Commission Regulation (EU) No 283/2013 of 1 March 2013 setting out the data requirements for active substances, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market Text with EEA relevance

The microbial biomass of soils used for laboratory degradation studies shall be determined immediately before the commencement and at the end of the study.

The soils used for degradation, adsorption and desorption or mobility studies shall be representative of the range of agricultural soils typical of the various regions of the Union where use exists or is anticipated.

The soils shall fulfil the following conditions:

• — they shall cover a range of organic carbon content, particle size distribution and pH(preferably CaCl2) values, and

• — where on the basis of other information, degradation or mobility are expected to be pH dependent, for example solubility and hydrolysis rate (see points 2.7 and 2.8), they shall cover approximately the following pH(preferably CaCl2) ranges: 5 to 6, 6 to 7 and 7 to 8.

Soils used shall, wherever possible, be freshly sampled. If use of stored soils is unavoidable, storage shall be carried out for a limited time (at the most three months) under defined and reported conditions, which are adequate to maintain soil microbial viability. Soils stored for longer periods of time may only be used for adsorption/desorption studies.

A soil having extreme characteristics with respect to parameters such as particle size distribution, organic carbon content and pH shall not be used.

Field studies shall be carried out in conditions as close to normal agricultural practice as possible on a range of soils and climatic conditions representative of the areas of use. Weather conditions shall be reported in cases where field studies are conducted.

7.1.1.   Route of degradation in soil

The data and information provided, together with other relevant data and information, shall be sufficient to:

• (a) identify, if possible, the relative importance of the types of processes involved (balance between chemical and biological degradation);

• (b) identify the individual components present which at any time account for more than 10 % of the amount of active substance added, including, if possible, non-extractable residues;

• (c) identify, if possible, the individual components which in at least two sequential measurements, account for more than 5 % of the amount of active substance added;

• (d) identify, if possible, the individual components (> 5 %) for which at the end of the study the maximum of formation is not yet reached;

• (e) identify or characterise, if possible, other individual components present;

• (f) establish the relative proportions of the components present (mass balance); and

• (g) permit the soil residue of concern to which non-target species are or may be exposed, to be defined.

For the purposes of this Section non-extractable residues means chemical species originating from active substances contained in plant protection products used in accordance with good agricultural practice that cannot be extracted by methods which do not significantly change the chemical nature of these residues or the nature of the soil matrix. These non-extractable residues are not considered to include fragments through metabolic pathways leading to natural products.

7.1.1.1.   Aerobic degradation

Circumstances in which required

The pathway or pathways of aerobic degradation shall be reported except where the nature and manner of use of plant protection products containing the active substance precludes soil contamination, such as indoor uses on stored products or brush applied wound healing treatments for trees.

Test conditions

Studies on the degradation pathway or pathways shall be reported for at least one soil. Oxygen levels shall be maintained at levels that do not restrict micro-organisms ability to metabolise aerobically. If there is reason to believe that the route of degradation is dependent on one or more properties of the soil, such as pH or clay content, the route of degradation shall be reported for at least one additional soil for which dependent properties are different.

Results obtained shall be presented in the form of schematic drawings showing the pathways involved, and in the form of balance sheets which show the distribution of radio-label as a function of time, as between:

• (a) active substance;

• (b) CO2;

• (c) volatile compounds other than CO2;

• (d) individual identified transformation products referred to in point 7.1.1;

• (e) extractable substances not identified; and

• (f) non-extractable residues in soil.

The investigation of degradation pathways shall include all possible steps to characterise and quantify non-extractable residues formed after 100 days when exceeding 70 % of the applied dose of the active substance. The techniques and methodologies applied shall be selected on a case-by-case basis. A justification shall be provided where the compounds involved are not characterised.

The duration of the study shall be at least 120 days, except where after a shorter period the levels of non-extractable residues and CO2 are such that they can be extrapolated in a reliable way to 100 days. It shall be longer where this is necessary to establish the degradation pathway of the active substance and its metabolites, breakdown or reaction products.

7.1.1.2.   Anaerobic degradation

Circumstances in which required

An anaerobic degradation study shall be submitted unless the applicant shows that exposure of the plant protection products containing the active substance to anaerobic conditions is unlikely to occur for the intended uses.

Test conditions

Point 7.1.1.1 shall apply as regards test conditions except oxygen levels which shall be minimised as to ensure that micro-organisms metabolise anaerobically.

7.1.1.3.   Soil photolysis

Circumstances in which required

A soil photolysis study shall be submitted unless the applicant shows that deposition of the active substance on the soil surface is unlikely to occur or that photolysis is not expected to contribute significantly to the degradation of the active substance in soil for example due to low light absorbance of the active substance.

7.1.2.   Rate of degradation in soil

7.1.2.1.   Laboratory studies

Laboratory studies on soil degradation shall provide best possible estimates of the time required for degradation of 50 % and 90 % (DegT50lab and DegT90lab) of the active substance, its metabolites, breakdown and reaction products under laboratory conditions.

7.1.2.1.1.   Aerobic degradation of the active substance

Circumstances in which required

The rate of degradation in soil shall be reported, except where the nature and manner of use of plant protection products containing the active substance preclude soil contamination such as indoor uses on stored products or brush applied wound healing treatments for trees.

Test conditions

Studies on the rate of aerobic degradation of the active substance shall be reported for three soils in addition to the one required under point 7.1.1.1. Reliable DegT50 and 90 values shall be available for a minimum of four different soils.

The duration of the study shall be at least 120 days. It shall be longer where this is necessary to establish the kinetic formation fractions of the metabolites, breakdown or reaction products. If more than 90 % of the active substance is degraded before the period of 120 days expires, the test duration may be shorter.

In order to assess the influence of temperature on degradation, a calculation with an adequate Q10 factor or an adequate number of additional studies at a range of temperatures shall be performed.

7.1.2.1.2.   Aerobic degradation of metabolites, breakdown and reaction products

Circumstances in which required

Aerobic degradation (DegT50 and 90 values) from a minimum of three different soils shall be provided for metabolites, breakdown and reaction products which occur in soil if one of the following conditions is fulfilled:

• (a) they account for more than 10 % of the amount of active substance added at any time during the studies;

• (b) they account for more than 5 % of the amount of active substance added in at least two sequential measurements;

• (c) the maximum of formation is not reached at the end of the study but accounts for at least 5 % of the active substance at the final measurement;

• (d) all metabolites found in lysimeter studies at annual average concentrations exceed 0.1 μg/L in the leachate.

Studies shall not be required where three DegT50 and 90 values can be reliably determined from the results of the degradation studies where the active substance is applied as test substance.

Test conditions

Test conditions shall be those indicated in Section 7.1.2.1.1 except the test substance applied will be the metabolite, breakdown or reaction product. Studies on metabolites, breakdown and reaction products shall be provided where these are necessary to obtain reliable DegT50 and 90 values for at least three different soils.

7.1.2.1.3.   Anaerobic degradation of the active substance

Circumstances in which required

The rate of anaerobic degradation of the active substance shall be reported where an anaerobic study has to be performed in accordance with point 7.1.1.2.

Test conditions

Anaerobic DegT50 and 90 values for the active substance are needed for the test conditions outlined in point 7.1.1.2.

7.1.2.1.4.   Anaerobic degradation of metabolites, breakdown and reaction products

Circumstances in which required

Anaerobic degradation studies shall be provided for metabolites, breakdown and reaction products which occur in soil if they fulfil one of the following conditions:

• (a) at any time during the studies account for more than 10 % of the amount of active substance added;

• (b) in at least two sequential measurements account for more than 5 % of the amount of active substance added, if feasible;

• (c) at the end of the study the maximum of formation is not yet reached but accounts for at least 5 % of the active substance at the final measurement, if feasible.

The applicant may deviate from such requirement by showing that DegT50 values for metabolites, breakdown and reaction products can be reliably determined from the results of the anaerobic degradation studies with the active substance.

Test conditions

Studies on metabolites, breakdown and reaction products shall be provided for one soil for the test conditions outlined at point 7.1.1.2.

7.1.2.2.   Field studies

7.1.2.2.1.   Soil dissipation studies

The soil dissipation studies shall provide estimates of the time required for dissipation of 50 % and 90 % (DisT50field and DisT90field) and, if possible, of the time required for degradation of 50 % and 90 % (DegT50field and DegT90field), of the active substance under field conditions. Where relevant, information on metabolites, breakdown and reaction products shall be provided.

Circumstances in which required

Such studies shall be conducted for the active substance, its metabolites, breakdown and reaction products if one of the following conditions is fulfilled:

• (a) DegT50lab for active substance, DegT50lab or DisT50lab for metabolites, breakdown and reaction products, in one or more soils determined at 20 °C and at a moisture content of the soil related to a pF value of 2 (suction pressure) is greater than 60 days; or

• (b) DegT90lab for active substance, DegT90lab or DisT90lab for metabolites, breakdown and reaction products, in one or more soils determined at 20 °C and at a moisture content of the soil related to a pF value of 2 (suction pressure) is greater than 200 days.

However, where plant protection products containing the active substance are intended for use in cold climatic conditions, the studies shall be conducted if one of the following conditions is fulfilled:

• (a) DegT50lab for active substance, DegT50lab or DisT50lab for metabolites, breakdown and reaction products, determined at 10 °C and at a moisture content of the soil related to a pF value of 2 (suction pressure) is greater than 90 days; or

• (b) DegT90lab for active substance, DegT90lab or DisT90lab for metabolites, breakdown and reaction products, in one or more soils, determined at 10 °C and at a moisture content of the soil related to a pF value of 2 (suction pressure) is greater than 300 days.

If during field studies metabolites, breakdown and reaction products which are present in laboratory studies are below the lowest technically feasible LOQ, which shall not exceed an equivalent of 5 % (molar basis) of the nominal concentration of active ingredient applied, no additional information on the fate and behaviour of these compounds shall be provided. In those cases, a scientifically valid justification for any discrepancy between laboratory and field appearance of metabolites shall be provided.

Test conditions

Individual studies on a range of representative soils (normally at least four different types at different geographical locations) shall be continued until at least 90% of the amount applied has dissipated from the soil or been transformed to substances that are not the subject of the investigation.

7.1.2.2.2.   Soil accumulation studies

Soil accumulation studies shall provide sufficient information to evaluate the possibility of accumulation of residues of the active substance and of metabolites, breakdown and reaction products. The soil accumulation studies shall provide estimates of the time required for dissipation of 50 % and 90 % (DisT50field and DisT90field) and, if possible, shall provide estimates of the time required for degradation of 50 % and 90 % (DegT50field and DegT90field), of the active substance under field conditions.

Circumstances in which required

Where on the basis of soil dissipation studies it is established that DisT90field, in one or more soils, is greater than one year and where repeated application is envisaged, whether in the same growing season or in succeeding years, the possibility of accumulation of residues in soil and the level at which a plateau concentration is achieved shall be investigated except where reliable information can be provided by a model calculation or another appropriate assessment.

Test conditions

Long-term field studies shall be performed on at least two relevant soils at different geographical locations and involve multiple applications.

In absence of guidance being included in the list referred to under point 6 of the introduction, the type and conditions of the study to be performed shall be discussed with the national competent authorities.

7.1.3.   Adsorption and desorption in soil

7.1.3.1.   Adsorption and desorption

The information provided, together with other relevant data, shall be sufficient to establish the adsorption coefficient of the active substance and of its metabolites, breakdown and reaction products.

7.1.3.1.1.   Adsorption and desorption of the active substance

Circumstances in which required

Studies on adsorption and desorption of the active substance shall be provided, except where the nature and manner of use of plant protection products containing the active substance preclude soil contamination such as indoor uses on stored products or brush applied wound healing treatments for trees.

Test conditions

Studies on the active substance shall be reported for at least four soils.

Where the batch equilibrium method cannot be applied due to fast degradation, methods such as studies with short equilibration times, QSPR (Quantitative Structure Property Relationship) or the HPLC (High-Performance Liquid Chromatography) method shall be considered as possible alternatives. Where the batch equilibrium method cannot be applied due to weak adsorption, column leaching studies (see point 7.1.4.1) shall be considered as an alternative.

7.1.3.1.2.   Adsorption and desorption of metabolites, breakdown and reaction products

Circumstances in which required

Studies on adsorption and desorption shall be provided for all metabolites, breakdown and reaction products, for which in soil degradation studies one of the following conditions is fulfilled:

• (a) they account for more than 10 % of the amount of active substance added, at any time during the studies;

• (b) they account for more than 5 % of the amount of active substance added in at least two sequential measurements;

• (c) the maximum of formation is not reached at the end of the study but accounts for at least 5 % of the active substance at the final measurement;

• (d) all metabolites found in lysimeter studies at annual average concentrations exceeding 0,1 μg/L in the leachate.

Test conditions

Studies on metabolites, breakdown and reaction products shall be provided for at least three soils.

Where the batch equilibrium method cannot be applied due to fast degradation, methods such as studies with short equilibration times, QSPR or the HPLC method shall be considered as an alternative. Where the batch equilibrium method cannot be applied due to weak adsorption, column leaching studies (see point 7.1.4.1) shall be considered as an alternative.

7.1.3.2.   Aged sorption

As a higher tier option, information on aged sorption may be provided.

Circumstances in which required

The need to carry out a study on aged sorption shall be discussed with the national competent authorities.

Test conditions

In absence of guidance being included in the list referred to under point 6 of the introduction, the type and conditions of the study to be performed shall be discussed with the national competent authorities. The influence on the rate of degradation shall also be considered. Aged sorption data shall be compatible with the model in which those values will be used.

7.1.4.   Mobility in soil

7.1.4.1.   Column leaching studies

7.1.4.1.1.   Column leaching of the active substance

Column leaching studies shall provide sufficient data to evaluate the mobility and leaching potential of the active substance.

Circumstances in which required

Studies in at least four soils shall be carried out where in the adsorption and desorption studies provided for under point 7.1.2 it is not possible to obtain reliable adsorption coefficient values due to weak adsorption (such as Koc < 25 L/Kg).

7.1.4.1.2.   Column leaching of metabolites, breakdown and reaction products

The test shall provide sufficient data to evaluate the mobility and leaching potential of metabolites, breakdown and reaction products.

Circumstances in which required

Studies in at least three soils shall be carried out where in the adsorption and desorption studies provided for under point 7.1.2 it is not possible to obtain reliable adsorption coefficient values due to weak adsorption (such as Koc < 25 L/Kg).

7.1.4.2.   Lysimeter studies

Lysimeter studies shall be performed, where necessary, to provide information on:

• — the mobility in soil,

• — the potential for leaching to ground water,

• — the potential distribution in soil.

Circumstances in which required

The decision whether lysimeter studies are to be carried out, as an experimental outdoor study in the framework of a tiered leaching assessment scheme shall take into account the results of degradation and other mobility studies and the predicted environmental concentrations in groundwater (PECGW), calculated in accordance with the provisions of Section 9 of Part A of the Annex to Regulation (EU) No 284/2013. The type and conditions of the study to be performed shall be discussed with the national competent authorities.

Test conditions

Studies shall cover the realistic worst case situation, and the duration necessary for observation of potential leaching, taking into account the soil type, climatic conditions, the application rate and the frequency and period of application.

Water percolating from soil columns shall be analysed at suitable intervals, while residues in plant material shall be determined at harvest. Residues in the soil profile in at least five layers shall be determined on termination of experimental work. Intermediate sampling shall be avoided, since removal of plants (except for harvesting in accordance with normal agricultural practice) and soil influence the leaching process.

Precipitation, soil and air temperatures shall be recorded at regular intervals, at least on a weekly base.

The depth of the lysimeters shall be at least 100 cm. The soil cores shall be undisturbed. Soil temperatures shall be similar to those pertaining in the field. Where necessary, supplementary irrigation shall be provided to ensure optimal plant growth and to ensure that the quantity of percolation water is similar to that in the regions for which authorisation is sought. When during the study the soil has to be disturbed for agricultural reasons it shall not be disturbed deeper than 25 cm.

7.1.4.3.   Field leaching studies

Field leaching studies shall be performed, where necessary, to provide information on:

• — the mobility in soil,

• — the potential for leaching to ground water,

• — the potential distribution in soil.

Circumstances in which required

The decision whether field leaching studies are to be carried out, as an experimental outdoor study in the framework of a tiered leaching assessment scheme shall take into account the results of degradation and other mobility studies and the predicted environmental concentrations in groundwater (PECGW), calculated in accordance with the provisions of Section 9 of Part A of the Annex to Regulation (EU) No 284/2013. The type and conditions of the study to be performed shall be discussed with the national competent authorities.

Test conditions

Studies shall cover the realistic worst case situation, taking into account the soil type, climatic conditions, the application rate and the frequency and period of application.

Water shall be analysed at suitable intervals. Residues in the soil profile in at least five layers shall be determined on termination of experimental work. Intermediate sampling of plant and soil material shall be avoided (except for harvesting in accordance with normal agricultural practice), since removal of plants and soil influence the leaching process.

Precipitation, soil and air temperatures shall be recorded at regular intervals (at least on a weekly base).

Information on the groundwater table in the experimental fields shall be submitted. Depending on the experimental design, a detailed hydrological characterisation of the test field shall be carried out. If soil cracking is observed during the study this shall be fully described.

Attention shall be given to the number and the location of water collection devices. The placement of these devices in the soil shall not result in preferential flow paths.

7.2.   Fate and behaviour in water and sediment

The information provided, taken together with that provided for one or more plant protection products containing the active substance, and other relevant information, shall be sufficient to establish or permit estimation of:

• (a) persistence in water systems (bottom sediment and water, including suspended particles);

• (b) the extent to which water and sediment organisms are at risk;

• (c) potential for contamination of surface water and groundwater.

7.2.1.   Route and rate of degradation in aquatic systems (chemical and photochemical degradation)

The data and information provided, together with other relevant data and information, shall be sufficient to:

• (a) identify the relative importance of the types of processes involved (balance between chemical and biological degradation);

• (b) where possible, identify the individual components present;

• (c) establish the relative proportions of the components present and their distribution as between water, including suspended particles, and sediment; and

• (d) permit the residue of concern to which non-target species are or may be exposed, to be defined.

7.2.1.1.   Hydrolytic degradation

Circumstances in which required

The hydrolysis rate of purified active substances shall be determined and reported at 20 °C or 25 °C. Studies on hydrolytic degradation shall also be performed for degradation and reaction products which account at any time for more than 10 % of the amount of active substance added in the hydrolysis study, unless sufficient information on their degradation is available from the test performed with the active substance. No additional hydrolysis information on degradates shall be required if they are considered to be stable in water.

Test conditions

The hydrolysis rate for pH 4, 7 and 9 under sterile conditions in the absence of light shall be determined and reported at 20 °C or 25 °C. For active substances that are stable or have a low rate of hydrolysis at 20-25 °C, the rate shall be determined at 50 °C, or another temperature above 50 °C. If degradation is observed at 50 °C or above, the degradation rate at at least three other temperatures shall be determined and an Arrhenius plot shall be constructed to permit an estimate to be made of hydrolysis rate at 20 °C and 25 °C. The identity of hydrolysis products formed and the rate constants observed, shall be reported. The estimated DegT50 values shall be reported for 20 °C or 25 °C.

7.2.1.2.   Direct photochemical degradation

Circumstances in which required

For compounds with a molar (decadic) absorption coefficient (ε) > 10 L × mol^–1 × cm^–1 at a wavelength (λ) ≥ 295 nm direct phototransformation of purified active substances shall be determined and reported unless the applicant shows that contamination of surface water will not occur.

Studies on direct photochemical degradation shall also be performed for metabolites, breakdown and reaction products which account at any time for more than 10 % of the amount of active substance added in the photolysis study, unless sufficient information on their degradation is available from the test performed with the active substance.

No additional photolysis information on degradates shall be required if they are considered to be stable under photolytic conditions.

Test conditions

The direct phototransformation in purified, (for example distilled) buffered water using artificial light under sterile conditions, if necessary using a solubiliser, shall be determined and reported. In the first theoretical step a maximum possible photolysis rate shall be estimated based on the molar extinction coefficient of the active substance. If photolysis is considered to be a potentially significant degradation pathway, photolysis experiments for range finding shall be carried out (tier 2). Determination of quantum yield and direct photolysis route/rate (tiers 3 and 4) shall be carried out for active substances where tier 2 indicates significant photolysis. The identity of breakdown products formed which exceed 10 % of the applied test substance at any time during the study, a mass balance to account for at least 90 % of the applied radioactivity, as well as photochemical half-life (DT50) shall be reported.

7.2.1.3.   Indirect photochemical degradation

Circumstances in which required

Studies on indirect photochemical degradation may be submitted where there are indications from other available data that route and rate of degradation in the water phase can be significantly influenced by indirect photodegradation.

Test conditions

Studies shall be performed in an aqueous system containing organic (humic substances) and inorganic (salts) compounds in a composition that is typical for natural surface waters.

7.2.2.   Route and rate of biological degradation in aquatic systems

7.2.2.1.   ‘Ready biodegradability’

Circumstances in which required

The ‘ready biodegradability’ test shall be performed. If no such test is provided, the active substance shall by default be considered not ‘readily biodegradable’.

7.2.2.2.   Aerobic mineralisation in surface water

The data and information provided, together with other relevant data and information, shall be sufficient to:

• (a) identify individual components present, which at any time account for more than 10 % of the amount of active substance added, including, where possible, non-extractable residues;

• (b) identify individual components present, which account for more than 5 % of the amount of active substance added in at least two sequential measurements, where possible;

• (c) identify individual components (> 5 %) for which at the end of the study the maximum of formation is not yet reached, where possible;

• (d) identify or characterise, where possible, other individual components;

• (e) establish, where relevant, the relative proportions of the components (mass balance); and

• (f) permit, where relevant, the sediment residue of concern and to which non-target species are or may be exposed, to be defined.

Circumstances in which required

Studies on aerobic mineralisation in surface water shall be provided unless the applicant shows that contamination of open water (freshwater, estuarine and marine) will not occur.

Test conditions

The rate of degradation and the pathway or pathways shall be reported either for a ‘pelagic’ test system or for a ‘suspended sediment’ system. Where relevant, additional test systems, which differ with respect to organic carbon content, texture or pH shall be used.

Results obtained shall be presented in the form of schematic drawings showing the pathways involved, and in the form of balance sheets which show the distribution of radio-label in water and, where relevant, sediment as a function of time, as between:

• (a) active substance;

• (b) CO2;

• (c) volatile compounds other than CO2; and

• (d) individual identified transformation products.

The duration of the study shall not exceed 60 days unless the semi-continuous procedure with periodical renewal of the test suspension is applied. However, the period for the batch test may be extended to a maximum of 90 days, if the degradation of the test substance has started within the first 60 days.

7.2.2.3.   Water/sediment study

The information provided, together with other relevant information, shall be sufficient to:

• (a) identify individual components present which at any time account for more than 10 % of the amount of active substance added, including, where possible, non-extractable residues;

• (b) identify individual components present which account for more than 5 % of the amount of active substance added in at least two sequential measurements, where possible;

• (c) identify individual components (> 5 %) for which at the end of the study the maximum of formation is not yet reached, where possible;

• (d) identify or characterise, where possible, also other individual components present;

• (e) establish the relative proportions of the components (mass balance); and

• (f) define the sediment residue of concern, to which non-target species are or may be exposed.

Where a reference is made to non-extractable residues these shall be defined as chemical species originating from active substances used in accordance with good agricultural practice that cannot be extracted by methods which do not significantly change the chemical nature of these residues or the nature of the sediment matrix. These non-extractable residues are not considered to include fragments through metabolic pathways leading to natural products.

Circumstances in which required

The water/sediment study shall be reported unless the applicant shows that contamination of surface water will not occur.

Test conditions

The degradation pathway or pathways shall be reported for two water/sediment systems. The two sediments selected shall differ with respect to organic carbon content and texture, and where relevant, with respect to pH.

Results obtained shall be presented in the form of schematic drawings showing the pathways involved, and in the form of balance sheets which show the distribution of radio-label in water and sediment as a function of time, as between:

• (a) active substance;

• (b) CO2;

• (c) volatile compounds other than CO2;

• (d) individual identified transformation products;

• (e) extractable substances not identified; and

• (f) non-extractable residues in sediment.

The duration of the study shall be at least 100 days. It shall be longer where this is necessary to establish the degradation pathway and water/sediment distribution pattern of the active substance and its metabolites, breakdown and reaction products. If more than 90 % of the active substance is degraded before the period of 100 days expires, the test duration may be shorter.

The degradation pattern of potentially relevant metabolites occurring within the water/sediment study shall be established either by extension of the study for the active substance, or by conducting a separate study for potentially relevant metabolites.

7.2.2.4.   Irradiated water/sediment study

The same general provisions as provided under point 7.2.2.3 apply.

Circumstances in which required

If photochemical degradation is of importance a water/sediment study under influence of a light/dark regime may additionally be provided.

Test conditions

The type and conditions of the study to be performed shall be discussed with the national competent authorities.

7.2.3.   Degradation in the saturated zone

The type and conditions of the study to be performed shall be discussed with the national competent authorities.

7.3.   Fate and behaviour in air

7.3.1.   Route and rate of degradation in air

The vapour pressure of purified active substance, as provided under point 2.2, shall be reported. An estimate of the half-life in the upper atmosphere of the active substance and any volatile metabolites, breakdown and reaction products, formed in soil or natural water systems, shall be calculated and reported.

Estimates of active substance upper atmospheric half-lives, based on monitoring data shall also be calculated, when monitoring data that enable this to be done, are available.

7.3.2.   Transport via air

The type and conditions of the study to be performed shall be discussed with the national competent authorities.

Circumstances in which required

If the trigger for volatilisation, Vp = 10^–5 Pa (plant) or 10^–4 Pa (soil) at a temperature of 20 °C, is exceeded and (drift) mitigation measures are required, data from confined experiments may be reported.

If needed, experiments to determine deposition following volatilisation may be provided.

The national competent authorities shall be consulted to decide whether this information is necessary.

7.3.3.   Local and global effects

For substances that are applied in high amounts, the following effects shall be considered:

• — global warming potential (GWP);

• — ozone depleting potential (OPD);

• — photochemical ozone creation potential (POCP);

• — accumulation in the troposphere;

• — acidification potential (AP);

• — eutrophication potential (EP).

7.4.   Definition of the residue

7.4.1.   Definition of the residue for risk assessment

The residue definition relevant for risk assessment for each compartment shall be defined to include all components (active substance, metabolites, breakdown and reaction products) that were identified in accordance with the criteria referred to in this Section.

The chemical composition of residues occurring in soil, groundwater, surface water (freshwater, estuarine and marine), sediment and air, resulting from use, or proposed use, of a plant protection product containing the active substance, shall be taken into account.

7.4.2.   Definition of the residue for monitoring

Considering the results of toxicological and ecotoxicological testing, the residue for monitoring shall be defined to include those components from the definition of the residue for risk assessment, which are considered relevant when assessing the results in those tests.

7.5.   Monitoring data

Available monitoring data concerning fate and behaviour of the active substance and relevant metabolites, breakdown and reaction products in soil, groundwater, surface water, sediment and air shall be reported.

SECTION 8

Ecotoxicological studies

Introduction

• 1.All available biological data and information which is relevant to the assessment of the ecotoxicological profile of the active substance shall be reported. This shall include all potentially adverse effects found during routine ecotoxicological investigations. Where required by the national competent authorities, additional studies, necessary to investigate the probable mechanisms involved and to assess the significance of these effects, shall be carried out and reported on.

• 2.The ecotoxicological assessment shall be based on the risk that the proposed active substance used in a plant protection product poses to non-target organisms. In carrying out a risk assessment, toxicity shall be compared with exposure. The general term for the output from such a comparison is ‘risk quotient’ or RQ. It shall be noted that RQ can be expressed in several ways, for example, toxicity:exposure ratio (TER) and as a hazard quotient (HQ). The applicant shall take into account the information from Sections 2, 5, 6, 7 and 8.

• 3.It may be necessary to conduct separate studies for metabolites, breakdown or reaction products derived from the active substance where non-target organisms may be exposed and where their effects cannot be evaluated by the available results relating to the active substance. Before such studies are performed, the applicant shall take into account the information from Sections 5, 6 and 7.

• Studies undertaken shall permit characterisation of metabolites, breakdown or reaction products as being significant or not, and reflect the nature and extent of the effects judged likely to arise.

• 4.In the case of certain study types, the use of a representative plant protection product instead of the active substance as manufactured may be more appropriate, for example testing of non-target arthropods, bees, earthworm reproduction, soil micro-flora and non-target terrestrial plants. In the case of certain plant protection product types (for example encapsulated suspension) testing with the plant protection product is more appropriate to testing with active substance when these organisms will be exposed to the plant protection product itself. For plant protection products where the active substance is always intended to be used together with a safener and/or synergist and/or in conjunction with other active substances, plant protection products containing these additional substances shall be used.

• 5.The potential impact of the active substance on biodiversity and the ecosystem, including potential indirect effects via alteration of the food web, shall be considered.

• 6.For those guidelines which allow for the study to be designed to determine an effective concentration (ECx), the study shall be conducted to determine an EC10, EC20 and EC50, when required, along with corresponding 95 % confidence intervals. If an ECx approach is used, a no observed effect concentration (NOEC) shall still be determined.

• Existing acceptable studies that have been designed to generate a NOEC shall not be repeated. An assessment of the statistical power of the NOEC derived from those studies shall be carried out.

• 7.All of the aquatic toxicity data shall be used when developing a proposal for environmental quality standards (Annual Average EQS, AA-EQS; Maximum Acceptable Concentration EQS, MAC-EQS). The methodology for derivation of these endpoints is outlined in the ‘Technical Guidance for Deriving Environmental Quality Standards ([^E0016])’ for the Water Framework Directive 2000/60/EC of the European Parliament and of the Council ([^E0017]).

• 8.In order to facilitate the assessment of the significance of test results obtained, including the estimation of intrinsic toxicity and the factors affecting toxicity, the same strain (or recorded origin) of each relevant species shall, where possible, be used in the various toxicity tests specified.

• 9.Higher tier studies shall be designed and data analysed using suitable statistical methods. Full details of the statistical methods shall be reported. Where appropriate and necessary, higher tier studies shall be supported by chemical analysis to verify exposure has occurred at an appropriate level.

• 10.Pending the validation and adoption of new studies and of a new risk assessment scheme, existing protocols shall be used to address the acute and chronic risk to bees, including those on colony survival and development, and the identification and measurement of relevant sub-lethal effects in the risk assessment.

8.1.   Effects on birds and other terrestrial vertebrates

For all avian and mammalian feeding studies, average achieved dose shall be reported, including where possible the dose in mg substance/kg body weight. Where dosing via the diet is utilised, the active substance shall be distributed uniformly in the diet.

8.1.1.   Effects on birds

8.1.1.1.   Acute oral toxicity to birds

The acute oral toxicity of the active substance to birds shall be determined.

Circumstances in which required

The effects of the active substance on birds shall be investigated except where the substance is included in plant protection products used, for example, in enclosed spaces and wound healing treatments where birds will experience neither direct nor secondary exposure.

Test conditions

A study shall be provided establishing the acute oral toxicity (LD50) of the active substance. Where available, the study shall be performed with a quail species (Japanese quail (Coturnix coturnix japonica) or Bobwhite quail (Colinus virginianus)), since regurgitation is rare in these species. The study shall provide, where possible, LD50 values. The lethal threshold dose, time courses of response and recovery, the LD10 and LD20 shall be reported together with the no observed effect level (NOEL) and gross pathological findings. Where LD10 and LD20 cannot be estimated, an explanation shall be provided. Study design shall be optimised for the achievement of an accurate LD50.

The highest dose used in tests shall not exceed 2 000  mg substance/kg body weight, however, depending on the expected exposure levels in the field following the intended use of the compound, higher doses may be required.

8.1.1.2.   Short-term dietary toxicity to birds

A study shall be provided establishing the short-term dietary toxicity. LC50 values, lowest lethal concentration (LLC), where possible, NOEC values, time courses of response and recovery and pathological findings shall be reported in such study. LC50 and NOEC values shall be converted to daily dietary dose (LD50) expressed in mg substance/kg bw/day and NOEL expressed in mg substance/kg bw/day.

Circumstances in which required

A study on the dietary (five-day) toxicity of the active substance to birds shall only be required where the mode of action or results from mammalian studies indicate a potential for the dietary LD50 measured by the short-term dietary toxicity study to be lower than the LD50 based on an acute oral study. The short-term dietary toxicity test shall not be conducted for any other purpose than to determine intrinsic toxicity through dietary exposure, unless a justification of the need to do so is supplied.

Test conditions

The test species shall be the same as tested under point 8.1.1.1.

8.1.1.3.   Sub-chronic and reproductive toxicity to birds

A study shall be provided establishing the sub-chronic and reproductive toxicity of the substance to birds. The EC10 and EC20 shall be reported. Where they cannot be estimated, an explanation shall be provided together with the NOEC expressed in mg substance/kg bw/day.

Circumstances in which required

The sub-chronic and reproductive toxicity of the active substance to birds shall be investigated, unless the applicant shows that exposure of adults, or exposure of nest sites during the breeding season is unlikely to occur. Such a justification shall be supported by information showing that no exposure or delayed effects will occur during the breeding season.

Test conditions

The study shall be conducted on the same species as tested under point 8.1.1.1.

8.1.2.   Effects on terrestrial vertebrates other than birds

The following information shall be derived from the mammalian toxicological assessment based on the studies referred to in Section 5.

8.1.2.1.   Acute oral toxicity to mammals

The acute oral toxicity of the active substance to mammals shall be determined and the LD50 expressed mg substance/kg bw/day.

Circumstances in which required

The effects of the active substance on mammals shall be investigated except when the substance is included in plant protection products used, for example, in enclosed spaces and wound healing treatments where mammals will experience neither direct nor secondary exposure.

8.1.2.2.   Long-term and reproductive toxicity to mammals

Circumstances in which required

The reproductive toxicity of the active substance to mammals shall be investigated, unless a justification is provided by the applicant showing that exposure of adults, during the breeding season is unlikely to occur. Such a justification shall be supported by information showing that no exposure or delayed effects will occur during the breeding season.

The most sensitive ecotoxicologically relevant mammalian long-term toxicological endpoint (NOAEL) expressed as mg substance/kg bw/day shall be reported. The EC10 and EC20 shall be reported together with the NOEC expressed in mg substance/kg bw/day. Where EC10 and EC20 cannot be estimated an explanation shall be provided.

8.1.3.   Active substance bioconcentration in prey of birds and mammals

For active substances with a log Pow > 3, an assessment of the risk posed by bioconcentration of the substance in the prey of birds and mammals shall be provided.

8.1.4.   Effects on terrestrial vertebrate wildlife (birds, mammals, reptiles and amphibians)

Available and relevant data, including data from the open literature for the active substance of concern, regarding the potential effects to birds, mammals, reptiles and amphibians (see point 8.2.3) shall be presented and taken into account in the risk assessment.

8.1.5.   Endocrine disrupting properties

Consideration shall be given to whether the active substance is a potential endocrine disruptor according to Union or internationally agreed guidelines. This may be done in consulting the mammalian toxicology section (see Section 5). In addition, other available information on toxicity profile and mode of action shall be taken into account. If as a result of this assessment, the active substance is identified as a potential endocrine disruptor, the type and conditions of the study to be performed shall be discussed with the national competent authorities.

8.2.   Effects on aquatic organisms

Reports of the tests referred to in points 8.2.1, 8.2.4 and 8.2.6 shall be submitted for every active substance and supported with analytical data on concentrations of the substance in the test media.

When aquatic toxicity studies are conducted with a poorly soluble substance, limit concentrations lower than 100 mg substance/L may be acceptable, however precipitation of the substance in the test medium shall be avoided and a solubiliser, auxiliary solvent or dispersing agent shall be used when appropriate. Testing using the plant protection product may be required by the national competent authorities if no biological effects occur at the solubility limit of the active substance.

Toxicity endpoints (such as LC50, EC10, EC20, EC50 and NOEC) shall be calculated on the basis of nominal or mean/initial measured concentrations.

8.2.1.   Acute toxicity to fish

A study shall be provided on the acute toxicity to fish (LC50) and details of observed effects.

Circumstances in which required

A test on rainbow trout (Oncorhynchus mykiss) shall be carried out.

Test conditions

The acute toxicity of the active substance to fish shall be determined. In order to minimise fish testing, a threshold approach to acute toxicity testing on fish shall be considered. An acute toxicity fish limit test shall be conducted at 100 mg substance/L or at an appropriate concentration selected from aquatic endpoints (points 8.2.4, 8.2.6 or 8.2.7) following consideration of the threshold exposure. When mortality is detected in the fish limit test an acute fish dose-response toxicity study shall be required to determine an LC50 for use in the risk assessment conducted in accordance with the relevant risk quotient analysis (see point 2 of the introduction of this Section).

8.2.2.   Long-term and chronic toxicity to fish

Circumstances in which required

A long-term or chronic toxicity study on fish shall be provided for all active substances where exposure of surface water is likely and the substance is deemed to be stable in water, that is to say there is less than 90 % loss of the original substance over 24 hours via hydrolysis (see point 7.2.1.1). A fish early life stage study shall be provided in these circumstances. However, if a fish full life cycle study is provided an early life stage study shall not be required.

8.2.2.1.   Fish early life stage toxicity test

A fish early life stage toxicity test shall determine effects on development, growth and behaviour, and details of observed effects on fish early life stages. The EC10 and EC20 shall be reported together with the NOEC. Where EC10 and EC20 cannot be estimated, an explanation shall be provided.

8.2.2.2.   Fish full life cycle test

A fish full life cycle test shall provide information on the effects on reproduction of the parental and the viability of the filial generation. The EC10 and EC20 shall be reported together with the NOEC.

For active substances that are not considered as potential endocrine disruptors, a fish full life cycle test may be required depending upon the persistence and bioaccumulative potential of the substance.

For active substances that fulfil the screening criteria on either of the fish screening assays, or for which there are other indications of endocrine disruption (see point 8.2.3), appropriate additional endpoints shall be included in the test and discussed with the national competent authorities.

Test conditions

Studies shall be designed to reflect concerns identified through lower tier testing, mammalian and bird toxicology studies and other information. The exposure regime shall be selected accordingly, taking account of the rates of application proposed.

8.2.2.3.   Bioconcentration in fish

The test on bioconcentration in fish shall provide the steady-state bioconcentration factors, uptake rate constants and depuration rate constants, incomplete excretion, metabolites formed in fish and, if available, information on organ-specific accumulation.

All data shall be provided with confidence limits for each test substance. Bioconcentration factors shall be expressed as a function of both total wet weight and of the lipid content of the fish.

Data provided under point 6.2.5 shall be considered, where relevant, in addressing this point.

Circumstances in which required

The bioconcentration of the substance, shall be assessed where:

• — the log Pow is greater than 3 (see point 2.7) or there are other indications of bioconcentration, and

• — the substance is considered stable, that is to say there is less than 90 % loss of the original substance over 24 hours via hydrolysis (see point 7.2.1.1).

8.2.3.   Endocrine disrupting properties

Consideration shall be given to whether the active substance is a potential endocrine disruptor in aquatic non-target organisms according to Union or internationally agreed guidelines. In addition, other available information on toxicity profile and mode of action shall be taken into account. If as a result of this assessment, the active substance is identified as a potential endocrine disruptor, the type and conditions of the studies to be performed shall be discussed with the national competent authorities.

8.2.4.   Acute toxicity to aquatic invertebrates

Circumstances in which required

The acute toxicity shall be determined for a Daphnia species (preferably Daphnia magna). For active substances with an insecticidal mode of action or which show insecticidal activity a second species shall be tested, for example Chironomid larvae or Mysid shrimps (Americamysis bahia).

8.2.4.1.   Acute toxicity to Daphnia magna

A test shall be provided on the 24- and 48-hour acute toxicity of the active substance to Daphnia magna, expressed as the median effective concentration (EC50) for immobilisation, and where possible, the highest concentration causing no immobilisation.

Test conditions

Concentrations up to 100 mg substance/L shall be tested. A limit test at 100 mg substance/L may be performed where the results of a range finding test indicate that no effects are to be expected.

8.2.4.2.   Acute toxicity to an additional aquatic invertebrate species

A test shall be provided on the 24- and 48-hour acute toxicity of the active substance to an additional aquatic invertebrate species, expressed as the median effective concentration (EC50) for immobilisation, and where possible, the highest concentration causing no immobilisation.

Test conditions

The conditions as set out in point 8.2.4.1 shall apply.

8.2.5.   Long-term and chronic toxicity to aquatic invertebrates

Circumstances in which required

A long-term or chronic toxicity study on aquatic invertebrates shall be provided for all active substances where exposure of surface water is likely and the substance is deemed to be stable in water, that is to say there is less than 90 % loss of the original substance over 24 hours via hydrolysis (see point 7.2.1.1).

A chronic toxicity study shall be submitted on one aquatic invertebrate species. If acute toxicity tests have been conducted on two aquatic invertebrate species the acute endpoints shall be taken into account (see point 8.2.4) in order to determine the appropriate species to be tested in the chronic toxicity study.

If the active substance is an insect growth regulator, an additional study on chronic toxicity shall be carried out using relevant non-crustacean species such as Chironomus spp.

8.2.5.1.   Reproductive and development toxicity to Daphnia magna

The aim of the test on reproductive and development toxicity to Daphnia magna shall be to measure adverse effects such as immobilisation and loss of reproductive capacity and to provide details of observed effects. The EC10, and EC20 shall be reported together with the NOEC. Where EC10 and EC20 cannot be estimated, an explanation shall be provided.

8.2.5.2.   Reproductive and development toxicity to an additional aquatic invertebrate species

The test on reproductive and development toxicity to an additional aquatic invertebrate species shall measure adverse effects such as immobilisation and loss of reproductive capacity and provide details of observed effects. The EC10, and EC20 shall be reported together with the NOEC. Where EC10 and EC20 cannot be estimated, an explanation shall be provided.

8.2.5.3.   Development and emergence in Chironomus riparius

The active substance shall be applied to the water overlying sediment and effects on survival and development of Chironomus riparius, including effects on emergence of adults, shall be measured to provide endpoints for those substances considered to interfere with insect moulting hormones or that have other effects on insect growth and development. The EC10 and EC20 shall be reported together with the NOEC.

Test conditions

Concentrations of active substance in the overlying water and the sediment shall be measured to establish an EC10, EC20 and a NOEC. The active substance shall be measured often enough to allow the calculation of test endpoints based on nominal as well as time-weighted average concentrations.

8.2.5.4.   Sediment dwelling organisms

When accumulation of an active substance in aquatic sediment is indicated or predicted by environmental fate studies, the impact on a sediment-dwelling organism shall be assessed. The chronic risk to Chironomus riparius or Lumbriculus spp. shall be determined. An appropriate alternative test species may be used where a recognised guideline is available. The active substance shall be applied to either the water or the sediment phase of a water/sediment system and the test shall take account of the major route of exposure. The key endpoint from the study shall be presented in terms of mg substance/kg dry sediment and mg substance/L water and the EC10 and EC20 shall be reported together with the NOEC.

Test conditions

Concentrations of active substance in the overlying water and the sediment shall be measured to establish an EC10, EC20 and a NOEC.

8.2.6.   Effects on algal growth

Circumstances in which required

Testing shall be carried out on one green alga (such as Pseudokirchneriella subcapitata, synonym Selenastrum capricornutum).

For active substances that exhibit herbicidal activity a test on a second species from a different taxonomic group shall be performed such as a diatom, for example Navicula pelliculosa.

The EC10, EC20, EC50 and corresponding NOEC values shall be provided.

8.2.6.1.   Effects on growth of green algae

A test shall be provided establishing EC10, EC20, EC50 for green algae and corresponding NOEC values for algal growth rate and yield, based on measurements of biomass or surrogate measurement variables.

Test conditions

Concentrations up to 100 mg substance/L shall be tested. A limit test at 100 mg substance/L may be performed when results of a range-finding test indicate that no effects are to be expected at lower concentrations.

8.2.6.2.   Effects on growth of an additional algal species

A test shall be provided establishing EC10, EC20, EC50 for an additional algal species and corresponding NOEC values for algal growth rate and yield, based on measurements of biomass (or surrogate measurement variables).

Test conditions

The test conditions as set out in point 8.2.6.1 shall apply.

8.2.7.   Effects on aquatic macrophytes

A test shall be provided establishing EC10, EC20, EC50 and corresponding NOEC values for Lemna species growth rate and yield, based on measurements of number of fronds and at least one additional measurement variable (dry weight, fresh weight or frond area).

For other species of aquatic macrophytes, a test shall provide sufficient information to evaluate impact on aquatic plants and provide EC10, EC20, EC50 and corresponding NOEC values based on measurement of appropriate biomass parameters.

Circumstances in which required

A laboratory test with Lemna species shall be performed for herbicides and plant growth regulators and for substances where there is evidence from information submitted under point 8.6 of Part A of this Annex or point 10.6 of Part A of the Annex to Regulation (EU) No 284/2013 that the test substance has herbicidal activity. Additional testing may be required by the national competent authorities on other macrophyte species depending on the mode of action of the substance, or if clear indications of higher toxicity are apparent to dicotyledonous (for example auxin inhibitor, broad leaf herbicides) or other monocotyledonous (for example grass herbicides) plant species from efficacy or terrestrial non-target plants tests (see point 8.6 of Part A of this Annex and point 10.6 of Part A of the Annex to Regulation (EU) No 284/2013).

Additional aquatic macrophyte species tests may be undertaken on a dicotyledonous species, such as Myriophyllum spicatum, Myriophyllum aquaticum or a monocotyledonous species, such as aquatic grass Glyceria maxima, as appropriate. The need to perform such studies shall be discussed with the national competent authorities.

Test conditions

Concentrations up to 100 mg substance/L shall be tested. A limit test at 100 mg substance/L may be performed when results of a range-finding test indicate that no effects are to be expected.

8.2.8.   Further testing on aquatic organisms

Further studies on aquatic organisms may be conducted to refine the identified risk and shall provide sufficient information and data to evaluate potential impact on aquatic organisms under field conditions.

Studies undertaken may take the form of additional species testing, modified exposure testing, microcosm or mesocosm studies.

Circumstances in which required

The need to perform such studies shall be discussed with the national competent authorities.

Test conditions

The type and conditions of the study to be performed shall be discussed with the national competent authorities.

8.3.   Effect on arthropods

8.3.1.   Effects on bees

Effects on bees shall be assessed and the risk evaluated, including the risk deriving from residues of the active substance or its metabolites in nectar, pollen and water, including guttation. Reports of the tests referred to in points 8.3.1.1, 8.3.1.2 and 8.3.1.3 shall be submitted, except where plant protection products containing the active substance are for exclusive use in situations where bees are not likely to be exposed such as:

• (a) food storage in enclosed spaces;

• (b) non-systemic preparations for application to soil, except granules;

• (c) non-systemic dipping treatments for transplanted crops and bulbs;

• (d) wound sealing and healing treatments;

• (e) non systemic rodenticidal baits;

• (f) use in greenhouses without bees as pollinators.

For seed treatments the risk from drift of dust during drilling of the treated seed shall be taken into account. As regards granules and slug pellets the risk from drift of dust during application shall be taken into account. If an active substance is systemic and to be used on seeds, bulbs, roots, applied directly to soil, irrigation water, or applied directly to or into the plant, for example by spraying or stem injection, the risk to bees foraging those plants shall be assessed, including the risk deriving from residues of the plant protection product in nectar, pollen and water, including guttation.

Where bees are likely to be exposed, testing by both acute (oral and contact) and chronic toxicity, including sub-lethal effects, shall be conducted.

Where exposure of bees to residues in nectar, pollen or water resulting from systemic properties of the active substance may occur and where the acute oral toxicity is < 100 μg/bee or a considerable toxicity for larvae occurs, residues concentrations in these matrices shall be provided and the risk assessment shall be based on a comparison of the relevant endpoint with those residue concentrations. If this comparison indicates that an exposure to toxic levels cannot be excluded, effects shall be investigated with higher tier tests.

8.3.1.1.   Acute toxicity to bees

Where bees are likely to be exposed, testing for acute oral and contact toxicity shall be performed.

8.3.1.1.1.   Acute oral toxicity

A test for acute oral toxicity shall be provided establishing the acute LD50 values together with the NOEC. Sub-lethal effects, if observed, shall be reported.

Test conditions

The test shall be conducted with the active substance. Results shall be presented in terms of μg active substance/bee.

8.3.1.1.2.   Acute contact toxicity

A test for acute contact toxicity shall be provided establishing the acute LD50 values together with the NOEC. Sub-lethal effects, if observed, shall be reported.

Test conditions

The test shall be conducted with the active substance. Results shall be presented in terms of μg active substance/bee.

8.3.1.2.   Chronic toxicity to bees

A test for chronic toxicity to bees shall be provided establishing the chronic oral EC10, EC20, EC50 together with the NOEC. Where the chronic oral EC10, EC20, EC50 cannot be estimated, an explanation shall be provided. Sub-lethal effects, if observed, shall be reported.

Circumstances in which required

The test shall be carried out where bees are likely to be exposed.

Test conditions

The test shall be conducted with the active substance. Results shall be presented in terms of μg active substance/bee.

8.3.1.3.   Effects on honeybee development and other honeybee life stages

A bee brood study shall be conducted to determine effects on honeybee development and brood activity. The bee brood study shall provide sufficient information to evaluate possible risks from the active substance on honeybee larvae.

The test shall provide the EC10, EC20 and EC50 for adult bees, where possible, and larvae together with the NOEC. Where EC10, EC20, EC50 cannot be estimated, an explanation shall be provided. Sub-lethal effects, if observed, shall be reported.

Circumstances in which required

The test shall be carried out for active substances for which sub-lethal effects on growth or development cannot be excluded, unless the applicant shows that it is not possible that honeybee brood will be exposed to the active substance.

8.3.1.4.   Sub-lethal effects

Tests investigating sub-lethal effects, such as behavioural and reproductive effects, on bees and, where applicable, on colonies may be required.

8.3.2.   Effects on non-target arthropods other than bees

Circumstances in which required

Effects on non-target terrestrial arthropods shall be investigated for all active substances except where plant protection products containing the active substance are for exclusive use in situations where non-target arthropods are not exposed such as:

• — food storage in enclosed spaces that preclude exposure,

• — wound sealing and healing treatments,

• — enclosed spaces with rodenticidal baits.

Two indicator species, the cereal aphid parasitoid Aphidius rhopalosiphi (Hymenoptera: Braconidae) and the predatory mite Typhlodromus pyri (Acari: Phytoseiidae) shall always be tested. Initial testing shall be performed using glass plates and mortality (and reproduction effects if assessed) shall be reported. Testing shall determine a rate-response relationship and LR50 ([^E0018]), ER50 ([^E0019]) and NOEC endpoints shall be reported for assessment of the risk to these species in accordance with the relevant risk quotient analysis. If adverse effects can be clearly predicted from these studies then testing using higher tier studies may be required (see point 10.3 of Part A of the Annex to the Regulation (EU) No 284/2013 for further details).

With active substances suspected of having a special mode of action (such as insect growth regulators, insect feeding inhibitors) additional tests involving sensitive life stages, special routes of uptake or other modifications, may be required by the national competent authorities. The rationale for the choice of test species used shall be provided.

8.3.2.1.   Effects on Aphidius rhopalosiphi

A test shall provide sufficient information to evaluate the toxicity in terms of LR50 and NOEC of the active substance to Aphidius rhopalosiphi.

Test conditions

Initial testing shall be performed using glass plates.

8.3.2.2.   Effects on Typhlodromus pyri

A test shall provide sufficient information to evaluate the toxicity in terms of LR50 and NOEC of the active substance to Typhlodromus pyri.

Test conditions

Initial testing shall be performed using glass plates.

8.4.   Effects on non-target soil meso- and macrofauna

8.4.1.   Earthworm — sub-lethal effects

A test shall provide information on the effects on growth, reproduction and behaviour of the earthworm.

Circumstances in which required

Sub-lethal effects on earthworms shall be investigated where the active substance can contaminate soil.

Test conditions

Testing shall determine a dose-response relationship and the EC10, EC20 and NOEC shall enable the risk assessment to be conducted in accordance with the appropriate risk quotient analysis, taking into account likely exposure, the organic carbon content (foc) of the test medium and the lipophilic properties (Kow) of the test substance. The test substance shall be incorporated into the soil to obtain a homogenous soil concentration. Testing with soil metabolites may be avoided if there is analytical evidence to indicate that the metabolite is present at an adequate concentration and duration in the study conducted with the parent active substance.

8.4.2.   Effects on non-target soil meso- and macrofauna (other than earthworms)

Circumstances in which required

Effects on soil organisms, other than earthworms, shall be investigated for all test substances, except in situations where soil organisms are not exposed such as:

• (a) food storage in enclosed spaces that preclude exposure;

• (b) wound sealing and healing treatments;

• (c) enclosed spaces with rodenticidal baits.

For plant protection products applied as a foliar spray data on Folsomia candida and Hypoaspis aculeifer may be required by the national competent authorities. If data are available on both Aphidius rhopalosiphi and Typhlodromus pyri these may be used in an initial risk assessment. If concern is raised with either species tested under point 8.3.2, data on both Folsomia candida and Hypoaspis aculeifer shall be provided.

If data on Aphidius rhopalosiphi and Typhlodromus pyri are not available, then the data set out in point 8.4.2.1 shall be provided.

For plant protection products applied directly to soil as soil treatments either as a spray or as a solid formulation, testing shall be carried out on both on Folsomia candida and Hypoaspis aculeifer (see point 8.4.2.1).

8.4.2.1.   Species level testing

A test shall provide sufficient information to perform an assessment of the toxicity of the active substance to the soil invertebrate indicator species Folsomia candida and Hypoaspis aculeifer.

Test conditions