Výkon jako z partesu. Beksa nedala Opavě žádnou naději - partesu

 2024-08-09 07:39:09

A plateau or steady state is defined as the equilibrium between the uptake and elimination processes that occur simultaneously during the exposure phase. The steady state is reached in the plot of BAF against time when the curve becomes parallel to the time axis and three successive analyses of BAF made on samples taken at intervals of at least two days are within 20 % of each other, and there are no statistically significant differences among the three sampling periods. For test chemicals which are taken up slowly, more appropriate intervals would be seven days (49).

Description of pilot studies used in the experimental design of the follow-up studies, if applicable. Pilot study supporting data should be submitted;

Quantitative measure of the rate at which a test chemical is removed from the blood, plasma or a certain tissue per unit time.

ISO 9439 (1990; revised 1999). Water Quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium — Carbon dioxide evolution Test (Sturm).

OECD (2000). Guidance Document on Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation; Environmental Health and Safety Publications, Series on Testing and Assessment No 19, ENV/JM/MONO(2000), OECD, Paris.

OECD (1981). Subchronic Inhalation Toxicity Testing, Original Test Guideline No 412, Environment Directorate, OECD, Paris.

Holsapple MP, Pitot HC, Cohen SN, Boobis AR, Klaunig JE, Pastoor T, Dellarco VL, Dragan YP (2006). Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk. Toxicol. Sci. 89: 51-56.

Tabulation of particle size data including analytical sample collection data, particle size distribution, and calculations of the MMAD and σg;

Environment Canada (1995). Guidance document on measurement of toxicity test precision using control sediments spiked with a reference toxicant. Environmental Protection Series Report EPS 1/RM/30.

Increase of the amount of a test chemical over time within tissues (usually fatty tissues, following repeated exposure); if the input of a test chemical into the body is greater than the rate at which it is eliminated, the organism accumulates the test chemical and toxic concentrations of a test chemical might be achieved.

Actual test chemical concentrations collected from the animals’ breathing zone; for test mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately;

Suedel BC and JH Rodgers (1994). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13: 1163-1175.

OECD (2008), Bioaccumulation in Sediment-dwelling Benthic Oligochates, Test Guideline No 315, Guidelines for the testing of chemicals, OECD, Paris

EPPO (2003). Environmental Risk Assessment scheme for plant protection products. Soil organisms and functions, EPPO (European Plant Protection Organization) Standards, Bull, OEPP/EPPO 33: 195-208.

ISO 14593 (1998). Water Quality-Evaluation in an aqueous medium of the ultimate biodegradability of organic substances. Method by analysis of released inorganic carbon in sealed vessels.

The corrosivity evaluation could be based on expert judgment using such evidence as: human and animal experience, existing (in vitro) data, e.g. chapter B.40 (10), B.40 bis (11) of this annex or OECD TG 435 (12), pH values, information from similar chemicals or any other pertinent data.

Elendt BP and Bias W-R (1990). Trace Nutrient Deficiency in Daphnia magna Cultured in Standard Medium for Toxicity Testing. Effects on the Optimization of Culture Conditions on Life History Parameters of D. magna. Water Research 24 (9): 1157-1167.

Berg UT and Nyholm N (1996). Biodegradability simulation Studies in semi-continuous activated sludge reactors with low (μg/l range) and standard (ppm range) chemical concentrations. Chemosphere 33 (4): 711-735.

OECD (1981). Subchronic Inhalation Toxicity Testing, Original Test Guideline No 413, Environment Directorate, OECD, Paris.

Expose five groups of animals at a lower concentration(14) (1/4L) with slightly longer exposure durations (factor √2 spaced; see Figure 1).

Painter HA and King EF (1978a). WRc porous-pot method for assessing biodegradability. Technical Report No 70, Water Research Centre, Medmenham, UK.

Dungworth DL, Tyler WS, Plopper CE (1985). Morphological Methods for Gross and Microscopic Pathology (Chapter 9) in Toxicology of Inhaled Material, Witschi, H.P. and Brain, J.D. (eds), Springer Verlag Heidelberg, pp. 229-258.

GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, December, 1989). Publication on the Planning and Structure of Animal Facilities for Institutes Performing Animal Experiments. ISBN 3-906255-06-9.

This timeline shows the different versions taken from EUR-Lex before exit day and during the implementation period as well as any subsequent versions created after the implementation period as a result of changes made by UK legislation.

Suedel BC and Rodgers JH (1994). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13: 1163-1175.

Belfroid A, Sikkenk M, Seinen W, Van Gestel C, Hermens J (1994). The toxicokinetic behavior of chlorobenzenes in earthworms (Eisenia andrei): Experiments in soil. Environ. Toxicol. Chem. 13: 93-99.

Emergence traps are placed on the test beakers. These traps are needed from day 20 to the end of the test. Example of trap used is drawn below:

Details of the equipment used to monitor chamber temperature, humidity, and chamber airflow (i.e. development of a calibration curve).

Nendza M (1991). QSARs of bioaccumulation: Validity assessment of log Kow/log BCF correlations, In: R. Nagel and R. Loskill (eds.): Bioaccumulation in aquatic systems, Contributions to the assessment, Proceedings of an international workshop, Berlin 1990, VCH, Weinheim.

All air concentrations should be reported in units of mass (e.g. mg/l, mg/m3, etc.), units of volume (e.g. ppm, ppb) may also be reported parenthetically;

Tabulation of response data and concentration level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity, time of onset, and duration of effects).

Original (As adopted by EU): The original version of the legislation as it stood when it was first adopted in the EU. No changes have been applied to the text.

Actual test chemical concentrations collected from the animals’ breathing zone; for mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately.

The bioaccumulation factor (BAF) at any time during the uptake phase of this bioaccumulation test is the concentration of test chemical in/on the test organism (Ca in g·kg-1 dry weight of worm) divided by the concentration of the chemical in the surrounding medium (Cs as g·kg-1 of dry weight of soil); the BAF has the units of kg soil·kg-1 worm.

If, for example, half of the volume of the aeration tank is exchanged (a = 0,5) and the mean hydraulic retention time is 6 h, the correction formula is:

Biodegradation kinetics: Generation and use of data for regulatory decision making (1997). Workshop at Port Sunlight, UK. Eds. Hales, SG. Feitjel, T. King, H. Fox, K. and Verstraete, W. 4-6th Sept. 1996. SETAC- Europe, Brussels.

Particle size distribution, mass median aerodynamic diameter (MMAD), and geometric standard deviation (σg), including their methods of calculation. Individual particle size analyses should be reported.

A description (preferably including a diagram) of the equipment used to generate the test atmosphere and to expose the animals to the test atmosphere;

Mechanism of action refers to specific biochemical interactions through which a test chemical produces its effect. Mode of action refers to more general pathways leading to the toxicity of a test chemical.

NF T 20-046 (AFNOR) (September 1985). Chemical products for industrial use — Determination of water solubility of solids and liquids with high solubility — Flask method’.

The elimination kinetics can be modelled using the data from the elimination phase and applying the following model equation and a computer-based non-linear parameter estimation method. If the data points plotted against time indicate a constant exponential decline of the test item concentration in the animals, a one-compartment model (equation 9) can be used to describe the time course of elimination.

Barlow SM, Greig JB, Bridges JW et al (2002). Hazard identification by methods of animal-based toxicology. Food. Chem. Toxicol. 40: 145-191

Enchytraeus crypticus (Westheide & Graefe 1992) is a species belonging to the same group as Enchytraeus luxuriosus. It has not been found to exist with certainty in the field, having only been described from earthworm cultures and compost heaps (Römbke 2003). Its original ecological requirements are therefore not known. However, recent laboratory studies in various field soils have confirmed that this species has a broad tolerance towards soil properties like pH and texture (Jänsch et al. 2005). In recent years, this species has often been used in ecotoxicological studies because of the simplicity of its breeding and testing, e.g. Kuperman et al. 2003). However, it is small (3-12 mm; 7 mm on average (Westheide & Müller 1996), and this makes handling more difficult compared with Enchytraeus albidus. When using this species instead of Enchytraeus albidus, the size of the test vessel can but needs not to be smaller. In addition, it should be considered that this species reproduces very rapidly having a generation time of less than 20 days at 20 ± 2 °C (Achazi et al. 1999) and even quicker at higher temperatures.

Actual test chemical concentrations collected from the animals’ breathing zone; for mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately.

Formulated sediment or reconstituted, artificial or synthetic sediment, is a mixture of materials used to mimic the physical components of a natural sediment.

OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment No 19, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

the water temperature should not differ by more than ± 1,0 °C. The water temperature could be controlled by isothermal room and in that case the room temperature should be confirmed in an appropriate time intervals.

Loizou G, Spendiff M, Barton HA, Bessems J, Bois FY, d’Yvoire MB, Buist H, Clewell HJ 3rd, Meek B, Gundert-Remy U, Goerlitz G, Schmitt W. (2008). Development of good modelling practice for physiologically based pharmacokinetic models for use in risk assessment: The first steps. Regulatory Toxicology and Pharmacology 50: 400 – 411.

The consistency of methods used to determine nominal and actual concentrations, and the relation of actual concentration to nominal concentration should be included in the overall assessment of the study.

Fleming R et al. (1994). Sediment Toxicity Tests for Poorly Water-Soluble Substances. Final Report to them European Commission. Report No: EC 3738. August 1994. WRc, UK.

Regions of the dose–response curve where particularly robust estimation is needed, e.g. in the range of the anticipated BMD or a suspected threshold;

Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (OJ L 396, 30.12.2006, p. 1).

Meller M, Egeler P, Rombke J, Schallnass H, Nagel R and Streit B (1998). Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulfate on Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. Safety 39: 10-20.

BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H.Köpp. Berlin 1995.

Kuperman RG, Checkai RT, Simini M, Phillips CT, Kolakowski JE, Kurnas CW, Sunahara GI (2003). Survival and reproduction of Enchytraeus crypticus (Oligochaeta, Enchytraeidae) in a natural sandy loam soil amended with the nitro-heterocyclic explosives RDX and HMX. Pedobiologia 47: 651-656.

Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity characterization of environmental chemicals by the US National Toxicology Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445.

OECD (2012).Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals. http://www.oecd.org/document/58/0,3343,fr_2649_37407_2348794_1_1_1_37407,00.html

The results of metabolite identification studies show identification of a potentially toxic metabolite that might have been produced by an enzyme pathway induced by the test chemical;

Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33).

If a solvent control is used, there is no need for negative control. The solvent control should contain the highest concentration of solvent added to the soil and should use solvent from the same batch used to make the stock solution. Toxicity and volatility of the solvent, and solubility of the test chemical in the chosen solvent should be the main criteria used for the selection of a suitable solubilising agent.

Jager T, Fleuren RLJ, Hoogendoorn E, de Korte G (2003b). Elucidating the routes of exposure for organic chemicals in the earthworm, Eisenia andrei (Oligochaeta). Environ. Sci. Technol. 37: 3399-3404.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

In the following tables reference is made to GHS (Globally Harmonised System of Classification and Labelling of Chemicals (GHS). The EU equivalent is Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity, the Regulation (EC) No 1272/2008 (9) does not implement Category 5.

Tabulation of particle size data including analytical sample collection data, particle size distribution, and calculations of the MMAD and σg.

Nyholm N, Jacobsen BN, Pedersen BM, Poulsen O, Dambourg A and Schultz B (1992). Removal of micropollutants in laboratory activated sludge reactors. Biodegradability. Wat. Res. 26: 339-353.

all measured data (DOC, COD, specific analyses, pH, temperature, oxygen concentration, suspended solids, N chemicals, if relevant;

OECD (2004a), Enchytraeid reproduction test, Test Guideline No 220, Guidelines for the testing of chemicals, OECD, Paris.

Van Gestel CA and Ma W-C (1990). An approach to quantitative structure-activity relationships (QSARs) in earthworm toxicity studies. Chemosphere 21: 1023-1033.

OECD (2002). Detailed Review Paper on the Appraisal of Test Methods for Sex Hormone Disrupting Chemicals. Series on Testing and Assessment No 21, ENV/JM/MONO(2002)8.

ISO 10634 (1996) Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.

Jager T, Sanchez PA, Muijs B, van der Welde E, Posthuma L (2000). Toxicokinetics of polycyclic aromatic hydrocarbons in Eisenia andrei (Oligochaeta) using spiked soil. Environ. Toxicol. Chem. 19: 953-961.

Belfroid A, Meiling J, Drenth H, Hermens J, Seinen W, Van Gestel C (1995). Dietary uptake of superlipophilic compounds by earthworms (Eisenia andrei). Ecotox. Environ. Safety 31: 185-191.

ISO 14593, (1999) Water Quality — Evaluation of ultimate aerobic biodegradability of organic compounds in an aerobic medium-method by analysis of inorganic carbon in sealed vessels (CO2 headspace test).

Harkema JR (1990). Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 85: 231-238.

What areEEAsizers used for

US EPA (1996). Fate, Transport and Transformation Test Guideline. 835. 3100. Aerobic aquatic biodegradation. Office, Prevention Pesticides and Toxic Substances Washington, DC.

BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H.Köpp. Berlin 1995.

The elimination phase is the time, following the transfer of the test organisms from a contaminated medium to a medium free of the test item, during which the elimination (or the net loss) of the chemical from the test organisms is studied.

Sims R W and Gerard BM (1985). Earthworms, In: Kermack, D. M. & Barnes, R. S. K. (Hrsg.): Synopses of the British Fauna (New Series) No 31.171 S. London: E. J. Brill/Dr W. Backhuys.

the graph of percentage degradation against time for the test and reference chemicals, the lag phase, degradation phase, 10-d window and slope;

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

Battersby NS, Ciccognani D, Evans MR, King D, Painter HA, Peterson DR and Starkey M (1999). An “inherent” biodegradability test for oil products: description and results of an international ring test. Chemosphere 38: 3219-3235.

(2) It is necessary to update Regulation (EC) No 440/2008 to include with priority new and updated alternative test methods recently adopted by the OECD, in order to obtain a reduction of the number of animals to be used for experimental purposes, in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes(3) and Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes(4).

concentration of DOC (or COD) in the influent due to the test chemical, preferably estimated from the concentration in, and volume added, of the stock solution (mg/l);

Romijn CA.FM, Luttik R, Van De Meent D, Slooff W, Canton JH (1993). Presentation of a General Algorithm to Include Effect Assessment on Secondary Poisoning in the Derivation of Environmental Quality Criteria, Part 2: Terrestrial food chains. Ecotox. Envir. Safety 27: 107-127.

An explanation should be provided if there was a need to humanely sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (7).

Sample BE, Suter DW, Beauchamp JJ, Efroymson RA (1999). LITERATURE-derived bioaccumulation models for earthworms: Development and validation. Environ. Toxicol. Chem. 18: 2110-2120.

The steady state bioaccumulation factor (BAFss) is the BAF at steady state and does not change significantly over a prolonged period of time, the concentration of the test chemical in the surrounding medium (Cs as g.kg-1 of dry weight of soil) being constant during this period of time.

Schlosser H-J and Riepert F (1992). Entwicklung eines Prüfverfahrens für Chemikalien an Bodenraubmilben (Gamasina), Teil 2: Erste Ergebnisse mit Lindan und Kaliumdichromat in subletaler Dosierung. Zool. Beitr. NF 34: 413-433.

Weingand K, Brown G, Hall R et al. (1996). Harmonisation of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. 29: 198-201.

For dermal studies, also include data on test chemical recovery from treated skin, skin washes, and residual radioactivity in the skin covering apparatus and metabolic unit as well as results of the dermal washing study. For further discussion, see paragraphs 74-77.

A process is linear in terms of kinetics when all transfer rates between compartments are proportional to the amounts or concentrations present, i.e. first order. Consequently, clearance and distribution volumes are constant, as well as half-lives. The concentrations achieved are proportional to the dosing rate (exposure), and accumulation is more easily predictable. Linearity/Non-linearity can be assessed by comparing the relevant parameters, e.g. AUC, after different doses or after single and repeated exposure. Lack of dose dependency may be indicative of saturation of enzymes involved in the metabolism of the compound, an increase of AUC after repeated exposure as compared to single exposure may be an indication for inhibition of metabolism and a decrease in AUC may be an indication for induction of metabolism [see also (11)].

GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, 2006). Microbiological monitoring of laboratory animals in various housing systems.

Legislation.gov.uk publishes the UK version. EUR-Lex publishes the EU version. The EU Exit Web Archive holds a snapshot of EUR-Lex’s version from IP completion day (31 December 2020 11.00 p.m.).

Plateau phase is the phase in which the maximal degradation has been reached and the biodegradation curve has levelled out.

The peat is air dried and ground to a fine powder. A suspension of the required amount of peat powder in deionised water is prepared using a high-performance homogenising device. The pH of this suspension is adjusted to 5,5 ± 0,5 with CaCO3. The suspension is conditioned for at least two days with gentle stirring at 20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is measured again and should be 6,0 ± 0,5. Then the peat suspension is mixed with the other constituents (sand and kaolin clay) and deionised water to obtain a homogeneous sediment with a water content in a range of 30-50 per cent of dry weight of the sediment. The pH of the final mixture is measured once again and is adjusted to 6,5 to 7,5 with CaCO3 if necessary. Samples of the sediment are taken to determine the dry weight and the organic carbon content. Then, before it is used in the chironomid toxicity test, it is recommended that the formulated sediment be conditioned for seven days under the same conditions which prevail in the subsequent test.

Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283.

Carmichael NG, Barton HA, Boobis AR et al (2006). Agricultural Chemical Safety Assessment: A Multisector Approach to the Modernization of Human Safety Requirements. Critical Reviews in Toxicology 36: 1-7.

Statistical relation, including estimate for the exponent n (C × t protocol). The name of the statistical software used should be provided;

the nominal test concentrations, the means of the measured values and their standard deviations in the test vessels, and the method by which these values were obtained;

A description (preferably including a diagram) of the equipment used to generate the test atmosphere and to expose the animals to the test atmosphere.

Boobis AR, Cohen SM, Dellarco V, McGregor D, Meek ME, Vickers C, Willcocks D, Farland W (2006). IPCS Framework for analyzing the Relevance of a Cancer Mode of Action for Humans. Crit. Rev. in Toxicol, 36: 793-801.

Stephenson GL, Kaushik A, Kaushik NK, Solomon KR, Steele T, Scroggins RP (1998). Use of an avoidance-response test to assess the toxicity of contaminated soils to earthworms. In: Advances in earthworm ecotoxicology. S. Sheppard, J. Bembridge, M. Holmstrup, L. Posthuma (eds.). Setac Press, Pensacola, 67-81.

Discuss any potential species and sex differences regarding the disposition and/or biotransformation of the test chemical;

source of the constituents of the artificial soil or – if natural media are used – origin of the soil, description of any pre-treatment, results of the controls (survival, biomass development, reproduction), soil characteristics (pH, total organic carbon content, particle size distribution (percent sand, silt, and clay), WHCmax, percent water content at start and at end of the test, and any other measurements made);

Details of the equipment used to collect samples for determination of chamber concentration and particle size distribution.

Details of the chemical analytical method used and method validation (including efficiency of recovery of test chemical from the sampling medium)

Access essential accompanying documents and information for this legislation item from this tab. Dependent on the legislation item being viewed this may include:

Kuper CF, Koornstra PJ, Hameleers DMH, Biewenga J, Spit BJ, Duijvestijn AM, Breda Vriesman van PJC, Sminia T (1992). The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219-224.

Nominal concentrations (total mass of test chemical generated into the inhalation chamber divided by the volume of air passed through the chamber);

4-5 % (dry weight) peat: as close to pH 5,5 to 6,0 as possible; it is important to use peat in powder form, finely ground (particle size ≤ 1 mm) and only air dried.

Details of test chemical preparation, including details of any procedures used to reduce the particle size of solids or to prepare solutions of the test chemical.

Birch RR and Fletcher RJ (1991). The application of dissolved inorganic carbon measurements to the study of aerobic biodegradability. Chemosphere 23: 507-524.

SETAC (1993). Guidance Document on Sediment toxicity Tests and Bioassays for Freshwater and Marine Environments. From the WOSTA Workshop held in the Netherlands.

Details of test chemical preparation, including details of any procedures used to reduce the particle size of solid materials or to prepare solutions of the test chemical.

for natural sediment, location and description of sediment sampling site, including, if possible, contamination history; characteristics: pH, organic carbon content, C/N ratio and granulometry (if appropriate).

(5) The measures provided for in this Regulation are in accordance with the opinion of the Committee established under Article 133 of Regulation (EC) No 1907/2006,

Egeler Ph, Gilberg D, Scheffczyk A, Moser Th and Römbke J (2009). Validation of a Soil Bioaccumulation Test with Terrestrial Oligochaetes by an International Ring Test (Validierung einer Methode zur standardisierten Messung der Bioakkumulation mit terrestrischen Oligochaeten). Report to the Federal Environmental Agency (Umweltbundesamt Dessau-Rosslau), R & D No.: 20467458: 149 pp. Available for download at: http://www.oecd.org/dataoecd/12/20/42552727.pdf.

The elimination of a test chemical is the loss of this chemical from the test organism tissue by active or passive processes that occurs independently of presence or absence of the test item in the surrounding medium.

the use of formulated sediment allows for comparisons of toxicity and ranking substances accordingly: toxicity data from tests with natural and artificial sediments were comparable for several chemicals (2).

Individual body weights of animals collected on study; date and time of death if prior to scheduled euthanasia, time course of onset of signs of toxicity and whether these were reversible for each animal

Combes RD, Gaunt I, Balls M (2004). A Scientific and Animal Welfare Assessment of the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union REACH System. ATLA 32: 163-208.

Nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT))

Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity characterization of environmental chemicals by the US National Toxicology Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445.

Weingand K, Brown G, Hall R et al. (1996). Harmonisation of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. 29: 198-201.

Carmichael NG, Enzmann H, Pate I, Waechter F (1997). The Significance of Mouse Liver Tumor Formation for Carcinogenic Risk Assessment: Results and Conclusions from a Survey of 10 Years of Testing by the Agrochemical Industry. Environ Health Perspect. 105:1196-1203.

ISO 14592 (ISO/TC 147/SC5/WG4, N264) (1998). Water Quality — Evaluation of the aerobic biodegradability of organic compounds at low concentrations in water.

Stumpf WE (2005). Drug localization and targeting with receptor microscopic autoradiography. J. Pharmacological and Toxicological Methods 51: 25-40.

Regions of the dose–response curve where particularly robust estimation is needed, e.g. in the range of the anticipated BMD or a suspected threshold;

When the UK left the EU, legislation.gov.uk published EU legislation that had been published by the EU up to IP completion day (31 December 2020 11.00 p.m.). On legislation.gov.uk, these items of legislation are kept up-to-date with any amendments made by the UK since then.

Amorim M (2000). Chronic and toxicokinetic behavior of Lindane (γ-HCH) in the Enchytraeid Enchytraeus albidus. Master thesis, University Coimbra.

IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.

The effluent concentration is independent of that in the influent (S0); hence, the percentage biodegradation varies with the influent concentration, S0.

Ten Berge WF and Zwart A (1989). More Efficient Use of Animals in Acute Inhalation Toxicity Testing. J. Haz. Mat. 21: 65-71.

Ca and Cs (with standard deviation and range, if appropriate) for all sampling times (Ca expressed in g kg–1 wet and dry weight of whole body, Cs expressed in g kg–1 wet and dry weight of soil). If a biota-soil accumulation factor (BSAF) is required (e.g. for comparison of results from two or more tests performed with animals of differing lipid content), Ca may additionally be expressed as g kg–1 lipid content of the organism, and Cs may be expressed as g kg–1 organic carbon (OC) of the soil;

Phillips DJH (1993). Bioaccumulation. In: Handbook of Ecotoxicology Vol. 1. Calow P. (ed.). Blackwell Scientific Publ., Oxford. 378-396.

Naylor C and C Rodrigues (1995). Development of a test method for Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303.

(Usually enzymatic) chemical conversion of a test chemical of interest into a different chemical within the body. Synonymous with “metabolism”.

OECD (2009). Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured Nanomaterials, Series on the Safety of Manufactured Nanomaterials No 15, ENV/JM/MONO(2009)21, OECD, Paris.

Williams DA (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics, 27: 103-117.

Dunnett CW (1964). A multiple comparisons procedure for comparing several treatments with a control. J. Amer. Statis. Assoc. 50: 1096-1121.

Proteins that catalyse chemical reactions. Isozymes are enzymes that catalyse similar chemical reactions but differ in their amino acid sequence.

normally gives a concentration of 4 mg/l suspended solids in the final mixture when activated sludge is used, concentrations up to 30 mg/l may be used but may significantly increase CO2 production of the blanks (26);

This information is only given for the convenience of users. Other equivalent computer programmes may be used if they can be shown to produce the same results.

The time taken for the concentration of the test chemical to decrease by one-half in a compartment. It typically refers to plasma concentration or the amount of the test chemical in the whole body.

the solution is given sufficient time for the suspended solid substance to settle out. During the settling period, the concentration of the test substance is monitored;

GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, 2006). Microbiological monitoring of laboratory animals in various housing systems.

Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM.(2002) Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol. Pathol. 30, 524-533.

Ideally, exposure of animals at the next concentration level should be delayed until there is reasonable confidence of survival for previously treated animals. This allows the study director to adjust the target concentration and durations for the next exposure session.

Biodegradation kinetics: Generation and use of data for regulatory decision making (1997). Workshop at Port Sunlight, UK. Eds. Hales SG, Feitjel T, King H, Fox K, Verstraete W. 4-6th Sept. 1996. SETAC- Europe, Brussels.

OECD (2005), In Vitro Membrane Barrier Test Method For Skin Corrosion. OECD Guideline for Testing of Chemicals No 435, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

Directive 2010/63/EU of the European parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33).

Other species of the Enchytraeus genus are also suitable, especially Enchytraeus luxuriosus. This species is a true soil inhabitant, which has been newly described in (65). If other species of Enchytraeus are used, they should be clearly identified and the rationale for the selection of the species should be reported.

Jager T (1998). Mechanistic approach for estimating bioconcentration of organic chemicals in earthworms (Oligochaeta). Environ. Toxicol. Chem. 17: 2080-2090.

Agents or solvents used in (c), (d) and (e) have to be tested for any stimulatory or inhibitory effect on microbial activity (see paragraph 42(b).)

Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal J-M, van de Vorstenbosch C. 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology 21:15-23.

ASTM International/E1706-00 (2002). Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates. pp 1125-1241. In ASTM International 2002 Annual Book of Standards. Volume 11.05. Biological Effects and Environmental Fate; Biotechnology; Pesticides. ASTM International, West Conshohocken, PA.

Your payment information is processed securely. We do not store credit card details nor have access to your credit card information.

preparation of the test water (if reconstituted water is used) and characteristics (oxygen concentration, pH, conductivity, hardness, etc. at the start of the test);

A supply of CO2 free air — this can be prepared by passing air through soda lime granules or by using an 80 % N2/20 % 02 gas mixture (optional) (see paragraph 28);

The dry constituents for preparation of the artificial sediment may be stored in a dry and cool place at room temperature. The formulated (wet) sediment should not be stored prior to its use in the test. It should be used immediately after the 7 days conditioning period that ends its preparation.

The elimination rate constant (ke) is usually determined from the elimination curve (i.e. a plot of the concentration of the test item in the worms during the elimination phase). The uptake rate constant ks is then calculated given ke and a value of Ca which is derived from the uptake curve – See Appendix 2 for a description of these methods. The preferred method for obtaining BAFK and the rate constants, ks, and ke, is to use non-linear parameter estimation methods on a computer. If the elimination is obviously not first-order, then more complex models should be employed.

Testing with 6 animals of the more susceptible sex only i.e. the lower boundary estimates of the toxic class should be based on 6 animals per test concentration group, regardless of sex.

the air-lift pump could be replaced by a peristaltic pump, thus enabling a sludge recirculation flow which about equals the influent flow to be used and allowing development of an anaerobic zone in the settled sludge (the geometry of the air-lift pump limits the minimum flow rate of returned sludge to be about 12-fold that of the influent);

Woutersen RA, Garderen-Hoetmer A, van Slootweg PJ, Feron VJ (1994). Upper respiratory tract carcinogenesis in experimental animals and in humans. In: Waalkes MP and Ward JM (eds) Carcinogenesis. Target Organ Toxicology Series, Raven Press, New York, 215-263.

Carmichael NG, Barton HA, Boobis AR et al. (2006). Agricultural Chemical Safety Assessment: A Multisector Approach to the Modernization of Human Safety Requirements. Critical Reviews in Toxicology 36: 1-7.

Sousa JP, Loureiro S, Pieper S, Frost M, Kratz W, Nogueira AJA, Soares AMVM (2000). Soil and plant diet exposure routes and toxicokinetics of lindane in a terrestrial isopod. Environ. Toxicol. Chem. 19: 2557–2563.

Barton HA, et al. (2006). The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments, Critical Reviews in Toxicology 36: 9-35.

Details of the equipment used to collect samples for determination of chamber concentration and particle size distribution.

Tabulate and discuss the identification and magnitude of metabolites, rates of clearance, bioaccumulation potential, and level of tissue residues of parent, and/or metabolite(s), as well as possible dose-dependent changes in TK parameters, as appropriate;

(Whole-body autoradiography): Used to determine qualitatively and/or quantitatively the tissue localisation of a radioactive test chemical, this technique uses X-ray film or more recently digital phosphorimaging to visualize radioactively labelled molecules or fragments of molecules by recording the radiation emitted within the object under study. Quantitative whole-body autoradiography, compared to organ dissection, may have some advantages for the evaluation of test chemical distribution and the assessment of overall recovery and resolution of radioactive material in tissues. One significant advantage, for example, is it can be used in a pigmented animal model to assess possible association of the test chemical with melanin, which can bind certain molecules. However, while it may provide convenient whole body overviews of the high-capacity-low-affinity binding sites, this technique might be limited in recognising specific target sites such as receptor-binding sites where relatively high-resolution and high-sensitivity are needed for detection. When autoradiography is used, experiments intended to determine mass balance of administered compound should be conducted as a separate group or in a separate study from the tissue distribution experiment, where all excreta (which may also include expired air) and whole carcasses are homogenised and assayed by liquid scintillation counting.

The dry constituents of the artificial soil may be stored at room temperature until use. The prepared, pre-moistened soil may be stored in a cool place for up to three days prior to spiking; care should be taken to minimise evaporation of water. Soil spiked with the test item should be used immediately unless there is information indicating that the particular soil can be stored without affecting the toxicity and bioavailability of the test item. Samples of spiked soil may then be stored under the conditions recommended for the particular test item until analysis.

“It should be noted that the term ‘chemical’ is used broadly in the UNCED agreements and subsequent documents to include substances, products, mixtures, preparations, or any other terms that may be used in existing systems to denote coverage”.”

Toxicokinetics, and dose ranges where metabolic induction, saturation, or nonlinearity between external and internal doses does or does not occur;

Validation is a scientific process designed to characterise the operational requirements and limitations of a test method and to demonstrate its reliability and relevance for a particular purpose.

For a number of measurements in serum and plasma, most notably for glucose, overnight fasting would be preferable. The major reason for this preference is that the increased variability which would inevitably result from non-fasting, would tend to mask more subtle effects and make interpretation difficult. On the other hand, however, overnight fasting may interfere with the general metabolism of the animals and, particularly in feeding studies, may disturb the daily exposure to the test chemical. If overnight fasting is adopted, clinical biochemical determinations should be performed after the conduct of functional observations in week 4 of the study.

Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1: 233-236.

Spacie A and Hamelink JL (1982). Alternative models for describing the bioconcentration of organics in fish. Environ. Toxicol. Chem. 1, 309-320.

Griffiths SA, Parkinson C, McAuslane JAN and Lumley CE (1994). The utility of the second rodent species in the carcinogenicity testing of pharmaceuticals. The Toxicologist 14(1):214.

OECD (2005). In Vitro Membrane Barrier Test Method for Skin Corrosion. OECD Guideline for testing of chemicals No 435, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

OECD. (2006). Report of the Validation of the Updated Test Guideline 407: Repeat Dose 28-day Oral Toxicity Study in Laboratory Rats. Series on Testing and Assessment No 59, ENV/JM/MONO(2006)26.

Thyroid activity is the capability of a chemical to act like a natural thyroid hormone (e.g. T3) in a mammalian organism.

Either maximal (peak) concentration in blood (plasma/serum) after administration or maximal (peak) excretion (in urine or faeces) after administration.

Petersen H and Luxton M (1982). A comparative analysis of soil fauna populations and their role in decomposition processes. Oikos 39: 287-388.

preparation of the test water (if reconstituted water is used) and characteristics (oxygen concentration, pH, conductivity, hardness, etc. at the start of the test);

Regions of the dose–response curve where particularly robust estimation is needed, e.g. in the range of the anticipated BMD or a suspected threshold;

all information relevant for the interpretation of the results, in particular with regard to impurities and physical state of the test substance.

fresh sludge or flocculant (for example 2 ml/vessel of 50 g/l FeCl3) could be added at regular intervals, e.g. weekly, but ascertain that no reaction or precipitation of the test chemical occurs with FeCl3;

OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment No 19. Available at: [http://www.oecd.org/env/testguidelines]

Details of test chemical preparation, including details of any procedures used to reduce the particle size of solid materials or to prepare solutions of the test chemical. In cases where mechanical processes may have altered test chemical composition, include the results of analyses to verify the composition of the test chemical

Kawai K (1986). Fundamental studies on Chironomid allergy. I. Culture methods of some Japanese Chironomids (Chironomidae, Diptera). Jp. J. Sanit. Zool. 37(1): 47-57.

National Research Council, 1985. Guide for the care and use of laboratory animals. NIH Publication No 86-23. Washington D.C., US. Dept. of Health and Human Services.

Crofton KM, Howard JL, Moser VC, Gill MW, Reiter LW, Tilson HA, MacPhail RC (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13: 599-609.

OECD (1995). Report of the Consultation Meeting on Sub-chronic and Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working document, Environment Directorate, OECD, Paris.

Garlon 4 Ultra Herbicide is the industry standard for herbicide control. Made by Dow AgroSciences, this product keeps right-of-ways, roadsides and other noncrop sites clear of unwanted weeds. Using triclopyr as its active ingredient, Garlon 4 Ultra Herbicide controls 80 different plants that other methods of control would have a difficult time dealing with.

OECD (2012). Guidance Document on the Design and Conduct of Chronic Toxicity and Carcinogenicity Studies, Supporting Test Guidelines 451, 452 and 453 — Second edition. Series on Testing and Assessment No 116, available on the OECD public website for Test Guideline at www.oecd.org/env/testguidelines.

Birch RR (1991). Prediction of the fate of detergent chemicals during sewage treatment. J. Chem. Tech. Biotechnol., 50: 411-422.

Römbke J and Moser Th (1999). Organisation and performance of an international ring-test for the validation of the Enchytraeid reproduction test. UBA-Texte 4/99: 373 pp.

OECD (Paris, 1992). Chairman’s Report of the Meeting of the ad hoc Working Group of Experts on Systemic Short-term and (Delayed) Neurotoxicity.

IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.

Crissman JW, Goodman DG, Hildebrandt PK et al. (2004). Best Practices Guideline: Toxicological Histopathology. Toxicologic Pathology 32: 126-131.

Venter JM and Reinecke AJ (1988). The life-cycle of the compost-worm Eisenia fetida (Oligochaeta). South African J. Zool. 23: 161-165.

Enchytraeids of the species Enchytraeus albidus (as well as other Enchytraeus species) can be bred in large plastic boxes (e.g. 30 × 60 × 10 cm or 20 × 12 × 8 cm which is suitable for culture of worms of small size) filled with a mixture of artificial soil and commercially available, uncontaminated garden soil free of additives. Compost material should be avoided since it could contain toxic chemicals like heavy metals. Fauna should be removed from the breeding soil before use by three times deep-freezing. Pure artificial soil can also be used but the reproduction rate could be slower compared to that obtained with mixed substrates. The substrate should have a pH of 6,0 ± 0,5. The culture is kept in an incubator at a temperature of 15 ± 2 °C without light. In any case, a temperature higher than 23 °C should be avoided. The artificial/natural soil moisture should be moist but not wet. When the soil is gently pressed by hand, only small drops of water should appear. In any case, anoxic conditions should be avoided (e.g. if a lid is used, the number of lid holes should be high enough to provide sufficient exchange of air). The breeding soil should be aerated by carefully mixing it once per week.

Enchytraeus albidus is commercially available, sold as food for fish. It should be checked whether the culture is contaminated by other, usually smaller species (60). If contamination occurs, all worms should be washed with water in a Petri dish. Large adult specimens of Enchytraeus albidus are then selected (by using a stereomicroscope) to start a new culture. All other worms are discarded. Its life cycle is short as maturity is reached between 33 days (at 18 °C) and 74 days (at 12 °C). Only cultures which have been kept in the laboratory for at least 5 weeks (one generation) without problems should be used for a test.

where foc is the fraction of soil organic carbon, and flip is the fraction of worm lipid, both preferably determined on samples taken from the test, and based either on dry weight or on wet weight, respectively.

In the case of paired organs, e.g. kidney, adrenal, both organs should be preserved. The clinical and other findings may suggest the need to examine additional tissues. Also, any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as set out for the oral route should be examined, and specific sampling and preservation of the skin from the site of application is necessary. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (8) and Chapter B.29 of this Annex (9). For other organs/tissues (and in addition to the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.

actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test chemical concentration (mg/kg or ppm) to the actual dose, if applicable;

OECD (1998). Report of the First Meeting of the OECD Endocrine Disrupter Testing and Assessment (EDTA) Task Force, 10th-11 March 1998, ENV/MC/CHEM/RA(98)5.

Identification of a no-observed-adverse-effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD);

Process(es) of uptake of chemicals into or across tissues. Absorption refers to parent compound and all its metabolites. Not to be confused with “bioavailability”.

The intermediate concentration level(s) should be spaced to produce a gradation of toxic effects between that of the low and high concentration.

Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet;

The study should not be extended beyond the point when the data available from the study are no longer sufficient to enable a statistically valid evaluation to be made.

Tabulation of response data and concentration level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity, and duration of effects);

Expose five groups of animals at a lower concentration(14) (1/2L) with slightly longer exposure durations (factor √2 spaced; see Figure 1).

Organic carbon content of the final mixture should be 2 % (± 0,5 %) and is to be adjusted by the use of appropriate amounts of peat and sand, according to (a) and (c).

Doe JE, Boobis AR, Blacker A et al (2006). A Tiered Approach to Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36: 37-68.

the tests can be initiated at any time without encountering seasonal variability in the test sediment and there is no need to pre-treat the sediment to remove indigenous fauna; the use of formulated sediment also reduces the cost associated with the field collection of sufficient amounts of sediment for routine testing;

Since natural soils from a particular source may not be available throughout the year, and indigenous organisms as well as the presence of micro-pollutants can influence the test, an artificial substrate, the artificial soil according to Chapter C.8 of this Annex, Toxicity for Earthworms (48), is recommended for use in this test. Several test species can survive, grow, and reproduce in this soil, and maximum standardisation as well as intra- and interlaboratory comparability of test and culture conditions are provided.

OECD (2000). Guidance Document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation, Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris.

The respirability of particles in light of the overall findings should be addressed, especially if the particle-size criteria could not be met

Widianarko B and Van Straalen NM (1996). Toxicokinetics-based survival analysis in bioassays using nonpersistent chemicals, Environ. Toxicol. Chem. 15: 402–406.

Arnold CG, Widenhaupt A, David MM, Müller SR, Haderlein SB, Schwarzenbach RP (1997). Aqueous speciation and 1-octanol-water partitioning of tributyl- and triphenyltin: effect of pH and ion composition. Environ. Sci. Technol. 31: 2596-2602.

De Bruijn JHM, Busser F, Seinen W, Hermens J. (1989). Determination of octanol/water partition coefficients with the “slow-stirring” method. Environ. Toxicol. Chem. 8: 499-512.

Rhomberg LR, Baetcke K, Blancato J, Bus J, Cohen S, Conolly R, Dixit R, Doe J, Ekelman K, Fenner-Crisp P, Harvey P, Hattis D, Jacobs A, Jacobson-Kram D, Lewandowski T, Liteplo R, Pelkonen O, Rice J, Somers D, Turturro A, West W, Olin S (2007). Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose Selection Crit Rev. Toxicol. 37 (9): 729 – 837.

Combes RD, Gaunt, I, Balls M (2004). A Scientific and Animal Welfare Assessment of the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union REACH System. ATLA 32: 163-208.

Emergence traps are placed on the test beakers. These traps are needed from day 20 to the end of the test. Example of trap used is drawn below:

Tomlin AD (1984). The earthworm bait market in North America. In: Earthworm Ecology — from Darwin to vermiculture. Satchell, J.E. (ed.), Chapman & Hall, London. 331-338 pp.

Bruce and Versteeg (1992). A statistical procedure for modelling continuous toxicity data. Environmental Toxicology and Chemistry 11: 1485-1494.

Then the normal biological test (with sludge) is performed under operating conditions identical to those in the stripping study. DOC or COD measurements are also made to check that the units are performing efficiently. Occasional analyses are made to determine the test chemical in the influent, effluent and exit gas in the first part of the test; after acclimation more frequent analyses are made. Again, from the data in the steady state, the percentage of removal of the test chemical from the liquid phase by all processes (RT) (physical and biological) may be calculated, as well as the proportion (RV) stripped from the system.

conversion from diet/drinking water test chemical concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable;

Landrum PF (1989). Bioavailability and toxicokinetics of polycyclic aromatic hydrocarbons sorbed to sediments for the amphipod Pontoporeia hoyi. Environ. Sci. Toxicol. 23: 588-595.

Fischer W, Gerike P, Holtmann W (1975). Biodegradability Determinations via Unspecific Analyses (Chemical Oxygen Demand, DOC) in Coupled Units of the OECD Confirmatory Test. I The test. Wat. Res. 9: 1131-1135.

However, it should be noted that if there is an interaction suspected between hardness ions and the test substance, lower hardness water should be used (and thus, Elendt Medium M4 must not be used in this situation).

Directive 2010/63/EU of the European parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33).

In certain cases it may be necessary to increase the concentration (2L) over a new time range with a still finer grid using a geometric division of exposure periods with a factor 1,4 (√2) around the time according to the minimum lethal concentration level observed during the first exposure. The minimum exposure duration should preferably exceed 5 minutes; the maximum exposure duration should not exceed 8 hours.

Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet.

US EPA (1996). Fate, Transport and Transformation Test Guideline. 835.3110 Carbon dioxide evolution test. Office, Prevention Pesticides and Toxic Substances Washington, DC.

This version of this Regulation was derived from EUR-Lex on IP completion day (31 December 2020 11:00 p.m.). It has not been amended by the UK since then. Find out more about legislation originating from the EU as published on legislation.gov.uk.

OECD (2002). Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies, Series on Testing and Assessment No 35 and Series on Pesticides No 14, ENV/JM/MONO(2002)19, OECD, Paris.

OECD. Draft Summary record of the meeting of the Task Force on Endocrine Disrupters Testing and Assessment. ENV/JM/TG/EDTA/M(2006)3.

The respirability of particles in light of the overall findings should be addressed, especially if the particle-size criteria could not be met.

Glass serum bottles, sealed with butyl rubber stoppers and crimp-on aluminium seals. The recommended size is “125 ml” which have a total volume of around 160 ml (in this case the volume of each bottle should be known to be 160 ± 1 ml). A smaller size of vessel may be used when the results fulfil the conditions described in paragraph 66 and 67;

the concentration of the diluted stock solution is measured. If the measured concentration is consistent with the dilution, the diluted stock solution can be employed in the slow-stirring experiment.

Birch RR, Biver C, Campagna R, Gledhill WE, Pagga U, Steber J, Reust H, and Bontinck WJ (1989). Screening of chemicals for anaerobic biodegradation. Chemosphere 19: 1527-1550.

ISO 10634 (1995) Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.

Jager T, Baerselman R, Dijkman E, De Groot AC, Hogendoorn EA, DeJong A, Kruitbosch JAW, Peijnenburg W J G. M (2003a). Availability of polycyclic aromatic hydrocarbons to earthworms (Eisenia andrei, Oligochaeta) in field-polluted soils and soil-sediment mixtures. Environ. Toxicol. Chem. 22: 767-775.

Optionally, calculate the percentage elimination DOC or COD due to the organic medium plus test chemical in the test unit from the equation:

The recommended test species is Eisenia fetida (Savigny 1826), belonging to the family Lumbricidae. Since 1972 it is divided into two subspecies (Eisenia fetida and Eisenia andrei (10)). According to Jaenike (36), they are true, separate species. Eisenia fetida is easily recognised by its bright intersegmental yellow stripes whereas Eisenia andrei has a uniform, dark red colour. Originating probably from the region of the Black Sea, they are distributed worldwide today, especially in anthropogenically modified habitats like compost heaps. Both can be used for ecotoxicological as well as bioaccumulation tests.

Tabulation of particle size data including analytical sample collection data, particle size distribution and calculations of the MMAD and σg

Barton HA, Pastoor TP, Baetcke T et al. (2006). The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments. Critical Reviews in Toxicology 36: 9-35.

Particular emphasis should be made to the description of methods used to meet the criteria of this Test Method, e.g. the limit concentration or the particle size.

BAF (expressed in kg soil·kg–1 worm), soil uptake rate constant ks (expressed in g soil kg–1 of worm day–1), and elimination rate constant ke (expressed in day–1); BSAF (expressed in kg soil OC kg–1 worm lipid content) may be reported additionally;

C. riparius and C. yoshimatsui emergence to adults from control vessels should occur between 12 and 23 days after their insertion into the vessels; for C. tentans, a period of 20 to 65 days is necessary.

Environment Canada (1997). Test for Growth and Survival in Sediment using Larvae of Freshwater Midges (Chironomus tentans or Chironomus riparius). Biological Test Method. Report SPE 1/RM/32. December 1997.

Latest Available (revised):The latest available updated version of the legislation incorporating changes made by subsequent legislation and applied by our editorial team. Changes we have not yet applied to the text, can be found in the ‘Changes to Legislation’ area.

Painter HA and Bealing D (1989). Experience and data from the OECD activated sludge simulation test. pp. 113-138, In: Laboratory tests for simulation of water treatment processes. CEC Water Pollution Report 18. Eds. Jacobsen BN, Muntau H, Angeletti G.

the experimental variability is reduced because it forms a reproducible “standardised matrix” and the need to find uncontaminated and clean sediment sources is eliminated;

Blank controls (denoted FB) containing only the test medium plus inoculum; any chemicals, solvents, agents or glass fibre filters used to introduce the test chemical into the test vessels must also be added;

Mery S, Gross EA, Joyner DR, Godo M, Morgan KT (1994). Nasal diagrams: A tool for recording the distribution of nasal lesions in rats and mice. Toxicol. Pathol. 22: 353-372.

Identification of a no-observed-adverse-effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD),

Nominal concentrations (total mass of test chemical generated into the inhalation chamber divided by the volume of air passed through the chamber).

This revised Test Method B.8 has been designed to fully characterise test chemical toxicity by the inhalation route following repeated exposure for a limited period of time (28 days), and to provide data for quantitative inhalation risk assessments. Groups of at least 5 male and 5 female rodents are exposed 6 hours per day for 28 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. This method allows the study director the flexibility to include satellite (reversibility) groups, bronchoalveolar lavage (BAL), neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterise the toxicity of a test chemical.

Beek B, Boehling S, Bruckmann U, Franke C, Joehncke U, Studinger G (2000). The assessment of bioaccumulation. In Hutzinger, O. (editor), The Handbook of Environmental Chemistry, Vol. 2 Part J (Vol. editor: B. Beek): Bioaccumulation — New Aspects and Developments. Springer-Verlag Berlin Heidelberg: 235-276.

The identification of the carcinogenic properties of a test chemical, resulting in an increased incidence of neoplasms, increased proportion of malignant neoplasms or a reduction in the time to appearance of neoplasms, compared with concurrent control groups;

Römbke J, Egeler P, Füll C (1997). Literaturstudie über Bioakkumulationstests mit Oligochaeten im terrestrischen Medium. Bericht für das UBA F + E 206 03 909, 86 S.

Hess RA, Moore BJ (1993). Histological Methods for the Evaluation of the Testis. In: Methods in Reproductive Toxicology, Chapin RE and Heindel JJ (eds). Academic Press: San Diego, CA, pp. 52-85.

Non-equilibrium state of an open system in which all forces acting on the system are exactly counter-balanced by opposing forces, in such a manner that all its components are stationary in concentration although matter is flowing through the system.

Dose is the amount of test chemical administered. The dose is expressed as weight of test chemical per unit body weight of test animal per day (e.g. mg/kg body weight/day), or as a constant dietary concentration.

Termination of the study should be considered when the number of survivors in the lower dose groups or the control group falls below 25 per cent.

The Whole Regulation you have selected contains over 200 provisions and might take some time to download. You may also experience some issues with your browser, such as an alert box that a script is taking a long time to run.

Hund-Rinke K, Römbke J, Riepert F, Achazi R (2000). Beurteilung der Lebensraumfunktion von Böden mit Hilfe von Regenwurmtests. In: Toxikologische Beurteilung von Böden. Heiden, S., Erb, R., Dott, W. & Eisentraeger, A. (eds.), Spektrum Verl., Heidelberg, 59-81.

Refers to the means by which chemicals are administered to the body (e.g. orally by gavage, orally by diet, dermal, by inhalation, intravenously, etc.).

Geographical Extent: Indicates the geographical area that this provision applies to. For further information see ‘Frequently Asked Questions’.

For inhalation studies, also include data on recovery of test chemical from lungs and nasal tissues (8). For further discussion, see paragraph 78.

Age-related sensitivity due to differences in the status of the blood-brain barrier, the kidney and/or detoxification capacities;

Pauluhn J and Thiel A (2007). A Simple Approach to Validation of Directed-Flow Nose-Only Inhalation Chambers. J. Appl. Toxicol. 27: 160-167.

Lyman WJ (1990). Octanol/water partition coefficient. In Lyman WJ, Reehl WF, Rosenblatt DH, eds, Handbook of chemical property estimation, American Chemical Society, Washington, D.C.

target tissues, or tissues which showed treatment-related changes in the high dose group, from all animals in all other dose groups,

Bruns E, Egeler Ph, Römbke J Scheffczyk A, Spörlein P (2001b). Bioaccumulation of lindane and hexachlorobenzene by the oligochaetes Enchytraeus luxuriosus and Enchytraeus albidus (Enchytraeidae, Oligochaeta, Annelida). Hydrobiologia 463: 185-196.

Worms of the species described above can be considered healthy if they move through the substrate, do not try to leave the substrate, and reproduce continuously. Very slow motioning or a yellow posterior end (in the case of Eisenia fetida) indicates substrate exhaustion. In this case, fresh substrate and/or a lower number of animals per box is recommended.

Details of test chemical preparation, including details of any procedures used to reduce the particle size of solid substances or to prepare solutions of the test chemical. In cases where mechanical processes may have altered test chemical composition, include the results of analyses to verify the composition of the test chemical;

analytical instrumentation, suitable for determination of the concentration of the test substance at the expected concentrations;

examples of raw data figures of representative analysis (all raw data have to be stored in accordance with GLP standards), including recoveries of surrogates, and the number of levels used in the calibration (along with the criteria for the correlation coefficient of the calibration curve), and results of quality assurance/quality control (QA/QC)

The results show that generally, the variability was higher for the less well-degraded surfactants. Within-test variability was less than 15 % for over 90 % of cases, the highest reaching 30-40 %.

Since the other parameters in equation (2) are associated with growth kinetics, temperature is likely to affect the effluent substrate level and the critical sludge age, ie. the sludge retention time needed to obtain a certain degree of treatment would increase with decreasing temperature.

Barlow SM, Greig JB, Bridges JW et al (2002). Hazard identification by methods of animal-based toxicology. Food. Chem. Toxicol. 40: 145-191.

Christensen ER (1984). Dose-response functions in aquatic toxicity testing and the Weibull model. Water Research 18: 213-221.

Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity characterization of environmental chemicals by the US National Toxicology Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445

When the background concentration in the non-exposed worms e.g. on day 0 differs significantly from zero (this may e.g. be the case for metals), this background concentration (Ca,0) should be included in these equations, to make them read:

EEAStapler

In the case where only the high dose group dies prematurely due to toxicity, this should not trigger termination of the study.

Antiandrogenicity is the capability of a chemical to suppress the action of a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Oestrogenicity is the capability of a chemical to act like a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

where AUC is the area under the plasma concentration-time curve, and exp is the experimental route (oral, dermal or via inhalation).

Bell AW (1958). The anatomy of Enchytraeus albidus, with a key to the species of the genus Enchytraeus. Ann. Mus. Novitat. 1902: 1-13.

Zwart JHE, Arts JM, ten Berge WF, Appelman LM (1992). Alternative Acute Inhalation Toxicity Testing by Determination of the Concentration-Time-Mortality Relationship: Experimental Comparison with Standard LC50 Testing. Reg. Toxicol. Pharmacol. 15: 278-290.

Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal J-M, van de Vorstenbosch C. (2001). A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology 21:15-23.

physical nature and, where relevant, physical-chemical properties (water solubility, vapour pressure, partition coefficient in soil (or in sediment if available), stability in water, etc.);

Crissman J, Goodman D, Hildebrandt P, et al. (2004). Best Practices Guideline: Toxicological Histopathology. Toxicologic Pathology 32: 126-131.

ISO 11733 (1995; revised 2004). Evaluation of the elimination and biodegradability of organic substances in an aqueous medium — activated sludge simulation test.

The recommended test species is Enchytraeus albidus Henle 1837 (white potworm). Enchytraeus albidus is one of the biggest (up to 15 mm) species of the annelid oligochaete family Enchytraeidae and it is worldwide distributed e.g. (8). Enchytraeus albidus is found in marine, limnic and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and rarely in meadows (42). This broad ecological tolerance and some morphological variations indicate that there might be different races for this species.

Chemicals that indicate the potential for bioaccumulation from structural features, e.g. analogues of chemicals with known bioaccumulation potential; and

US-EPA (2000). Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates. Second edition. EPA 600/R-99/064. March 2000. Revision to the first edition dated June 1994.

Füll C (1996). Bioakkumulation und Metabolismus von -1,2,3,4,5,6-Hexachlorcyclohexan (Lindan) und 2-(2,4-Dichlorphenoxy)-propionsäure (Dichlorprop) beim Regenwurm Lumbricus rubellus (Oligochaeta, Lumbricidae). Dissertation University Mainz, 156 pp.

If these limits are not met, the test should be repeated with an inoculum from another source and/or the procedures used should be reviewed. For example, if high blank IC production is a problem the procedure given in paragraphs 27 to 32 should be followed.

the analytical specificity and sensitivity of the method used with non-radioactive test chemical is equal to or greater than that which could be obtained with the radiolabelled test chemical,

Reversible movement of a test chemical from one location in the body to another. Tissue distribution can be studied by organ dissection, homogenisation, combustion and liquid scintillation counting or by qualitative and/or quantitative whole body autoradiography. The former is useful to obtain concentration and percent of recovery from tissues and remaining carcass of the same animals, but may lack resolution for all tissues and may have less than ideal overall recovery (< 90 %). See definition for the latter above.

stirring-vessel with a tap at the bottom. Dependent on the estimate of log POW and the Limit of Detection (LOD) of the test compound, the use of a reaction vessel of the same geometry larger than one litre has to be considered, so that a sufficient volume of water can be obtained for chemical extraction and analysis. This will result in higher concentrations in the water extract and thus a more reliable analytical determination. A table giving estimates of the minimum volume needed, the LOD of the compound, its estimated log POW and its water solubility is given in Appendix 1. The table is based on the relationship between log POW and the ratio between the solubilities in octanol and water, as presented by Pinsuwan et al. (14):

Key (or suspected) aspects of mode of action, such as doses at which cytotoxicity begins to arise, hormone levels are perturbed, homeostatic mechanisms are overwhelmed, etc.;

In the case of paired organs, e.g. kidney, adrenal, both organs should be preserved. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as set out for the oral route should be preserved, and specific sampling and preservation of the skin from the site of application is essential. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (8) and Chapter B.29 of this Annex (9). For other organs/tissues (and in addition to the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.

If a solvent other than water is used, it should be one that is water-miscible and/or can be driven off (for example, evaporated), leaving only the test chemical on the soil.

Cohen SM, Meek ME, Klaunig JE, Patton DE, and Fenner-Crisp PA (2003). The human relevance of information on carcinogenic Modes of Action: An Overview. Crit. Rev. Toxicol. 33: 581-589.

test design (e.g. number and size of test vessels, soil mass and height of soil layer, number of replicates, number of worms per replicate, number of test concentrations, duration of uptake and elimination phases, sampling frequency);

Elimination processes sometimes appear to be biphasic, showing a rapid decline of Ca during the early phases, that changes to a slower loss of test items in the later phases of the elimination, e.g. (27) (68). The two phases can be interpreted by the assumption, that there are two different compartments in the organism, from which the test item is lost with different velocities. In these cases, specific LITERATURE should be studied e.g. (38) (39) (40) (78).

The type or description of any vehicle, diluents, suspending agents, and emulsifiers or other materials used in administering the test chemical should be stated.

Her Majesty’s Stationery Office (1982). Assessment of biodegradability. Methods for the examination of waters and associated materials. pp. 91-98 ISBN 0117516619.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 39, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

Truesdale GA, Jones K, Vandyke KG (1959). Removal of synthetic detergents in sewage treatment processes: Trials of a new biologically attackable material.Wat. Waste Tr. J. 7: 441-444.

Nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue(NALT)

test chemical stock solution: DOC and TOC content; how prepared, if suspension; test concentration used; reasons if outside range of 10-20 mg/l DOC; method of addition; date first added; any changes;

Elendt, B.P. & W.-R. Bias (1990). Trace Nutrient Deficiency in Daphnia magna Cultured in Standard Medium for Toxicity Testing. Effects on the Optimization of Culture Conditions on Life History Parameters of D. magna. Water Research 24 (9): 1157-1167.

GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, 1988). Publication on the Planning and Structure of Animal Facilities for Institutes Performing Animal Experiments. ISBN 3-906255-04-2.

The respirability of particles in light of the overall findings should be addressed, especially if the particle-size criteria could not be met.

Meek EM, Bucher JR, Cohen SM, Dellarco V, Hill RN, Lehman-McKemmon LD, Longfellow DG, Pastoor T, Seed J, Patton DE (2003). A Framework for Human Relevance analysis of Information on Carcinogenic Modes of Action. Crit. Rev. Toxicol. 33: 591-653.

test chemical stock solution — DOC (dissolved organic carbon) and TOC (total organic carbon) content; how prepared, if suspension; test concentration(s) used, reasons if outside range 10-20 mg/l DOC; method of addition; date first added; any changes in concentration;

Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the RoutineAssessment of Fore- and Hind-limb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1: 233-236.

Biomagnification is the increase in concentration of the test chemical in or on an organism, resulting mainly from uptake from contaminated food or prey, relative to the concentration of the test chemical in the food or prey. Biomagnification can lead to a transfer or accumulation of the test item within food webs.

estimates of toxic endpoints e.g. ECx (and associated confidence intervals), NOEC and/or LOEC, and the statistical methods used for their determination;

Evident toxicity is a general term describing clear signs of toxicity following administration of test chemical. These should be sufficient for hazard assessment and should be such that an increase in the dose administered can be expected to result in the development of severe toxic signs and probable mortality.

The study should not be extended beyond the point when the data available from the study are no longer sufficient to enable a statistically valid evaluation to be made.

Mount DR, Dawson TD, Burkhard LP (1999). Implications of gut purging for tissue residues determined in bioaccumulation testing of sediment with Lumbriculus variegates. Environ. Toxicol. Chem. 18: 1244-1249.

sludge could be pumped intermittently from the separator to the aeration vessel (e.g. 5 min. every 2,5 h to recycle 1 l/h to 1,5 l/h;

OECD (2000). OECD Draft Guideline for the Testing of Chemicals: 122 Partition Coefficient (n-Octanol/Water): pH-Metric Method for Ionisable Substances. Paris.

Mery S, Gross EA, Joyner DR, Godo M, Morgan KT (1994). Nasal diagrams: A tool for recording the distribution of nasal lesions in rats and mice. Toxicol. Pathol. 22: 353-372.

ASTM International/E1706-00 (2002). Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates. pp 1125-1241. In ASTM International 2002 Annual Book of Standards. Volume 11.05. Biological Effects and Environmental Fate;Biotechnology; Pesticides. ASTM. International, West Conshohocken, PA.

Horn JA, Moyer JE, Hale JH (1970). Biological degradation of tertiary butyl alcohol. Proc. 25th Ind. Wastes Conference Purdue Univ.: 939-854.

EEA sizermeaning

As an option, the organic carbon content of the artificial soil may be reduced, e.g. by lowering the peat content to 4-5 % of dry soil and increasing the sand content accordingly. By such a reduction in organic carbon content, the possibilities of adsorption of test chemical to the soil (organic carbon) may be decreased, and the availability of the test chemical to the worms may increase (74). It has been demonstrated that Enchytraeus albidus and Eisenia fetida can comply with the validity criteria on reproduction when tested in field soils with lower organic carbon content, e.g. 2,7 % (33), (61), and there is experience that this can also be achieved in artificial soil with 5 % peat.

GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, 2006). Microbiological monitoring of laboratory animals in various housing systems.

If the dispersions are stable, investigate how the test chemical can be determined in the dispersed form. It is unlikely that DOC will be suitable, so that a specific analytical method for the test chemical would have to be established which could be applied to effluents, effluent solids and activated sludge. The fate of the test chemical in the simulation of the activated sludge process would then be determined in liquid and solid phases. Thus, a “mass balance” would be established to decide whether the test chemical had been biodegraded. However, this would indicate only primary biodegradation. Demonstration of ultimate biodegradation should be attempted by applying a respirometric test for ready biodegradability (chapter C.4 of this Annex (5) C, F or D) using as inoculum sludge exposed to the test chemical in the simulation test.

Conder JM and Lanno RP (2003). Lethal critical body residues as measures of Cd, Pb, and Zn bioavailability and toxicity in the earthworm Eisenia fetida. J. Soils Sediments 3: 13-20.

common name, chemical name, CAS number, structural formula (indicating position of label when radiolabelled substance is used) and relevant physical-chemical properties (see paragraph 17)

IPCS (2010). Characterization and application of Physiologically-Based Pharmacokinetic Models in Risk Assessment. IPCS Harmonization Project Document No 9. Geneva, World Health Organization, International Programme on Chemical Safety.

All air concentrations should be reported in units of mass (mg/l mg/m3, etc.) rather than in units of volume (ppm, ppb, etc.).

Nevertheless, these model equations should be used with caution, especially when changes in the test chemical's bioavailability, or (bio)degradation occur during the test (see e.g. (79)).

Definitive identification of thyroid-active chemicals is more reliable by histopathological analysis rather than hormone levels.

SETAC (1993). Guidance Document on Sediment toxicity Tests and Bioassays for Freshwater and Marine Environments. From the WOSTA Workshop held in the Netherlands.

Römbke J, Riepert F, Achazi R (2000). Enchytraeen als Testorganismen, In: Toxikologische Beurteilung von Böden. Heiden, S., Erb, R., Dott, W. & Eisentraeger, A. (eds.). Spektrum Verl., Heidelberg. 105-129.

Zwart JHE, Arts JM, Klokman-Houweling ED, Schoen ED (1990). Determination of Concentration-Time-Mortality Relationships to Replace LC50 Values. Inhal. Toxicol. 2: 105-117.

Milani D, Day KE, McLeay DJ, and Kirby RS (1996). Recent intra- and inter-laboratory studies related to the development and standardisation of Environment Canada’s biological test methods for measuring sediment toxicity using freshwater amphipods (Hyalella azteca) and midge larvae (Chironomus riparius). Technical Report. Environment Canada. National Water Research Institute. Burlington, Ontario, Canada.

Usui T, Griffiths SA and Lumley CE (1996). The utility of the mouse for the assessment of the carcinogenic potential of pharmaceuticals. In D’Arcy POF & Harron DWG (eds). Proceedings of the Third International Conference on Harmonisation. Queen’s University Press, Belfast. pp 279-284.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

Dunnett CW (1964). A multiple comparisons procedure for comparing several treatments with a control. J. Amer. Statis. Assoc., 50: 1096-1121.

the mean percentage degradation in vessels FC containing the reference chemical is > 60 % by the 14th day of incubation; and

Tabulation of particle size data including analytical sample collection data, particle size distribution, and calculations of the MMAD and σg.

In the case of systems modelling studies such as PBTK, presentation of models should include a full description of the model to allow independent reconstruction and validation of the model (see paragraph 65 and Appendix: Definitions).

Sturm RN (1973). Biodegradability of Nonionic surfactants: screening test for predicting rate and ultimate biodegradation. J.A,.Oil Chem Soc. 50: 159-167.

NF T 20-045 (AFNOR) (September 1985). Chemical products for industrial use — Determination of water solubility of solids and liquids with low solubility — Column elution method.

Grady CPL, Smets BF and Barbeau DS (1996). Variability in kinetic parameter estimates: A review of possible causes and a proposed terminology. Wat. Res., 30 (3): 742-748.

Larson RJ (1979). Estimation of biodegradation potential of xenobiotic organic chemicals. Appl Env. Microbiol. 38: 1153-1161.

mortality of the control worms and the worms in each test vessel and any observed abnormal behaviour (e.g. soil avoidance, lack of reproduction in a bioaccumulation test with enchytraeids);

Barlow SM, Greig JB, Bridges JW et al. (2002). Hazard identification by methods of animal-based toxicology. Food. Chem. Toxicol. 40, 145-191.

The high concentration level should result in toxic effects but not cause lingering signs or lethality which would prevent a meaningful evaluation.

Doe JE, Boobis AR, Blacker A et al. (2006). A Tiered Approach to Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36: 37-68.

Boethling RS, Mackay D (eds.) (2000). Handbook of property estimation methods for chemicals. Lewis Publishers Boca Raton, FL, USA.

SOT (1992). Technical Committee of the Inhalation Specialty Section, Society of Toxicology (SOT). Recommendations for the Conduct of Acute Inhalation Limit Tests. Fund. Appl. Toxicol. 18: 321-327.

Gloyna EF, Comstock RF, Renn CE (1952). Rotary tubes as experimental trickling filters. Sewage ind. Waste 24: 1355-1357.

Expose five groups of animals at a higher concentration(15) (2L) with slightly shorter exposure durations (factor √2 spaced; see Figure 1).

Janssen MPM, Bruins A, De Vries TH, Van Straalen NM (1991). Comparison of cadmium kinetics in four soil arthropod species. Arch. Environ. Contam. Toxicol. 20: 305-312.

Commission Regulation (EU) No 260/2014 of 24 January 2014 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 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)

Cohen SM, Meek ME, Klaunig JE, Patton DE, Fenner-Crisp PA (2003). The human relevance of information on carcinogenic Modes of Action: An Overview. Crit. Rev. Toxicol. 33: 581-589.

Marinussen MPJC, Van der Zee SEATM, De Haan FA (1997). Cu accumulation in Lumbricus rubellus under laboratory conditions compared with accumulation under field conditions. Ecotox. Environ. Safety 36: 17-26.

OECD (2009). Guidance Document 106 on Histologic evaluation of Endocrine and Reproductive Tests in Rodents ENV/JM/Mono(2009)11.

Androgenicity is the capability of a chemical to act like a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

National Research Council, 1985. Guide for the care and use of laboratory animals. NIH Publication No 86-23. Washington D.C., US. Dept. of Health and Human Services.

Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283.

Milani D, Day KE, McLeay DJ, Kirby RS (1996). Recent intra- and inter-laboratory studies related to the development and standardisation of Environment Canada’s biological test methods for measuring sediment toxicity using freshwater amphipods (Hyalella azteca) and midge larvae (Chironomus riparius). Technical Report. Environment Canada. National Water Research Institute. Burlington, Ontario, Canada.

For each starting concentration, the respective testing schemes as included in this Appendix outline the procedure to be followed.

Doe JE, Boobis AR, Blacker A et al. (2006). A Tiered Approach to Systemic Toxicity Testing for Agricultural Chemical Safety Assessment. Crit. Rev. Toxicol. 36: 37-68.

Chemicals which belong to a class of organic chemicals known to have the potential to bioaccumulate in living organisms, e.g. surface active or highly adsorptive chemicals;

(1) Commission Regulation (EC) No 440/2008(2) contains the test methods for the purposes of the determination of the physico-chemical properties, toxicity and eco-toxicity of substances to be applied for the purposes of Regulation (EC) No 1907/2006.

Antithyroid activity is the capability of a chemical to suppress the action of a natural thyroid hormone (e.g. T3) in a mammalian organism.

Dietrich DR, Schmid P, Zweifel U, Schlatter C, Jenni-Eiermann S, Bachmann H, Bühler U, Zbinden N (1995). Mortality of birds of prey following field application of granular carbofuran: A Case Study. Arch. Environ. Contam. Toxicol. 29: 140-145.

In the case of paired organs, e.g. kidney, adrenal, both organs should be preserved. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as set out for the oral route should be examined, and specific sampling and preservation of the skin from the site of application is necessary. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (9) and Chapter B.29 of this Annex (10). For other organs/tissues (and in addition to the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.

The application of waste water treatment simulations to volatile chemicals is both debatable and problematic. As with poorly water-soluble test chemicals, very few reports seem to have been published describing simulation tests using volatile chemicals. A conventional type of complete-mixing apparatus is adapted by sealing the aeration and settling tanks, measuring and controlling the air flow using flow-meters and passing the exit gas through traps to collect volatile organic matter. In some cases, a vacuum pump is used to draw the exit gas through a “cold” trap or a purge-trap containing Tenax and silica gel for gas-chromatographic analyses. The test chemical present in the trap can be determined analytically.

BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H. Köpp. Berlin 1995.

Cooper RL, Lamb JS, Barlow SM et al. (2006). A Tiered Approach to Life Stages Testing for Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36: 69-98.

The endpoint information for one or more chemicals is used to make a prediction of the endpoint for the target chemical.

Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (OJ L 383, 29.12.1992, p. 113).

Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet

Gabric A.J, Connell DW, Bell PRF (1990). A kinetic model for bioconcentration of lipophilic compounds by oligochaetes. Wat. Res. 24: 1225-1231.

Nominal concentrations (total mass of test chemical generated into the inhalation chamber divided by the volume of air passed through the chamber).

if measured: percentages of parent chemical, metabolites, and bound residues (i.e. the percentage of test chemical that cannot be extracted with common extraction methods) detected in soil and test animals;

if radiolabelled test chemical is used, the precise position of the labelled atoms, the specific radioactivity, and the radiochemical purity.

For each starting concentration, the respective testing schemes as included in this Appendix outline the procedure to be followed.

Key (or suspected) aspects of mode of action, such as doses at which cytotoxicity begins to arise, hormone levels are perturbed, homeostatic mechanisms are overwhelmed, etc.;

information on lag and plateau phases, test duration, the degree of elimination of the test chemical, of the reference chemical (if tested) and of the organic medium (in the control unit), together with statistical data and statements of biodegradability and validity of the test;

actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test chemical concentration (mg/kg or ppm) to the actual dose, if applicable;

OECD (1995). Report of the Consultation Meeting on Sub-chronic and Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working document, Environment Directorate, OECD, Paris.

Ennis DM and Kramer A (1975). A rapid microtechnique for testing biodegradability of nylons and polyamides. J. Food Sci. 40: 181-185.

ISO 11268-2 (1998) Soil Quality – Effects of pollutants on earthworms (Eisenia fetida). Part 2: Determination of effects on reproduction.

Hund-Rinke K and Wiechering H (2000). Earthworm avoidance test for soil assessments: An alternative for acute and reproduction tests. J. Soils Sediments 1: 15-20.

Details of the equipment used to monitor chamber temperature, humidity, and chamber airflow (i.e. development of a calibration curve).

detailed information on the treatment of soil and worm samples, including details of preparation, storage, spiking procedures, extraction, and analytical procedures (and precision) for the test item in worms and soil, and lipid content (if measured), and recoveries of the test item.

Van Brummelen TC and Van Straalen NM (1996). Uptake and elimination of benzo(a)pyrene in the terrestrial isopod Porcellio scaber. Arch. Environ. Contam. Toxicol. 31: 277-285.

Struijs J and Stoltenkamp J (1990). Head space determination of evolved carbon dioxide in a biodegradability screening test. Ecotox. Env. Safety 19: 204-211.

Römbke J, Egele, P, Füll C (1998). Literaturstudie über Bioakkumulationstests mit Oligochaeten im terrestrischen Medium. UBA-Texte 28/98, 84 S.

Few reports seem to have been published on subjecting poorly water-soluble and insoluble chemicals to tests simulating waste water treatment (1)(2)(3).

Fraction of an administered dose that reaches the systemic circulation or is made available at the site of physiological activity. Usually, bioavailability of a test chemical refers to the parent compound, but it could refer to its metabolite. It considers only one chemical form. Nota Bene: bioavailability and absorption are not the same. The difference between e.g. oral absorption (i.e. presence in gut wall and portal circulation) and bioavailability (i.e. presence in systemic blood and in tissues) can arise from chemical degradation due to gut wall metabolism or efflux transport back to the intestinal lumen or presystemic metabolism in the liver, among other factors (10). Bioavailability of the toxic component (parent compound or a metabolite) is a critical parameter in human risk assessment (high-to-low dose extrapolation, route-to-route extrapolation) for derivation of an internal value from the external NOAEL or BMD (applied dose). For liver effects upon oral administration, it is the oral absorption that suffices. However, for every effect other than at the portal of entry, it is the bioavailability that is in general a more reliable parameter for further use in risk assessment, not the absorption.

A comparison between the proportion of test chemical stripped from the biological (Rv) and non-biological test (Rvp) systems indicates the overall effect that biological treatment has had on the emission of the test chemical into the atmosphere.

ILSI (International Life Sciences Institute) (1997). Principles for the Selection of Doses in Chronic Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC.

OECD (2000). Guidance document on the recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. Series on Testing and Assessment No 19. ENV/JM/MONO(2000)7.

Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283.

Harkema JR (1990). Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 85: 231-238.

preparation of the formulated sediment: ingredients and characteristics (organic carbon content, pH, moisture, etc. at the start of the test);

The maximum water holding capacity (WHC) of the artificial soil is determined according to ISO 11268-2 (35). At least two days before starting the test, the dry artificial soil is moistened by adding enough deionised or reconstituted water to obtain approximately half of the final water content. The final water content should be 40 % to 60 % of the maximum WHC. At the start of the test, the pre-moistened soil is divided into as many batches as the number of test concentrations and controls used for the test, and the moisture content is adjusted to 40-60 % of WHCmax by using the solution of the test item and/or by adding deionised or reconstituted water. The moisture content is determined at the beginning and at the end of the test (at 105 °C). It should be optimal for the species’ requirements (the moisture content can also be checked as follows: when the soil is gently squeezed in the hand, small drops of water should appear between the fingers).

In the case of paired organs, e.g. kidney, adrenal, both organs should be preserved. The clinical and other findings may suggest the need to examine additional tissues. Also, any organs considered likely to be target organs based on the known properties of the test chemical should be preserved. In studies involving the dermal route of administration, the list of organs as set out for the oral route should be preserved, and specific sampling and preservation of the skin from the site of application is essential. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 and B.29 of this Annex. For other organs/tissues (and in addition to the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.

UN (2007), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), ST/SG/AC.10/30, UN New York and Geneva. Available: [http://www.unece.org/trans/danger/publi/ghs/ghs_welcome_e.html]

EPA (2005). Guidelines for Carcinogen Risk Assessment Risk Assessment Forum U.S. Environmental Protection Agency Washington, DC.

Calcium carbonate of chemically pure quality (CaCO3) is added to adjust the pH of the final mixture of the sediment to 7,0 ± 0,5. Organic carbon content of the final mixture should be 2 % (± 0,5 %) and is to be adjusted by the use of appropriate amounts of peat and sand, according to (a) and (c).

Also known as the distribution coefficient, it is a measure of the differential solubility of a chemical in two solvents.

Lyman WJ (1990). Solubility in water. In: Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds, Lyman WJ, Reehl WF, Rosenblatt DH, Eds. American Chemical Society, Washington, DC, 2-1 to 2-52.

Vitamin stock solution for medium M4 and M7. All three vitamin solutions are combined to make a single vitamin stock solution

the experimental variability is reduced because it forms a reproducible “standardised matrix” and the need to find uncontaminated and clean sediment sources is eliminated;

discussion of the results, including any influence on the outcome of the test resulting from deviations from this Test Method.

An arbitrary classification of chemicals which have passed certain specified screening tests for ultimate biodegradability; these tests are so stringent that it is assumed that such chemicals will rapidly and completely biodegrade in aquatic environments under aerobic conditions.

Bouche M (1972). Lombriciens de France. Ecologie et Systematique. INRA, Annales de Zoologie-Ecologie animale, Paris, 671 p.

In the following tables reference is made to GHS (Globally Harmonised System of Classification and Labelling of Chemicals (GHS). The EU equivalent is Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity the Regulation (EC) No 1272/2008 (9) does not implement Category 5.

Environment Canada (1995). Guidance Document on Measurement of Toxicity Test Precision Using Control Sediments Spiked with a Reference Toxicant. Report EPS 1/RM/30. September 1995.

Painter HA, Bealing DJ (1989). Experience and Data from the OECD Activated Sludge Simulation Test. pp. 113-138. In: Laboratory Tests for Simulation of Water Treatment Processes CEC Water Pollution Report 18. Eds. Jacobsen BN, Muntau H, Angeletti G.

Because a lengthy fasting period can introduce bias in glucose measurements for the treated versus control animals, the study director should determine whether it is appropriate to fast the animals. If a fasting period is used, it should be appropriate to the species used; for the rat this may be 16 h (overnight fasting). Determination of fasting glucose may be carried out after overnight fasting during the last exposure week, or after overnight fasting prior to necropsy (in the latter case together with all other clinical pathology parameters).

and the relationship given by Lyman (15) for predicting water solubility. Water solubilities calculated with the equation given in Appendix 1 must be seen as a first estimate. It should be noted that the user is free to generate an estimate of water solubility by means of any relationship that is considered to better represent the relationship between hydrophobicity and solubility. For solid compounds, inclusion of melting point in the prediction of solubility is for instance recommended. In case a modified equation is used, it should be ascertained that the equation for calculation of solubility in octanol is still valid. A schematic drawing of a glass-jacketed stirring-vessel with a volume of ca. one litre is given in Appendix 2. The proportions of the vessel shown in Appendix 2 have proven favourable and should be maintained when apparatus of a different size is used;

The minimum dose (concentration x time) which resulted in mortality during testing at initial concentration (first exposure session) will be taken as a guide to establish the next combination of concentration and exposure durations. Typically, the concentration will be decreased two-fold (1/2L) and animals will be exposed over a new time range with a finer grid using a geometric division of exposure periods with a factor 1,4 (√2; see reference 11) around the time according to the minimum lethal dose level (time x concentration) observed during the first exposure. In this figure (Figure 1), mortality in Exposure session I was first observed at 15 min; the durations during session II are therefore centred around 30 min, and are 15, 21 30, 42 and 60 min. After the first two exposures, it is strongly advised to plot the data in a similar figure as indicated above, and to check whether the relationship between concentration and time has an angle of 45 degrees (n = 1) or if the concentration-time-response relationship is less steep (e.g. n = 2) or steeper (e.g. n = 0,8). In the latter cases it is strongly advised to adapt the next concentrations and durations accordingly.

Using the model equations above, the kinetic parameters (ks and ke) may also be calculated in one run by applying the first order kinetics model to all data from both the uptake and elimination phase simultaneously. For a description of a method that may allow for such a combined calculation of uptake and elimination rate constants, references (41), (73) and (70) may be consulted.

Nyholm N, Ingerslev F, Berg UT, Pedersen JP and Frimer-Larsen H (1996). Estimation of kinetic rate constants for biodegradation of chemicals in activated sludge waste water treatment plants using short-term batch experiments and μg/l range spiked concentrations Chemosphere 33 (5): 851-864.

US EPA (1996). Fate, Transport and Transportation. 835.3120. Sealed vessel carbon dioxide production test. Office, Prevention Pesticides and Toxic Substance, Washington, DC.

addition of known volume of a solution of the test chemical in an easily volatile solvent to an empty test vessel, followed by evaporation of the solvent.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Environmental Health and Safety Monograph Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

estimates of toxic endpoints e.g. ECx (and associated confidence intervals), NOEC and/or LOEC,, and the statistical methods used for their determination;

The percentage biodegradation % D is calculated for the test (FT), reference (FC) and, if included inhibition monitoring control (FI) bottles from the respective amounts of TIC produced up to each sampling time.

Vessels (denoted FS) for checking a possible abiotic degradation as (a) plus 50 mg/l HgCl2 or sterilised by some other means (e.g. by autoclaving).

Bruce and Versteeg (1992). A statistical procedure for modelling continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.

The high concentration level should result in toxic effects but not cause lingering signs or lethality which would prevent a meaningful evaluation.

Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the RoutineAssessment of Fore- and Hind-limb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1: 233-236.

Reference to appendix data which contain individual animal data for all measurement endpoints (e.g. dose administration, percent recovery, concentrations, TK parameters, etc.);

I and E must be corrected for the DOC due to the test chemical in the test units, otherwise the calculations of percentage inhibition will be incorrect.

Hawker DW and Connell DW (1988). Influence of partition coefficient of lipophilic compounds on bioconcentration kinetics with fish. Wat. Res. 22: 701-707.

Separate tests should be carried out to determine whether the test chemical is adsorbed; if it is, then a further correction may be made.

Holsapple MP, Pitot HC, Cohen SN, Boobis AR, Klaunig JE, Pastoor T, Dellarco VL, Dragan YP (2006). Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk. Toxicol. Sci. 89: 51-56.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Environmental Health and Safety Monograph Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

In general, the culturing conditions are sufficient if worms do not try to leave the substrate, move quickly through the soil, exhibit a shiny outer surface without soil particles clinging to it, are more or less whitish coloured, and if worms of different ages are visible. Actually, worms can be considered healthy if they reproduce continuously.

Plasma concentrations and toxicokinetic parameters (bioavailability, AUC, Cmax, Tmax, clearance, half-life) after administration by the relevant route(s) of exposure;

Westheide W and Müller MC (1996). Cinematographic documentation of enchytraeid morphology and reproductive biology. Hydrobiologia 334: 263-267.”

If the analytical method gives a positive value in unamended sewage equivalent to S c mg/l, calculate the percentage removal (DSC) from:

This subsection should include information on the test animals, including selection and justification for species, strain, and age at study initiation, sex as well as body weight, health status, and animal husbandry.

Lymph nodes from the hilar region of the lung, especially for poorly soluble particulate test chemicals, For more in depth examinations and/or studies with immunological focus, additional lymph nodes may be considered, e.g. those from the mediastinal, cervical/submandibular and/or auricular regions.

4-5 % (dry weight) peat: as close to pH 5,5 to 6,0 as possible; it is important to use peat in powder form, finely ground (particle size ≤ 1 mm) and only air dried.

This revised Test Method B.29 has been designed to fully characterise test chemical toxicity by the inhalation route for a subchronic duration (90 days), and to provide robust data for quantitative inhalation risk assessments. Groups of 10 male and 10 female rodents are exposed 6 hours per day during a 90 day (13 week) period to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. This method allows the study director the flexibility to include satellite (reversibility) groups, interim sacrifices, bronchoalveolar lavage (BAL), neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterise the toxicity of a test chemical.

Both species can be bred in a wide range of animal wastes. The breeding medium recommended by ISO (35) is a 50:50 mixture of horse or cattle manure and peat. The medium should have a pH value of about 6 to 7 (regulated with calcium carbonate), a low ionic conductivity (less than 6 mS/cm or less than 0,5 % salt concentration) and should not be contaminated excessively with ammonia or animal urine. Also, a commercial gardening soil free of additives, or artificial soil according to OECD (48), or a 50:50 mixture of both can be used. The substrate should be moist but not too wet. Breeding boxes of 10 litre to 50 litre volume are suitable.

Griffiths SA, Parkinson C, McAuslane JAN and Lumley CE (1994). The utility of the second rodent species in the carcinogenicity testing of pharmaceuticals. The Toxicologist 14(1):214.

National Research Council, 1985. Guide for the care and use of laboratory animals. NIH Publication No 86-23. Washington, D.C., US Dept. of Health and Human Services.

This technique may be used to probe xenobiotic interaction with specific tissue sites or cell populations as for instance in receptor binding or specific mode of action studies that may require high-resolution and high sensitivity which may not be feasible with other techniques such as whole-body autoradiography.

after the measured concentrations in the 1-octanol-solution have attained stable values, the stock solution is diluted with an appropriate volume of 1-octanol;

Painter HA and King EF (1978b). The effect of phosphate and temperature on growth of activated sludge and on biodegradation of surfactants. Wat. Res. 12: 909-915.

Sugaya Y (1997). Intra-specific variations of the susceptibility of insecticides in Chironomus yoshimatsui. Jp. J. Sanit. Zool. 48 (4): 345-350.

actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test chemical concentration (mg/kg or ppm) to the actual dose, if applicable;

Pinsuwan S, Li A and Yalkowsky S.H. (1995). Correlation of octanol/water solubility ratios and partition coefficients, J. Chem. Eng. Data. 40: 623-626.

Vijver MG, Vink JPM, Jager T, Wolterbeek HT, van Straalen NM, van Gestel CAM (2005). Biphasic elimination and uptake kinetics of Zn and Cd in the earthworm Lumbricus rubellus exposed to contaminated floodplain soil. Soil Biol, Biochem. 37: 1843-1851.

Particular emphasis should be made to the description of methods used to meet the criteria of this Test Method, e.g. the limit concentration or the particle size.

US EPA (2000). Methods for measuring the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates. Second Edition, EPA 600/R-99/064, US, Environmental Protection Agency, Duluth, MN, March 2000.

Kawai K (1986). Fundamental studies on Chironomid allergy. I. Culture methods of some Japanese Chironomids (Chironomidae, Diptera). Jp. J. Sanit. Zool. 37(1): 47-57.

Theoretical carbon dioxide (mg) is the quantity of carbon dioxide calculated to be produced from the known or measured carbon content of the test chemical when fully mineralised; also expressed as mg carbon dioxide evolved per mg test chemical.

Having regard to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC(1), and in particular Article 13(3) thereof,

As a ratio of concentrations it is dimensionless. Most frequently it is given as the logarithm to the base 10 (log POW). POW is temperature dependent and reported data should include the temperature of the measurement.

When treatment-related histopathological changes are observed in the high dose group, those same tissues are to be examined from all animals in all other dose groups;

Franke C, Studinger G, Berger G, Böhling S, Bruckmann U, Cohors-Fresenborg D, Jöhncke U (1994). The assessment of bioaccumulation. Chemosphere 29: 1501-1514.

The octanol-water partitioning coefficient (Kow) is the ratio of a chemical’s solubility in n-octanol and water at equilibrium, also sometimes expressed as Pow. The logarithm of Kow (log Kow) is used as an indication of a chemical's potential for bioaccumulation by aquatic organisms.

Actual test chemical concentrations collected from the animals’ breathing zone; for mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately

Sugaya Y (1997). Intra-specific variations of the susceptibility of insecticides in Chironomus yoshimatsui. Jp. J. Sanit. Zool. 48 (4): 345-350.

discussion of the results, including any influence on the outcome of the test resulting from deviations from this Test Method.

at the end of the test, pH and the dissolved oxygen concentration should be measured in each vessel. The oxygen concentration should be at least 60 % of the air saturation value (ASV) at the temperature used, and the pH of overlying water should be in the 6-9 range in all test vessels;

State whereby one or more of the kinetic (e.g. absorption, metabolism or clearance) process(es) are at a maximum (read “saturated”).

OECD (2006). Final Summary report of the meeting of the Validation Management Group for mammalian testing. ENV/JM/TG/EDTA/M(2006)2.

Holzhütter H-G, Genschow E, Diener W, and Schlede E (2003). Dermal and Inhalation Acute Toxicity Class Methods: Test Procedures and Biometric Evaluations for the Globally Harmonized Classification System. Arch. Toxicol. 77: 243-254.

For Eisenia fetida and Eisenia andrei, the mean mass loss as measured at the end of the uptake and at the end of the elimination phase should not exceed 20 % compared to the initial fresh weight (f.w.) at start of each phase.

The test is carried out in two parts. The units are first operated without sludge but with the synthetic waste water plus test chemical being pumped into the aeration tank. Influent, effluent and exit gas samples are collected and analysed for the test chemical for a few days. From the data collected, the percentage (Rvs) of the test chemical stripped from the system may be calculated.

Gardner WS, Frez WA, Cichocki EA, Parrish CC (1985). Micromethods for lipids in aquatic invertebrates. Limnology and Oceanography 30: 1099-1105.

Bioconcentration is the increase in concentration of the test chemical in or on an organism, resulting from the uptake of the chemical exclusively from the surrounding medium (i.e. via the body surface and ingested soil), relative to the concentration of the test chemical in the surrounding medium.

The percentage of the dose of test chemical absorbed from the site of administration (i.e. GI tract). This critical parameter can be used to understand the fraction of the administered test chemical that reaches the portal vein, and subsequently the liver.

This subsection should include identification of the test chemical: chemical name, molecular structure, qualitative and quantitative determination of its chemical composition, chemical purity and whenever possible, type and quantities of any impurities. It should also include information on physical/chemical properties including physical state, colour, gross solubility and/or partition coefficient, stability, and if appropriate, corrosivity. If applicable, information on isomers should be provided. If the test chemical is radiolabelled, information on the following should be included in this subsection: the type of radionuclide, position of label, specific activity, and radiochemical purity.

Baumann U, Kuhn G and Benz M. (1998) Einfache Versuchsanordnung zur Gewinnung gewässerökologisch relevanter Daten, UWSF — Z. Umweltchem. Ökotox. 10: 214-220.

Fleming R et al. (1994). Sediment Toxicity Tests for Poorly Water-Soluble Substances. Final Report to them European Commission. Report No: EC 3738. August 1994. WRc, UK.

OECD (2009). Acute Inhalation Toxicity Testing. OECD Guideline for Testing of Chemicals No 403, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

If no sex susceptibility information is available, rats of both sexes will be used, i.e. 1 animal/sex per concentration. Based on existing information, or if it becomes apparent during this exposure session that one sex is more susceptible, 10 animals of the susceptible sex will be used (2 animals per concentration/time point) at each concentration level during subsequent testing.

Schmelz R and Collado R (1999). Enchytraeus luxuriosus sp. nov., a new terrestrial oligochaete species (Enchytraeide, Clitellata, Annelida). Carolinea 57: 93–100.

Set up bottles containing 5 and 10 mg/l as IC using a solution of anhydrous sodium carbonate (Na2 CO3) in CO2-free water prepared by acidifying water to pH 6,5 with concentrated ortho-phosphoric acid (paragraph 20), sparging overnight with CO2-free air and raising the pH to neutrality with alkali. Ensure that the ratio of the headspace volume to the liquid volume is the same as in the tests (e.g. 1:2). Acidify and equilibrate as described in paragraph 52, and measure the IC concentrations of both the headspace and liquid phases. Check that the two concentrations are the same within experimental error. If they are not, the operator should review the procedures. This check on the distribution of IC between liquid and gaseous phases need not be made every time the test is performed; it could presumably be made while performing the calibration.

the nominal test concentrations, the measured test concentrations and the results of all analyses to determine the concentration of the test substance in the test vessel;

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

De Boer J, Smedes F, Wells D, Allan A (1999). Report on the QUASH interlaboratory study on the determination of total-lipid in fish and shellfish. Round 1 SBT-2, Exercise 1000, EU, Standards, Measurement and Testing Programme.

For each starting concentration, the respective testing schemes as included in this Appendix outline the procedure to be followed.

dispersion with the aid of emulsifying agents to be required to establish whether they have any inhibitory or stimulatory effects on microbial activity before addition;

chemical identification data (common name, chemical name, structural formula, CAS number, etc.) including purity and analytical method for quantification of test substance.

target tissues, or tissues which showed treatment-related changes in the high dose group, from all animals in all other dose groups;

physical nature and, where relevant, physical-chemical properties (water solubility, vapour pressure, partition coefficient in soil (or in sediment if available), stability in water, etc.);

German Government (1962). Ordinance of the degradability of detergents in washing and cleaning agents. Bundesgesetzblatt, Pt.1 No 49: 698-706.

Termination of the study should be considered when the number of survivors in the lower dose groups or the control group falls below 25 per cent.

The elimination rate constant (ke) is the numerical value defining the rate of reduction in the concentration of the test item in/on the test organism, following the transfer of the test organisms from a medium containing the test item to a chemical-free medium; ke is expressed in d-1.

Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet.

OECD (1988). OECD Ring-test of methods for determining ready biodegradability: Chairman’s report (M. Hashimoto; MITI) and final report (M. Kitano and M. Takatsuki; CITI). Paris.

When treatment-related histopathological changes are observed in the high dose group, those same tissues are to be examined from all animals in all other dose groups,

A classification of chemicals for which there is unequivocal evidence of biodegradation (primary or ultimate) in any test of biodegradability.

Details of the chemical analytical method used and method validation (including efficiency of recovery of test chemical from the sampling medium).

All air concentrations should be reported in units of mass (mg/l, mg/m3, etc.) rather than in units of volume (ppm, ppb, etc.).

(3) The adaptation contains two methods for the determination of physicochemical properties including an update of the water solubility test method and a new partition coefficient test method relevant for the persistent, bioaccumulative and toxic (PBT) assessment; four new and one updated method for the determination of ecotoxicity and environmental fate and behaviour; nine methods for the determination of toxicity and other health effects including four inhalation toxicity test methods, which include an update of three methods and one new method to reduce the number of animals used and to improve assessment of effects, an update of the repeat dose 28-day oral toxicity test method to include parameters for assessment of endocrine activity, an update of the toxicokinetics test method relevant for the design and understanding of toxicological studies and an update of chronic, carcinogenicity and combined chronic and carcinogenicity test methods.

Randall RC, Lee II H, Ozretich RJ, Lake JL, Pruell RJ (1991). Evaluation of selected lipid methods for normalising pollutant bioaccumulation. Environ.Toxicol. Chem. 10: 1431-1436.

Bioaccumulation factors calculated directly from the ratio of the soil uptake rate constant and the elimination rate constant (ks and ke, see below) are termed kinetic bioaccumulation factor (BAFK).

EU (1999). Ring-test of the ISO Headspace CO2 method: application to surfactants: Surfactant Ring Test-1, Report EU4697, Water Research Centre, May 1999, Medmenham, SL7 2HD, UK.

Capability of a method or instrument to discriminate between measurement responses representing different levels of a variable of interest.

description of the test vessels and stirring conditions (geometry of the stirring bar and of the test vessel, vortex height in mm, and when available: stirring rate) used

Van Gestel CAM. (1992). The influence of soil characteristics on the toxicity of chemicals for earthworms; a review, In: Ecotoxicology of Earthworms (Ed. Becker, H, Edwards, PJ, Greig-Smith, PW & Heimbach, F). Intercept Press, Andover (GB).

Nominal concentrations (total mass of test chemical generated into the inhalation chamber divided by the volume of air passed through the chamber)

OECD (2007). OECD Guideline for Testing of Chemicals No 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties.

Füll C, Schulte C, Kula C (2003). Bewertung der Auswirkungen von Pflanzenschutzmitteln auf Regenwürmer. UWSF — Z. Umweltchem, Ökotox. 15: 78-84.

EEAsizers Medtronic

Kasprzak K (1982). Review of enchytraeid community structure and function in agricultural ecosystems. Pedobiologia 23: 217-232.

Elmegaard N and Jagers op Akkerhuis GAJM (2000). Safety factors in pesticide risk assessment, Differences in species sensitivity and acute-chronic relations. National Environmental Research Institute, NERI Technical Report 325: 57 pp.

If toxicologically significant alterations in the metabolic profile of the test chemical are observed through either in vitro or in vivo experiments with different species or conditions, characterisation of the enzyme(s) involved may be needed (e.g. Phase I enzymes such as isoenzymes of the Cytochrome P450-dependent mono-oxygenase system, Phase II enzymes such as isoenzymes of sulfotransferase or uridine diphosphate glucuronosyl transferase, or any other relevant enzymes). This information might be used to evaluate the pertinence of species to species extrapolations.

The intermediate concentration level(s) should be spaced to produce a gradation of toxic effects between that of the low and high concentration.

Lymph nodes from the hilar region of the lung, especially for poorly soluble particulate test chemicals. For more in depth examinations and/or studies with immunological focus, additional lymph nodes may be considered, e.g. those from the mediastinal, cervical/submandibular and/or auricular regions.

There is no single method of dispersal of the test chemical which is applicable to all insoluble chemicals. Two of the four types of method described in ISO 10634 (4) would seem to be suitable for attempting to disperse test chemicals for simulation testing; they are the use of emulsifying agents and/or of ultrasonic energy. The stability over at least 24h periods of the resulting dispersion should be established. Suitably stabilised dispersions, contained in a constantly stirred reservoir (paragraph 38), would then be dosed to the aeration tank separately from the domestic (or synthetic) sewage.

Achazi RK, Fröhlich E, Henneken M, Pilz C (1999). The effect of soil from former irrigation fields and of sewage sludge on dispersal activity and colonizing success of the annelid Enchytraeus crypticus (Enchytraeidae, Oligochaeta). Newsletter on Enchytraeidae 6: 117-126.

Weingand K, et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fund. Appl. Toxicol. 29: 198-201.

If needed, vessels (denoted FI) for checking a possible inhibitory effect of the test chemical containing both the test chemical and reference chemical at the same concentrations (paragraph 24) as in bottles FT and FC, respectively;

preparation of the formulated sediment: ingredients and characteristics (organic carbon content, pH, moisture, etc. at the start of the test);

Toxicokinetics, and dose ranges where metabolic induction, saturation, or nonlinearity between external and internal doses does or does not occur;

Rhomberg LR, Baetcke K, Blancato J, Bus J, Cohen S, Conolly R, Dixit R, Doe J, Ekelman K, Fenner-Crisp P, Harvey P, Hattis D, Jacobs A, Jacobson-Kram D, Lewandowski T, Liteplo R, Pelkonen O, Rice J, Somers D, Turturro A, West, W, Olin S(2007). Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose Selection Crit Rev. Toxicol. 37 (9): 729 – 837.

OECD (2008), Bioaccumulation in Sediment-dwelling Benthic Oligochates, Test Guideline No 315, Guidelines for the testing of chemicals, OECD, Paris.

Whalan.E and Redden JC (1994). Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. Office of Pesticide Programs, United States Environmental Protection Agency.

The dates for the EU versions are taken from the document dates on EUR-Lex and may not always coincide with when the changes came into force for the document.

Boatman RJ, Cunningham SL and Ziegler DA (1986). A method for measuring the biodegradation of organic chemicals, Env. Toxicol. Chem. 5: 233-243.

Bruns E, Egeler Ph, Moser T, Römbke J, Scheffczyk A, Spörlein P (2001a). Standardisierung und Validierung eines Bioakkumulationstests mit terrestrischen Oligochaeten. Report to the German Federal Environmental Agency (Umweltbundesamt Berlin), R & D No.: 29864416.

Stover EL, Kincannon DF (1983). Biological treatability of specific organic compounds found in chemical industry waste waters. J. Wat. Pollut. Control Fed. 55: 97.

Using equation 1, the LC50 value can be calculated for a given time period (e.g. 4 hour, 1 hour, 30 minutes, or any time period within the range of time periods tested) using P = 5 (50 % response). Note that Haber’s rule is only applicable when n = 1. The LC01 can be calculated using P = 2,67.”

If testing with chapter B.52 of this Annex (4) was discontinued in favour of this Test Method B.2, justifications should be provided

When Regulation (EC) No 1272/2008 is used, the limit concentrations for gases, vapours, and aerosols are 20 000 ppm, 20 mg/l, and 5 mg/l, respectively. In case of expected toxicity or based on the results of the sighting study, lower starting concentrations should be chosen. In case of regulatory or scientific needs, higher concentrations may be used.

Baumann U, Kuhn G and Benz M. (1998). Einfache Versuchsanordnung zur Gewinnung gewässerökologisch relevanter Daten, UWSF — Z. Umweltchem. Ökotox. 10: 214-220.

(Alternatively, approximate values of KS and μm may be obtained using a simple computer program to fit the theoretical curve calculated from equation 2 (Appendix 6.2) to the experimental values obtained. Although any given solution will not be unique, a reasonable approximation of KS and μm can be obtained.)

Khalil AM (1990). Aufnahme und Metabolismus von 14C-Hexachlorbenzol und 14C-Pentachlornitrobenzol in Regenwürmern. Dissertation University München, 137 pp.

The dry constituents of the soil are mixed thoroughly (e.g. in a large-scale laboratory mixer). This should be done about one week before starting the test. The mixed dry soil constituents should be moistened with deionised water at least 48 h before application of the test item in order to equilibrate/stabilise the acidity. For the determination of pH a mixture of soil and 1 M KCl solution in a 1:5 ratio is used. If the pH value is not within the required range (6,0 ± 0,5), a sufficient amount of CaCO3 is added to the soil, or a new batch of soil is prepared.

Tissue distribution reported as percent of administered dose and concentration (microgram equivalents per gram of tissue), and tissue-to-blood or tissue-to-plasma ratios;

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

Identification of a no-observed-adverse-effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD);

Didden W (2003). Oligochaeta, In: Bioindicators and biomonitors. Markert, B.A., Breure, A.M. & Zechmeister, H.G. (eds.). Elsevier Science Ltd, The Netherlands, pp. 555-576.

Gerike P, Fischer W, Holtmann W (1980). Biodegradability determinations in trickling filter units compared with the OECD Confirmatory Test. Wat. Res. 14: 753-758.

For any versions created after the implementation period as a result of changes made by UK legislation the date will coincide with the earliest date on which the change (e.g an insertion, a repeal or a substitution) that was applied came into force. For further information see our guide to revised legislation on Understanding Legislation.

Danish Environmental Protection Agency. (1996). Activated sludge biodegradability simulation test. Environmental Project, No 337. Nyholm, N. Berg, UT. Ingerslev, F. Min. of Env. and Energy, Copenhagen.

Process of assessing the adequacy of a model to consistently describe the available toxicokinetic data. Models may be evaluated via statistical and visual comparison of model predictions with experimental values against a common independent variable (e.g. time). The extent of evaluation should be justified in relation to the intended use of the model.”

all information relevant for the interpretation of the results, in particular with regard to impurities and physical state of the test substance.

SOT (1992). Technical Committee of the Inhalation Specialty Section, Society of Toxicology (SOT). Recommendations for the Conduct of Acute Inhalation Limit Tests. Fund. Appl. Toxicol. 18: 321-327.

Mineralisation is the complete degradation of an organic chemical to CO2 and H2O under aerobic conditions, and CH4, CO2 and H2O under anaerobic conditions.

ISO 14593 (1998). Water Quality — Evaluation in an aqueous medium of the ultimate biodegradability of organic compounds. Method by the analysis of inorganic carbon in sealed vessels.

information on lag and plateau phases, test duration, the degree of elimination of the test chemical and that of the organic medium in the control unit, together with statistical information and statements of biodegradability and validity of the test;

Van Straalen NM, Donker MH, Vijver MG, van Gestel CAM (2005). Bioavailability of contaminants estimated from uptake rates into soil invertebrates. Environmental Pollution 136: 409-417.

OECD (2000). Guidance Document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation, No 19, ENV/JM/MONO(2000)7, OECD, Paris.

Posthuma L, Weltje L, Anton-Sanchez FA (1996). Joint toxic effects of cadmium and pyrene on reproduction and growth of the earthworm Eisenia fetida. RIVM Report No 607506001, Bilthoven.

The worms should be fed at least once per week ad libitum with rolled oats which are placed into a cavity on the soil surface and covered with soil. If food from the last feeding date remains in the container, the amount of food given should be adjusted accordingly. If fungi grow on the remaining food, it should be replaced by a new quantity of rolled oats. In order to stimulate reproduction, the rolled oats may be supplemented with commercially available, vitamin amended protein powder every two weeks. After three months, the animals are transferred to a freshly prepared culture or breeding substrate. The rolled oats, which have to be stored in sealed vessels, should be autoclaved or heated before use in order to avoid infections by flour mites (e.g. Glyzyphagus sp., Astigmata, Acarina) or predacious mites (e.g. Hypoaspis (Cosmolaelaps) miles, Gamasida, Acarina). After disinfecting, the food is ground up so that it can easily be strewn on the soil surface. Another possible food source is baker’s yeast or the fish food TetraMin®.

The biota-soil accumulation factor (BSAF) is the lipid-normalised concentration of the test chemical in/on the test organism divided by the organic carbon-normalised concentration of the test chemical in the soil at steady state. Ca is then expressed as g·kg-1 lipid content of the organism, and Cs as g·kg-1 organic content of the soil; the BSAF has the units of kg OC·kg-1 lipid.

adsorption of liquid test chemicals, or a solution in a suitable volatile solvent, on to an inert medium or support (e.g. glass fibre filter), followed by evaporation of the solvent, if used, and direct addition of known amounts;

Sludge exchanges can give the appearance of quite a considerable removal, since some of the test chemical in transferred and the concentrations of test chemical in the test and control effluents become more nearly equal. Thus, correcting factors have to be used, which depend on the fraction exchanged and the mean hydraulic retention time. More details of the calculation have been published (1).

Jacobsen BN, Nyholm N, Pedersen BM, Poulsen O, and Ostfeldt P (1993). Removal of organic micropollutants in laboratory activated sludge reactors under various operating conditions: Sorption. Wat. Res. 27: 1505-1510.

Tabulation of response data and concentration level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity, time of onset and duration of effects)

ILSI (International Life Sciences Institute) (1997). Principles for the Selection of Doses in Chronic Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC.

Tabulation of response data and concentration level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity, time of onset, and duration of effects).

A description (preferably including a diagram) of the equipment used to generate the test atmosphere and to expose the animals to the test atmosphere.

Christensen ER (1984). Dose-response functions in aquatic toxicity testing and the Weibull model. Water Research 18: 213-221.

Individual body weights of animals collected on study days, date and time of death if prior to scheduled euthanasia; time course of onset of signs of toxicity, and whether these were reversible for each animal;

Tolls J (2002). Partition Coefficient 1-Octanol/Water (Pow) Slow-Stirring Method for Highly Hydrophobic Chemicals, Validation Report. RIVM contract-Nrs 602730 M/602700/01.

the wet weights of the worms at each sampling time; for earthworms, the wet weights at start of the test, and at each sampling time before and after gut purging;

Weytjens D, Van Ginneken I and Painter HA (1994). The recovery of carbon dioxide in the Sturm test for ready biodegradability. Chemosphere 28: 801-812.

Gerike P and Fischer WK (1979). A correlation study of biodegradability determinations with various chemicals in various tests. Ecotox. Env. Saf. 3:157-173.

application of test chemical and reference chemical to test system: test concentration used and amount of carbon dosed into each test bottle, any use of solvents;

OECD (1995). Report of the Consultation Meeting on Sub-chronic and Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working document, Environment Directorate, OECD, Paris.

The calculations of TIC for the two analytical methods used for measuring IC in this test are described below in paragraphs 60 and 61. Percentage biodegradation (% D) in each case is given by:

At the end of the test, the overall mortality during uptake and elimination phase should not exceed 10 % (earthworms) or 20 % (enchytraeids) of the total number of the introduced worms.

EEA sizerhysterectomy

The respirability of particles in light of the overall findings should be addressed, especially if the particle-size criteria could not be met;

Barton HA, Pastoor TP, Baetcke T et al (2006). The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments. Critical Reviews in Toxicology 36: 9-35.

Within-test variability (replicability), using aniline, was low with coefficients of variability not greater than 5 % in nearly all test runs. In the two cases in which the replicability was worse, the greater variability was probably due to high IC production in the blanks. Replicability was worse with 1-octanol but was still less than 10 % for 79 % of test runs. This greater within-test variability may have been due to dosing errors, as a small volume (3 to 4 μl) of 1-octanol had to be injected into sealed test bottles. Higher coefficients of variation would result when lower concentrations of test chemical are used, especially at concentrations lower than 10 mg C/l. This could be partially overcome by reducing the concentration of total inorganic carbon (TIC) in the inoculum.

Meller M, Egeler P, Rombke J, Schallnass H, Nagel R, Streit B (1998). Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulfate on Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. Safety 39: 10-20.

Solon E G, Kraus L (2002). Quantitative whole-body autoradiography in the pharmaceutical industry; Survey results on study design, methods, and regulatory compliance, J Pharm and Tox Methods 46: 73-81.

Pflugmacher J (1992). Struktur-Aktivitätsbestimmungen (QSAR) zwischen der Konzentration von Pflanzenschutzmitteln und dem Octanol-Wasser-Koeffzienten UWSF- Z. Umweltchem. Ökotox. 4: 77-81.

Calcium carbonate of chemically pure quality (CaCO3) is added adjust the pH of the final mixture of the sediment to 7,0 ± 0,5.

Diener W, Kayser D and Schlede E (1997). The Inhalation Acute-Toxic-Class Method; Test Procedures and Biometric Evaluations. Arch. Toxicol. 71: 537-549.

The main endpoint of a bioaccumulation test is the bioaccumulation factor, BAF. The measured BAF can be calculated by dividing the concentration in the test organism, Ca, by the concentration in the soil, Cs, at steady state. If the steady state is not reached during the uptake phase, the BAFK is calculated from the rate constants instead of BAFss. However, it should be noted if the BAF is based on steady state concentrations or not.

For a number of measurements in serum and plasma, most notably for glucose, overnight fasting is preferable. The major reason for this preference is that the increased variability which would inevitably result from non-fasting, would tend to mask more subtle effects and make interpretation difficult. However it should be noted that overnight fasting may interfere with the general metabolism of the animals and, particularly in feeding studies, may disrupt the daily exposure to the test chemical. All animals should be assessed in the same physiological condition and preferably detailed or neurological assessments should therefore be scheduled for a different day than clinical biochemistry sampling.

Revised legislation carried on this site may not be fully up to date. At the current time any known changes or effects made by subsequent legislation have been applied to the text of the legislation you are viewing by the editorial team. Please see ‘Frequently Asked Questions’ for details regarding the timescales for which new effects are identified and recorded on this site.

Crissman JW, Goodman DG, Hildebrandt PK et al. (2004). Best Practices Guideline: Toxicological Histopathology. Toxicologic Pathology 32: 126-131.

NOAEL is the abbreviation for no-observed-adverse-effect level. This is the highest dose level where no adverse treatment-related findings are observed due to treatment.

The organic carbon-water partitioning coefficient (Koc) is the ratio of a chemical’s concentration in/on the organic carbon fraction of a soil and the chemical's concentration in water at equilibrium.

Jänsch S, Amorim MJB, Römbke J (2005). Identification of the ecological requirements of important terrestrial ecotoxicological test species. Environ. Reviews 13: 51-83.

OECD (2009). Report on Biostatistical Performance Assessment of the Draft TG 436 Acute Toxic Class Testing Method for Acute Inhalation Toxicity. Environmental Health and Safety Monograph Series on Testing and Assessment No 105, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

Williams DA (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117.

The Schedules you have selected contains over 200 provisions and might take some time to download. You may also experience some issues with your browser, such as an alert box that a script is taking a long time to run.

To obtain worms of standard age and mass, it is best to start the culture with cocoons. Therefore, adult worms are added to a breeding box containing fresh substrate to produce cocoons. Practical experience has shown that a population density of approximately 100 adult worms per kg substrate (wet weight) leads to good reproduction rates. After 28 days, the adult worms are removed. The earthworms hatched from the cocoons are used for testing when mature after at least 2 months but less than 12 months.

Hey there! We at Sunspot Supply are thrilled to provide professional-quality products to everyone - from wholesale dealers and end users to do-it-yourselfers.

If statistical analysis is used to analyse the study findings, then sufficient information on the method of analysis and the computer program employed should be included, so that an independent reviewer/statistician can re-evaluate and reconstruct the analysis.

the nominal test concentrations, the measured test concentrations and the results of all analyses to determine the concentration of the test substance in the test vessel;

Particular emphasis should be made to the description of methods used to meet this Test Method’s criteria, e.g. the limit concentration or the particle size

Cooper RL, Lamb JS, Barlow SM et al (2006). A Tiered Approach to Life Stages Testing for Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36: 69-98.

mean hydraulic retention time (with no growth); rotational speed of tube; approximate angle of inclination, if possible;

A description (preferably including a diagram) of the equipment used to generate the test atmosphere and to expose the animals to the test atmosphere

Gerike P, Fischer WK and Holtmann W (1980). Biodegradability determinations in trickling filter units compared with the OECD confirmatory test. Wat.Res. 14: 753-758.

Expose five groups of animals at this target concentration for durations of 15, 30, 60, 120 and 240 minutes, respectively.

OECD (2004b), Earthworm reproduction test (Eisenia fetida/Eisenia Andrei), Test Guideline No 222, Guidelines for the testing of chemicals, OECD, Paris.

Whalan JE and Redden JC (1994). Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. Office of Pesticide Programs, United States Environmental Protection Agency.

OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No 23.

OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation, Environmental Health and Safety Monograph Series on Testing and Assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris.

Thus, it is possible to control the sludge retention time at any pre-selected value by the control of the waste sludge flow rate, Q1.

Kuper CF, Koornstra PJ, Hameleers DMH, Biewenga J, Spit BJ, Duijvestijn AM, Breda Vriesman van PJC, Sminia T (1992). The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219-224.

Show Timeline of Changes: See how this legislation has or could change over time. Turning this feature on will show extra navigation options to go to these specific points in time. Return to the latest available version by using the controls above in the What Version box.

Eisenia fetida and Eisenia andrei are commercially available, e.g. as fish bait. In comparison to other lumbricid earthworms, they have a short life-cycle, reaching maturity within ca. 2-3 months (at room temperature). Their optimum temperature is approximately at 20-24 °C. They prefer relatively moist substrates with a nearly neutral pH and a high content of organic material. Since these species have been widely used in standardised ecotoxicological tests for about 25 years, their culturing is well established (48) (77).

Dissolved organic carbon is the organic carbon present in solution or that which passes through a 0,45 micrometre filter or remains in the supernatant after centrifuging at approx. 4 000 g (about 40 000 m sec-2) for 15 min.

Her Majesty’s Stationery Office (1984). Methods for the examination of waters and associated materials. Methods for assessing the treatability of chemicals and industrial waste waters and their toxicity to sewage treatment processes, 1982, London.

ASTM International (2004). Standard guide for conducting laboratory soil toxicity or bioaccumulation tests with the Lumbricid earthworm Eisenia fetida and the Enchytraeid potworm Enchytraeus albidus. ASTM International, E1676-04: 26 pp.

chemical identification data (common name, chemical name, structural formula, CAS number, etc.) including purity and analytical method for quantification of test substance.

In order to try to equalise the microbial populations in sludges in a test unit, receiving sewage plus a test chemical, and in a control unit, receiving only sewage, a daily interchange of sludge was introduced (1). The procedure was called coupling and the method is known as coupled units. Coupling was initially performed using Husmann activated sludge units but it has also been done with Porous Pot units (2)(3). No significant differences in results were found as between non-coupled and coupled units, whether Husmann or Porous Pot so there is no advantage in expending the time and energy needed in coupling the units.

Westheide W and Graefe U (1992). Two new terrestrial Enchytraeus species (Oligochaeta, Annelida). J. Nat. Hist. 26: 479-488.

Details of the chemical analytical method used and method validation (including efficiency of recovery of test chemical from the sampling medium).

The usual means for obtaining the kinetic bioaccumulation factor (BAFK), the soil uptake rate constant (ks) and the elimination rate constant (ke) is to use non-linear parameter estimation methods on a computer, e.g. based on the models described in (68). Given a set of sequential time concentration data and the model equations:

Species/strain used and justification for using a species other than the rat. Source and historical data may be provided, if they are from animals exposed under similar exposure, housing, and fasting conditions.

Key (or suspected) aspects of mode of action, such as doses at which cytotoxicity begins to arise, hormone levels are perturbed, homeostatic mechanisms are overwhelmed, etc.;

Cooper RL, Lamb JS, Barlow SM et al. (2006). A Tiered Approach to Life Stages Testing for Agricultural Chemical Safety Assessment. Crit. Rev. Toxicol. 36: 69-98.

Antioestrogenicity is the capability of a chemical to suppress the action of a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Sterenborg I, Vork NA, Verkade SK, Van Gestel CAM, Van Straalen NM (2003). Dietary zinc reduces uptake but not metallothionein binding and elimination of cadmium in the springtail Orchesella cincta. Environ. Toxicol. Chemistry 22: 1167-1171.

interest in seeing if a steady state level has been achieved in specific tissues (e.g. in repeated dosing studies, even though an apparent blood steady state level of test chemical may have been achieved, there may be interest in ascertaining that a steady state level has also been attained in target tissues).

ISO 8192 (2007) Water quality — Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation.

ISO 15522 (1999) Water Quality — Determination of the inhibitory effect of water constituents on activated sludge microorganisms.

Her Majesty’s Stationery Office (1982). Methods for the examination of waters and associated materials. Assessment of biodegradability, 1981, London.

In the case where only the high dose group dies prematurely due to toxicity, this should not trigger termination of the study.

All air concentrations should be reported in units of mass (e.g. mg/l, mg/m3, etc.); units of volume (e.g. ppm, ppb, etc.) may also be reported parenthetically

In cases where a significant decrease of the test chemical concentration in the soil is observed over time during the uptake phase, the following models can be used e.g. (67) (79):

IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.

Tomlinson TG, Snaddon DHM, (1966). Biological oxidation of sewage by films of micro-organisms. Int.J. Air Wat. Pollut. 10: 865-881.

OECD (2009). Performance Assessment: Comparison of 403 and C × t Protocols via Simulation and for Selected Real Data Sets. Environmental Health and Safety Monograph Series on Testing and Assessment No 104, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

The time from the start of a test until acclimatization and/or adaptation of the degrading microorganisms is achieved and the biodegradation degree of a test chemical or organic matter has increased to a detectable level (e.g. 10 % of the maximum theoretical biodegradation, or lower, dependent on the accuracy of the measuring technique).

at the end of the test, pH and the dissolved oxygen concentration should be measured in each vessel. The oxygen concentration should be at least 60 per cent of the air saturation value (ASV) at the temperature used, and the pH of overlying water should be in the 6-9 range in all test vessels;

Chapter C.19 of this Annex, Estimation of the Adsorption Coefficient (K OC ) on Soil and on Sewage Sludge Using High Performance Liquid Chromatography (HPLC).

Species/strain used and justification for using a species other than the rat. Source and historical data may be provided, if they are for animals exposed under similar exposure, housing, and fasting conditions.

Details of the chemical analytical method used and method validation (including efficiency of recovery of test chemical from the sampling medium);

Bioaccumulation is the increase in concentration of the test chemical in or on an organism relative to the concentration of the test chemical in the surrounding medium. Bioaccumulation results from both bioconcentration and biomagnification processes (see below).

BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H. Köpp. Berlin 1995.

(Area under the plasma concentration-time curve): Area under the curve in a plot of concentration of test chemical in plasma over time. It represents the total amount of test chemical absorbed by the body within a predetermined period of time. Under linear conditions, the AUC (from time zero to infinity) is proportional to the total amount of a test chemical absorbed by the body, irrespective of the rate of absorption.

75-76 % (dry weight) quartz sand (fine sand should predominate with more than 50 per cent of the particles between 50 and 200 μm).

UBA (Umweltbundesamt) (1991). Bioakkumulation — Bewertungskonzept und Strategien im Gesetzesvollzug. UBA-Texte 42/91. Berlin.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

Any available information on acute or long term toxicity (e.g. ECx, LCx„ NOEC) of the test chemical towards soil-dwelling oligochaetes;

ILSI (International Life Sciences Institute) (1997). Principles for the Selection of Doses in Chronic Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC.

ASTM (2000). Standard guide for the determination of the bioaccumulation of sediment-associated contaminants by benthic invertebrates. American Society for Testing and Materials, E 1688-00a.

Toxicokinetics, and dose ranges where metabolic induction, saturation, or nonlinearity between external and internal doses does or does not occur;

All content is available under the Open Government Licence v3.0 except where otherwise stated. This site additionally contains content derived from EUR-Lex, reused under the terms of the Commission Decision 2011/833/EU on the reuse of documents from the EU institutions. For more information see the EUR-Lex public statement on re-use.

Environment Canada (1995). Guidance Document on Measurement of Toxicity Test Precision Using Control Sediments Spiked with a Reference Toxicant. Report EPS 1/RM/30. September 1995.

OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No 23.

Use this menu to access essential accompanying documents and information for this legislation item. Dependent on the legislation item being viewed this may include:

Naylor C and Rodrigues C (1995). Development of a test method for Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303.

The level of degradation achieved when the test chemical is totally utilised by micro-organisms resulting in the production of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass).

Larson RJ, Hansmann MA and Bookland EA (1996). Carbon dioxide recovery in ready biodegradability tests: mass transfer and kinetic constants, Chemosphere 33: 1195-1210.

Painter HA, King EF (1978). Water Research Centre Porous Pot Method for Assessing Biodegradability. Technical Report TR70, Water Research Centre, Stevenage, UK.

Bligh EG and Dyer WJ (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Pysiol. 37: 911-917.

The soil uptake rate constant (ks) is the numerical value defining the rate of increase in the concentration of the test item in/on the test organism resulting from uptake from the soil phase. ks is expressed in g soil kg-1 of worm d-1.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing. Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

details of test chemical formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation;

Gledhill WE (1975). Screening test for assessment of biodegradability: Linear alkyl benzene sulfonate. Appl Microbiol. 30: 922-929.

Particular emphasis should be made to the description of methods used to meet this Test Method’s criteria, e.g. the limit concentration or the particle size;

Haley P, Perry R, Ennulat D, Frame S, Johnson C, Lapointe J-M, Nyska A, Snyder PW, Walker D, Walter G (2005). STP Position Paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System. Toxicol Pathol 33: 404-407.

Carmichael NG, Barton HA, Boobis AR et al. (2006). Agricultural Chemical Safety Assessment: A Multisector Approach to the Modernization of Human Safety Requirements. Crit. Rev. Toxicol. 36, 1-7.

then a radiolabelled test chemical does not need to be used. Furthermore, other radioactive and stable isotopes may be used, particularly if the element is responsible for or is a part of the toxic portion of the test chemical. If possible, the radiolabel should be located in a core portion of the molecule which is metabolically stable (it is not exchangeable, is not removed metabolically as CO2, and does not become part of the one-carbon pool of the organism). Labelling of multiple sites or specific regions of the molecule may be necessary to follow the metabolic fate of the test chemical.

Rekker RF, de Kort HM (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. Chim. Ther. 14: 479-488.

mean sludge age and mean hydraulic retention time; method of sludge wastage; methods of overcoming bulking, loss of sludge, etc.;

Belfroid A, Van Wezel A, Sikkenk M, Van Gestel C, Seinen W & Hermens J (1993). The toxicokinetic behavior of chlorobenzenes in earthworms (Eisenia andrei): Experiments in water. Ecotox. Environ. Safety 25: 154-165.

Expose five groups of animals at a lower concentration(14) (1/8L) with slightly longer exposure durations (factor √2 spaced; see Figure 1).

Dissolve in water and make up to 1 litre. The pH of this solution should be 7,4 (± 0,2). If this is not the case, then prepare a new solution.

Struijs J, Stoltenkamp-Wouterse MJ and Dekkers ALM (1995). A rationale for the appropriate amount of inoculum in ready biodegradability tests. Biodegradation 6: 319-327.

Formulated sediment or reconstituted, artificial or synthetic sediment, is a mixture of materials used to mimic the physical components of a natural sediment.

Pauluhn J and Thiel A (2007). A Simple Approach to Validation of Directed-Flow Nose-Only Inhalation Chambers. J. Appl. Toxicol. 27: 160-167

Reynolds L et al. (1987). Evaluation of the toxicity of substances to be assessed for biodegradability. Chemosphere 16: 2259.

News

Výkon jako z partesu. Beksa nedala Opavě žádnou naději - partesu

What areEEAsizers used for

EEAStapler

EEA sizermeaning

EEAsizers Medtronic

EEA sizerhysterectomy

You Might Also Be Interested In