEST
1.6. QUALITY CRITERIA
1.6.1. Accuracy
1.6.2. Repeatability
1.6.3. Reproducibility
1.7. DESCRIPTION OF THE TEST METHOD
1.7.1. Preliminary Estimation of the Adsorption Coefficient
1.7.2. Apparatus
1.7.3. Mobile phases
1.7.4. Solutes
1.8. PERFORMANCE OF THE TEST
1.8.1. Test condition
1.8.2. Determination of dead time to
1.8.2.1. Determination of the dead time to by means of a homologous series
1.8.2.2. Determination of the dead time to by inert substances which are not retained by the column
1.8.3. Determination of the retention times tR
1.8.4. Evaluation
2. DATA AND REPORTING
3. REFERENCES
Appendix
substance |
CAS-No |
log Koc sewage sludges |
log Koc HPLC |
Δ |
log Koc soils |
log Koc HPLC |
Δ |
Atrazine |
1912-24-9 |
1,66 |
2,14 |
0,48 |
1,81 |
2,20 |
0,39 |
Linuron |
330-55-2 |
2,43 |
2,96 |
0,53 |
2,59 |
2,89 |
0,30 |
Fenthion |
55-38-9 |
3,75 |
3,58 |
0,17 |
3,31 |
3,40 |
0,09 |
Monuron |
150-68-5 |
1,46 |
2,21 |
0,75 |
1,99 |
2,26 |
0,27 |
Phenanthrene |
85-01-8 |
4,35 |
3,72 |
0,63 |
4,09 |
3,52 |
0,57 |
Benzoic acid phenylester |
93-99-2 |
3,26 |
3,03 |
0,23 |
2,87 |
2,94 |
0,07 |
Benzamide |
55-21-0 |
1,60 |
1,00 |
0,60 |
1,26 |
1,25 |
0,01 |
4-Nitrobenzamide |
619-80-7 |
1,52 |
1,49 |
0,03 |
1,93 |
1,66 |
0,27 |
Acetanilide |
103-84-4 |
1,52 |
1,53 |
0,01 |
1,26 |
1,69 |
0,08 |
Aniline |
62-53-3 |
1,74 |
1,47 |
0,27 |
2,07 |
1,64 |
0,43 |
2,5-Dichloroaniline |
95-82-9 |
2,45 |
2,59 |
0,14 |
2,55 |
2,58 |
0,03 |
substance |
CAS-No |
log Koc |
Koc |
log Koc |
[OECD 106] |
[HPLC-method] |
|||
Atrazine |
1912-24-9 |
1,81 |
78 ± 16 |
1,89 |
Monuron |
150-68-5 |
1,99 |
100 ± 8 |
2,00 |
Triapenthenol |
77608-88-3 |
2,37 |
292 ± 58 |
2,47 |
Linuron |
330-55-2 |
2,59 |
465 ± 62 |
2,67 |
Fenthion |
55-38-9 |
3,31 |
2062 ± 648 |
3,31 |
Reference substance |
CAS-No |
log Koc mean values from batch equilibrium |
number of Koc data |
log S.D. |
source |
Acetanilide |
103-84-4 |
1,25 |
4 |
0,48 |
(34) |
Phenol |
108-95-2 |
1,32 |
4 |
0,70 |
(34) |
2-Nitrobenzamide |
610-15-1 |
1,45 |
3 |
0,90 |
(35) |
N.N-dimethylbenzamide |
611-74-5 |
1,52 |
2 |
0,45 |
(34) |
4-Methylbenzamide |
619-55-6 |
1,78 |
3 |
1,76 |
(34) |
Methylbenzoate |
93-58-3 |
1,80 |
4 |
1,08 |
(34) |
Atrazine |
1912-24-9 |
1,81 |
3 |
1,08 |
(36) |
Isoproturon |
34123-59-6 |
1,86 |
5 |
1,53 |
(36) |
3-Nitrobenzamide |
645-09-0 |
1,95 |
3 |
1,31 |
(35) |
Aniline |
62-53-3 |
2,07 |
4 |
1,73 |
(34) |
3,5-Dinitrobenzamide |
121-81-3 |
2,31 |
3 |
1,27 |
(35) |
Carbendazim |
10605-21-7 |
2,35 |
3 |
1,37 |
(36) |
Triadimenol |
55219-65-3 |
2,40 |
3 |
1,85 |
(36) |
Triazoxide |
72459-58-6 |
2,44 |
3 |
1,66 |
(36) |
Triazophos |
24017-47-8 |
2,55 |
3 |
1,78 |
(36) |
Linuron |
330-55-2 |
2,59 |
3 |
1,97 |
(36) |
Naphthalene |
91-20-3 |
2,75 |
4 |
2,20 |
(34) |
Endosulfan-diol |
2157-19-9 |
3,02 |
5 |
2,29 |
(36) |
Methiocarb |
2032-65-7 |
3,10 |
4 |
2,39 |
(36) |
Acid Yellow 219 |
63405-85-6 |
3,16 |
4 |
2,83 |
(34) |
1,2,3-Trichlorobenzene |
87-61-6 |
3,16 |
4 |
1,40 |
(34) |
γ-HCH |
58-89-9 |
3,23 |
5 |
2,94 |
(34) |
Fenthion |
55-38-9 |
3,31 |
3 |
2,49 |
(36) |
Direct Red 81 |
2610-11-9 |
3,43 |
4 |
2,68 |
(34) |
Pyrazophos |
13457-18-6 |
3,65 |
3 |
2,70 |
(36) |
α-Endosulfan |
959-98-8 |
4,09 |
5 |
3,74 |
(36) |
Diclofop-methyl |
51338-27-3 |
4,20 |
3 |
3,77 |
(36) |
Phenanthrene |
85-01-8 |
4,09 |
4 |
3,83 |
(34) |
Basic Blue 41 (mix) |
26850-47-5 12270-13-2 |
4,89 |
4 |
4,46 |
(34) |
DDT |
50-29-3 |
5,63 |
1 |
— |
(35) |
C.20
DAPHNIA MAGNA
REPRODUCTION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Test organism
1.6.3. Test medium
1.6.4. Test solutions
1.7. TEST DESIGN
1.8. PROCEDURE
1.8.1. Conditions of exposure
1.8.1.1. Duration
1.8.1.2. Loading
1.8.1.3. Number of animals
1.8.1.4. Feeding
1.8.1.5. Light
1.8.1.6. Temperature
1.8.1.7. Aeration
1.8.2. Test concentration
1.8.3. Controls
1.8.4. Test medium renewal
1.8.5. Observations
1.8.6. Offspring
1.8.7. Mortality
1.8.8. Other parameters
1.8.9. Frequency of analytical determinations and measurements
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
Appendix 1
PREPARATION OF FULLY DEFINED ELENDT M7 AND M4 MEDIA
Acclimation to Elendt M7 and M4 media
PREPARATION
Trace elements
Stock solutions I (single substance) |
Amount added to water mg/l |
Concentration (in relation to medium M4) fold |
To prepare the combined stock-solution II add the following amount of stock solution I to water ml/l |
|
M 4 |
M 7 |
|||
H3BO3 |
57 190 |
20 000 |
1,0 |
0,25 |
MnCl2 * 4 H2O |
7 210 |
20 000 |
1,0 |
0,25 |
LiCl |
6 120 |
20 000 |
1,0 |
0,25 |
RbCl |
1 420 |
20 000 |
1,0 |
0,25 |
SrCl2 * 6 H2O |
3 040 |
20 000 |
1,0 |
0,25 |
NaBr |
320 |
20 000 |
1,0 |
0,25 |
Na2MoO4 * 2 H2O |
1 260 |
20 000 |
1,0 |
0,25 |
CuCl2 * 2 H2O |
335 |
20 000 |
1,0 |
0,25 |
ZnCl2 |
260 |
20 000 |
1,0 |
1,0 |
CoCl2 * 6 H2O |
200 |
20 000 |
1,0 |
1,0 |
KI |
65 |
20 000 |
1,0 |
1,0 |
Na2SeO3 |
43,8 |
20 000 |
1,0 |
1,0 |
NH4VO3 |
11,5 |
20 000 |
1,0 |
1,0 |
Na2EDTA * 2 H2O |
5 000 |
2 000 |
— |
— |
FeSO4 * 7 H2O |
1 991 |
2 000 |
— |
— |
Both Na2EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. This gives: |
||||
21 Fe-EDTA solution |
|
1 000-fold |
20,0 |
5,0 |
M4 and M7 media
|
Amount added to water mg/l |
Concentration (related to medium M4) fold |
Amount of stock solution added to prepare medium ml/l |
|||||
M 4 |
M 7 |
|||||||
Stock solution II combined trace elements |
|
20 |
50 |
50 |
||||
Macro-nutrient stock solutions (single substance) |
||||||||
CaCl2 * 2 H2O |
293 800 |
1 000 |
1,0 |
1,0 |
||||
MgSO4 * 7 H2O |
246 600 |
2 000 |
0,5 |
0,5 |
||||
KCl |
58 000 |
10 000 |
0,1 |
0,1 |
||||
NaHCO3 |
64 800 |
1 000 |
1,0 |
1,0 |
||||
Na2SiO3 * 9 H2O |
50 000 |
5 000 |
0,2 |
0,2 |
||||
NaNO3 |
2 740 |
10 000 |
0,1 |
0,1 |
||||
KH2PO4 |
1 430 |
10 000 |
0,1 |
0,1 |
||||
K2HPO4 |
1 840 |
10 000 |
0,1 |
0,1 |
||||
Combined Vitamin stock |
— |
10 000 |
0,1 |
0,1 |
||||
The combined vitamin stock solution is prepared by adding the 3 vitamins to 1 litre water as show below: |
||||||||
Thiamine hydrochloride |
750 |
10 000 |
— |
— |
||||
Cyanocobalamine (B12) |
10 |
10 000 |
— |
— |
||||
Biotine |
7,5 |
10 000 |
— |
— |
||||
The combined vitamin stock is stored frozen in small aliquots. Vitamins are added to the media shortly before use.
|
Appendix 2
TOTAL ORGANIC CARBON (TOC) ANALYSIS AND PRODUCTION OF A NOMOGRAPH FOR TOC CONTENT OF ALGAL FEED
Appendix 3
EXAMPLE DATA SHEET RECORDING MEDIUM RENEWAL, PHISICAL/CHEMICAL MONITORING DATA, FEEDING,
DAPHNIA
REPRODUCTION AND ADULT MORTALITY
Experiment No: |
Data starded: |
Clone: |
Medium: |
Type of food: |
Test Substance: |
Nominal conc: |
||||||||||||||||||
Day |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
21 |
|
|
Medium renewal (tick) |
|
|
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|
PH(37) |
|
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new |
|
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|
old |
|
|
O2 mg/l(37) |
|
|
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new |
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old |
|
|
Temp ( °C)(37) |
|
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new |
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|
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|
old |
|
|
Food provided (tick) |
|
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|
|
No live offspring(38) |
|
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|
Total |
Vessel 1 |
|
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2 |
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3 |
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|
4 |
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|
5 |
|
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6 |
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7 |
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8 |
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9 |
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|
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|
|
|
|
10 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
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|
|
|
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|
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|
Total |
|
Cumulative adult mortality(39) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Appendix 4
EXAMPLE DATA SHEET FOR RECORDING RESULTS OF CHEMICAL ANALYSIS
(a) Measured concentrations
Nominal conc. |
Week 1 sample |
Week 2 sample |
Week 3 sample |
|||
Fresh |
Old |
Fresh |
Old |
Fresh |
Old |
|
|
|
|
|
|
|
|
(b) Measured concentrations as a percentage of nominal
Nominal conc. |
Week 1 sample |
Week 2 sample |
Week 3 sample |
|||
Fresh |
Old |
Fresh |
Old |
Fresh |
Old |
|
|
|
|
|
|
|
|
Appendix 5
CALCULATION OF A TIME-WEIGHTED MEAN
Time-weighted mean
Figure 1: Example of time-weighted mean
Renewal No |
Days |
Conc0 |
Conc1 |
Ln(Conc0) |
Ln(Conc1) |
Area |
1 |
2 |
10,000 |
4,493 |
2,303 |
1,503 |
13,767 |
2 |
2 |
11,000 |
6,037 |
2,398 |
1,798 |
16,544 |
3 |
3 |
10,000 |
4,066 |
2,303 |
1,403 |
19,781 |
Total Days: 7 |
Total Area |
50,091 |
||||
TW Mean |
7,156 |
|||||
Days is the number of days in the renewal period Conc0 is the measured concentration at the start of each renewal period Conc1 is the measured concentration at the end of each renewal period Ln(Conc0) is the natural logarithm of Conc0 Ln(Conc1) is the natural logarithm of Conc1 Area is the area under the exponential curve for each renewal period. It is calculated by: [Bild bitte in Originalquelle ansehen] |
C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Selection and number of soils
1.6.3. Collection and storage of soil samples
1.6.3.1. Collection
1.6.3.2. Storage
1.6.4. Handling and preparation of soil for the test
1.6.4.1. Pre-incubation
1.6.4.2. Physical-chemical characteristics
1.6.4.3. Amendment with organic substrate
1.6.5. Preparation of the test substance for the application to soil
1.6.6. Test concentrations
1.7. PERFORMANCE OF THE TEST
1.7.1. Conditions of exposure
1.7.1.1. Treatment and control
1.7.1.2. Incubation of soil samples
1.7.1.3. Test conditions and duration
1.7.2. Sampling and analysis of soils
1.7.2.1. Soil sampling schedule
1.7.2.2. Analysis of soil samples
2. DATA
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
3. REPORTING
4. REFERENCES
C.22. SOIL MICROORGANISMS: CARBON TRANSFORMATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Selection and number of soils
1.6.3. Collection and storage of soil samples
1.6.3.1. Collection
1.6.3.2. Storage
1.6.4. Handling and preparation of soil for the test
1.6.4.1. Pre-incubation
1.6.4.2. Physical-chemical characteristics
1.6.5. Preparation of the test substance for the application to soil
1.6.6. Test concentrations
1.7. PERFORMANCE OF THE TEST
1.7.1. Conditions of exposure
1.7.1.1. Treatment and control
1.7.1.2. Incubation of soil samples
1.7.1.3. Test conditions and duration
1.7.2. Sampling and analysis of soils
1.7.2.1. Soil sampling schedule
1.7.2.2. Measurement of glucose-induced respiration rates
2. DATA
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
C.23. AEROBIC AND ANAEROBIC TRANSFORMATION IN SOIL
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. APPLICABILITY OF THE TEST
1.5. INFORMATION ON THE SUBSTANCE
1.6. PRINCIPLE OF THE TEST METHOD
1.7. QUALITY CRITERIA
1.7.1. Recovery
1.7.2. Repeatability and sensitivity of analytical method
1.7.3. Accuracy of transformation data
1.8. DESCRIPTION OF THE METHOD
1.8.1. Equipment and chemical reagents
1.8.2. Test substance application
1.8.3. Soils
1.8.3.1. Soil selection
1.8.3.2. Collection, handling, and storage of soils
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
1.9.1.1. Test temperature
1.9.1.2. Moisture content
1.9.1.3. Aerobic incubation conditions
1.9.1.4. Sterile aerobic conditions
1.9.1.5. Anaerobic incubation conditions
1.9.1.6. Paddy incubation conditions
1.9.1.7. Test duration
1.9.2. Performance of the test
1.9.3. Sampling and measurement
1.9.4. Optional tests
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
Appendix 1
WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY (WHC)(47)
Height of Water Column [cm] |
pF(48) |
bar(49) |
Remarks |
107 |
7 |
104 |
Dry Soil |
1,6 · 104 |
4,2 |
16 |
Wilting point |
104 |
4 |
10 |
|
103 |
3 |
1 |
|
6· 102 |
2,8 |
0,6 |
|
3,3 · 102 |
2,5 |
0,33(50) |
Range of Field capacity(51) |
102 |
2 |
0,1 |
|
60 |
1,8 |
0,06 |
|
33 |
1,5 |
0,033 |
|
10 |
1 |
0,01 |
WHC (approximation) |
1 |
0 |
0,001 |
Water saturated soil |
Appendix 2
SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM VARIOUS COUNTRIES
|
|
Soil moisture content at |
||
Soil type |
Country |
|||
|
|
WHC(52) |
pF = 1,8 |
pF = 2,5 |
Sand |
Germany |
28,7 |
8,8 |
3,9 |
Loamy sand |
Germany |
50,4 |
17,9 |
12,1 |
Loamy sand |
Switzerland |
44,0 |
35,3 |
9,2 |
Silt loam |
Switzerland |
72,8 |
56,6 |
28,4 |
Clay loam |
Brazil |
69,7 |
38,4 |
27,3 |
Clay loam |
Japan |
74,4 |
57,8 |
31,4 |
Sandy loam |
Japan |
82,4 |
59,2 |
36,0 |
Silt loam |
USA |
47,2 |
33,2 |
18,8 |
Sandy loam |
USA |
40,4 |
25,2 |
13,3 |
Appendix 3
Figure 1
Example of a flow-through apparatus to study transformation of chemicals in soil
(53) (54)
Figure 2
Example of a biometer-type flask for studying the transformation of chemicals in soil
(55)
C.24. AEROBIC AND ANAEROBIC TRANSFORMATION IN AQUATIC SEDIMENT SYSTEMS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. PRINCIPLE OF THE TEST METHOD
1.6. APPLICABILITY OF THE TEST
1.7. QUALITY CRITERIA
1.7.1. Recovery
1.7.2. Repeatability and sensitivity of analytical method
1.7.3. Accuracy of transformation data
1.8. DESCRIPTION OF THE METHOD
1.8.1. Test system and apparatus
1.8.2. Selection and number of aquatic sediments
1.8.2.1. Sediment selection
1.8.2.2. Characterisation of water-sediment samples
Parameter |
Stage of test procedure |
|||||
field sampling |
post- handling |
start of acclimation |
start of test |
during test |
end of test |
|
Water |
||||||
Origin/source |
x |
|
|
|
|
|
Temperature |
x |
|
|
|
|
|
pH |
x |
|
x |
x |
x |
x |
TOC |
|
|
x |
x |
|
x |
O2 concentration* |
x |
|
x |
x |
x |
x |
Redox Potential* |
|
|
x |
x |
x |
x |
Sediment |
||||||
Origin/source |
x |
|
|
|
|
|
Depth of layer |
x |
|
|
|
|
|
pH |
|
x |
x |
x |
x |
x |
Particle size distribution |
|
x |
|
|
|
|
TOC |
|
x |
x |
x |
|
x |
Microbial biomass(59) |
|
x |
|
x |
|
x |
Redox potential(60) |
Observation (colour/smell) |
|
x |
x |
x |
x |
1.8.3. Collection, handling and storage
1.8.3.1. Collection
1.8.3.2. Handling
1.8.3.3. Storage
1.8.4. Preparation of the sediment/water samples for the test
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
1.9.2. Treatment and application of test substance
1.9.3. Test duration and sampling
1.9.4. Optional preliminary test
1.9.5. Measurements and analysis
2. DATA
2.1. TREATMENT OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
GUIDANCE ON THE AEROBIC AND THE ANAEROBIC TEST SYSTEMS
Aerobic test system
Anaerobic test system
Appendix 2
EXAMPLE OF A GAS FLOW-THROUGH APPARATUS
Appendix 3
EXAMPLE OF A BIOMETER APPARATUS
Appendix 4
EXAMPLE CALCULATION FOR APPLICATION DOSE TO TEST VESSELS
Cylinder internal diameter: |
= 8 cm |
Water column depth not including sediment: |
= 12 cm |
Surface area: 3,142 × 42 |
= 50,3 cm2 |
Application rate: 500 g test substance/ha corresponds to 5 μg/cm2 |
|
Total μg: 5 × 50,3 |
= 251,5 μg |
Adjust quantity in relation to a depth of 100 cm: 12 × 251,5 ÷ 100 |
= 30,18 μg |
Volume of water column: 50,3 × 12 |
= 603 ml |
Concentration in water: 30,18 ÷ 603 |
= 0,050 μg/ml or 50 μg/l |