Conclusion on the peer review of the pesticide risk assessment of the active substance Trichoderma asperellum strain T34 1

Transcription

1 CONCLUSION ON PESTICIDE PEER REVIEW Conclusion on the peer review of the pesticide risk assessment of the active substance 1 ABSTRACT European Food Safety Authority 2 European Food Safety Authority (EFSA), Parma, Italy The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State the United Kingdom, for the pesticide active substance are reported. The context of the peer review was that required by Commission Regulation (EU) No 188/2011. The conclusions were reached on the basis of the evaluation of the representative uses of as a fungicide in greenhouses and indoors on carnation plants. The reliable endpoints concluded as being appropriate for use in regulatory risk assessment, derived from the available studies and literature in the dossier peer reviewed, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified. European Food Safety Authority, 2012 KEY WORDS, peer review, risk assessment, pesticide, fungicide 1 On request from the European Commission, Question No EFSA-Q , approved on 20 April Correspondence: pesticides.peerreview@efsa.europa.eu Suggested citation: European Food Safety Authority; Conclusion on the peer review of the pesticide risk assessment of the active substance.. 37 pp. doi: /j.efsa Available online: European Food Safety Authority, 2012

2 SUMMARY is a new active substance for which in accordance with Article 6(2) of Council Directive 91/414/EEC 3 the United Kingdom (hereinafter referred to as the RMS ) received an application from Biocontrol Technologies S.L. for approval. Complying with Article 6(3) of Directive 91/414/EEC, the completeness of the dossier was checked by the RMS. The European Commission recognised in principle the completeness of the dossier by Commission Decision 2010/132/EU of 2 March The RMS provided its initial evaluation of the dossier on in the Draft Assessment Report (DAR), which was received by the EFSA on 16 May The peer review was initiated on 15 June 2011 by dispatching the DAR for consultation of the Member States and the applicant Biocontrol Technologies S.L. Following consideration of the comments received on the DAR, it was concluded that there was no need to conduct an expert consultation and EFSA should adopt a conclusion on whether Trichoderma asperellum strain T34 can be expected to meet the conditions provided for in Article 5 of Directive 91/414/EEC, in accordance with Article 8 of Commission Regulation (EU) No 188/2011. The conclusions laid down in this report were reached on the basis of the evaluation of the representative uses of as a fungicide in greenhouses and indoors on carnation plants, as proposed by the applicant. Full details of the representative uses can be found in Appendix A to this report. In the areas of identity of the microorganism, biological properties, physical and technical properties and methods of analysis, the following data gaps remain: level of contaminating microorganism, identity of toxins/secondary metabolites and additional validation data for the analytical method. In the area of mammalian toxicology a data gap was identified as the production of toxins/secondary metabolites cannot be excluded and therefore the risk assessment for human health cannot be finalised. The consumer risk assessment cannot be finalised because there may be a following crop issue, which is subject to a data gap in section on fate and behaviour in the environment. The information on fate and behaviour in the environment was not sufficient to characterise the competitiveness/persistence of T. asperellum strain T34 and its dispersion characteristics in the context of the representative uses assessed, therefore data gaps were identified. It was not shown that T. asperellum strain T34 will not persist in the environment in concentrations higher than the natural background levels, taking into account repeated applications over the years. Satisfactory information was missing to demonstrate that, under the conditions of use, any relevant toxins/secondary metabolites produced by T. asperellum strain T34 (which may include trichothecenes, viridofungins and peptaibols) will not occur in the environmental compartments in concentrations considerably higher than under natural conditions. Further data on the persistence, transformation and mobility of these compounds may be needed in order to assess the potential for groundwater contamination and possibly exposure levels in other environmental compartments. A data gap was identified in the ecotoxicology section for information to address the risk to organisms involved in biological methods for sewage treatment. 3 4 OJ No L 230, , p. 1. Directive as last amended by L 20, , p.19 and by L309, , p.1 OJ No L 52, , p. 51 2

3 Table of contents Abstract... 1 Summary... 2 Background... 4 The identity of the microorganism and the properties of the formulated product... 6 Conclusions of the evaluation Identity of the microorganism/biological properties/physical and technical properties and methods of analysis Mammalian toxicity Residues Environmental fate and behaviour Fate and behaviour in the environment of the microorganism Fate and behaviour in the environment of any relevant metabolite formed by the microorganism under relevant environmental conditions Ecotoxicology Overview of the risk assessment of compounds listed in residue definitions triggering assessment of effects data for the environmental compartments Soil Ground water Surface water and sediment Air List of studies to be generated, still ongoing or available but not peer reviewed Particular conditions proposed to be taken into account to manage the risk(s) identified Concerns Issues that could not be finalised Critical areas of concern Overview of the concerns identified for each representative use considered References Appendices

4 BACKGROUND In accordance with Article 80(1)(a) of Regulation (EC) No 1107/2009 5, Council Directive 91/414/EEC 6 continues to apply with respect to the procedure and conditions for approval for active substances for which a decision recognising in principle the completeness of the dossier was adopted in accordance with Article 6(3) of that Directive before 14 June Commission Regulation (EU) No 188/ (hereinafter referred to as the Regulation ) lays down the detailed rules for the implementation of Council Directive 91/414/EEC as regards the procedure for the assessment of active substances which were not on the market on 26 July This regulates for the European Food Safety Authority (EFSA) the procedure for organising the consultation of Member States and the applicant for comments on the initial evaluation in the Draft Assessment Report (DAR) provided by the rapporteur Member State (RMS), and the organisation of an expert consultation, where appropriate. In accordance with Article 8 of the Regulation, EFSA is required to adopt a conclusion on whether the active substance is expected to meet the conditions provided for in Article 5 of Directive 91/414/EEC within 4 months from the end of the period provided for the submission of written comments, subject to an extension of 2 months where an expert consultation is necessary, and a further extension of up to 8 months where additional information is required to be submitted by the applicant(s) in accordance with Article 8(3). In accordance with Article 6(2) of Council Directive 91/414/EEC the United Kingdom (hereinafter referred to as the RMS ) received an application from Biocontrol Technologies S.L. for approval of the active substance. Complying with Article 6(3) of Directive 91/414/EEC, the completeness of the dossier was checked by the RMS. The European Commission recognised in principle the completeness of the dossier by Commission Decision 2010/132/EU of 2 March The RMS provided its initial evaluation of the dossier on in the DAR, which was received by the EFSA on 16 May 2011 (United Kingdom, 2011). The peer review was initiated on 15 June 2011 by dispatching the DAR to Member States and the applicant Biocontrol Technologies S.L. for consultation and comments. In addition, the EFSA conducted a public consultation on the DAR. The comments received were collated by the EFSA and forwarded to the RMS for compilation and evaluation in the format of a Reporting Table. The comments were evaluated by the RMS in column 3 of the Reporting Table. The applicant was invited to respond to the comments in column 3 of the Reporting Table. The comments and the applicant s response were evaluated by the RMS in column 3. The need for expert consultation and the necessity for additional information to be submitted by the applicant in accordance with Article 8(3) of the Regulation were considered in a telephone conference between the EFSA, the RMS, and the European Commission on 10 October On the basis of the comments received, the applicant s response to the comments and the RMS s evaluation thereof it was concluded that additional information should be requested from applicant and that there was no need to conduct an expert consultation. The outcome of the telephone conference, together with EFSA s further consideration of the comments is reflected in the conclusions set out in column 4 of the Reporting Table. All points that were identified as unresolved at the end of the comment evaluation phase and which required further consideration, including the additional information to be submitted by the applicant, were compiled by the EFSA in the format of an Evaluation Table. 5 OJ No L 309, , p OJ No L 230, , p. 1. Directive as last amended by L 20, , p.19 and by L309, , p.1 7 OJ No L 53, , p OJ No L 52, , p. 51 4

5 The conclusions arising from the consideration by the EFSA, and as appropriate by the RMS, of the points identified in the Evaluation Table were reported in the final column of the Evaluation Table. A final consultation on the conclusions arising from the peer review of the risk assessment took place with Member States via a written procedure in March - April This conclusion report summarises the outcome of the peer review of the risk assessment on the active substance and the representative formulation evaluated on the basis of the representative use as a fungicide in greenhouses and indoors on carnation plants, as proposed by the applicant. A list of the relevant end points for the active substance as well as the formulation is provided in Appendix A. In addition, a key supporting document to this conclusion is the Peer Review Report, which is a compilation of the documentation developed to evaluate and address all issues raised in the peer review, from the initial commenting phase to the conclusion. The Peer Review Report (EFSA, 2012) comprises the following documents, in which all views expressed during the course of the peer review, including minority views, can be found: the comments received on the DAR, the Reporting Table (17 October 2011), the Evaluation Table (26 March 2012), the comments received on the assessment of the additional information (where relevant), the comments received on the draft EFSA conclusion. Given the importance of the DAR including its addendum (compiled version of March 2012 containing all individually submitted addenda (United Kingdom, 2012)) and the Peer Review Report, both documents are considered respectively as background documents A and B to this conclusion. 5

6 THE IDENTITY OF THE MICROORGANISM AND THE PROPERTIES OF THE FORMULATED PRODUCT was isolated from a suppressive compost-peat mix in Spain. The representative formulation evaluated is T34 Biocontrol, a wettable powder formulation (WP) containing a minimum of 1 x 10 9 CFU/g. T34 Biocontrol is used in greenhouses to control Fusarium oxysporum in carnation plants. Full details of the GAP can be found in the list of end points in Appendix A. CONCLUSIONS OF THE EVALUATION 1. Identity of the microorganism/biological properties/physical and technical properties and methods of analysis. is kept in the CECT (Spanish Collection of Type Cultures) culture collection under the accession code The strain is not a human pathogen and is not related to known human pathogens. The strain is not expected to grow at 37 C and above. Trichoderma species have been reported to synthesize many toxins/secondary metabolites. Data were available on the production of trichothecenes, which were not found in detectable quantities. It may be possible that this strain produces some other toxins/secondary metabolites and a data gap has been identified to address this issue. The content of contaminating microorganisms was not fully addressed and a data gap has been identified. The assessment of the data package revealed no issues that need to be included as critical areas of concern with respect to the identity, physical, chemical and technical properties of the representative formulation. A well validated method was available for the identification of this strain. A method is available for the determination of the microorganism in the technical material, however additional validation data have been identified as a data gap. Acceptable methods are available for determination of the content of contaminating microorganisms. 2. Mammalian toxicity No detailed analysis of the batches used in the toxicological studies is available. However, further information is not required, provided that adequate quality control is undertaken on the batches produced, certifying that toxicologically relevant pathogenic microbial contaminants are kept below levels internationally recognized for microbial contaminants (see data gap in section 1). Considering the development stages and life cycle of T. asperellum strain T34, as well as the absence of a close relationship with pathogenic microorganisms, it is not expected that it will transfer genetic material (e.g. for resistance against antibiotic and antifungal compounds) to microorganisms of concern for human health. In the literature, some T. viride strains have been identified in infections of immunocompromised humans. It cannot be excluded that the current T. asperellum strain T34 could be involved in clinical cases. However, in most of the cases, the route of exposure is not relevant for the use of Trichoderma as a plant protection product. In the absence of a reliable test for sensitisation, as for other microorganisms, the following warning phrase is applicable, related to the potential to provoke allergic reactions by inhalation as well as by dermal exposure: Microorganisms may have the potential to provoke sensitising reactions. 6

7 In the DAR (United Kingdom, 2011), one acute oral, one intratracheal and two intraperitoneal (i.p.) toxicity/pathogenicity/infectivity studies were performed in rats with the Microbial Pest Control Product (MPCP; i.e. T34 Biocontrol ). The MPCP induced deaths after acute intratracheal exposure at 1 x 10 7 CFU/animal and in the two i.p. studies at ca 7.3 x 10 7 and 10 8 CFU/animal. No deaths were observed after oral exposure at ca 1.1 x 10 8 CFU/animal. Some pathological findings (i.e. red/pink discolouration) were observed in lungs (intratracheal exposure), adrenals (oral and intratracheal exposure) and pancreas (oral and intratracheal administration). Adhesions of abdominal organs, discolouration of organs and peritonitis were observed after i.p. exposure too. After oral, intratracheal and i.p. administration, the clearance from the different tissues was rapid and almost completed within 21 (oral and intratracheal exposure) and 22 days (i.p. administration) indicating the lack of infectivity. This is corroborated by the fact that T. asperellum strain T34 is not expected to grow at 37 C and above (see section 1). In the DAR (United Kingdom, 2011), skin and eye irritation studies, acute oral, dermal and inhalation toxicity studies were also provided for T34 Biocontrol. The results indicated that T34 Biocontrol is neither a skin nor eye irritant. It is not acutely toxic via oral (LD 50 > 2000 mg/kg bw; 3 x 10 9 CFU/kg bw), dermal (LD 50 > 2000 mg/kg bw; 3 x 10 9 CFU/kg bw) and inhalation routes (LC 50 > 2.03 mg/l; 3.65 x 10 6 CFU/animal) to rats. Given the results of the first two i.p and intratracheal studies, a data requirement was set after the commenting phase to clarify these findings. Two additional i.p. studies were conducted: no deaths and pathological findings were observed with the MPCP at ca 1.4 x 10 7 to 4.9 x 10 7 CFU/animal or with the active agent of the microbial pest control product (MCPA) at ca 4.9 x 10 7 CFU/animal (see United Kingdom, 2012). Trichoderma species have been reported to synthesize many toxins/secondary metabolites. Of these, trichothecenes (e.g. T2 toxin, harzianum or trichodermin) are known to affect human health. Data on toxin/secondary metabolite production showed that T34 formulated product does not contain the trichothecene toxins T2 or harzianium A, and is unlikely to produce relevant levels of trichodermin. An Ames test performed with a supernatant from T. asperellum strain T34 product gave negative results. No adverse effects were observed in rats after i.p. exposure to sterile filtrate of the medium where T34 grew. Thus, these data provide an indication that if toxins are produced in the product, the absolute toxicity and/or the levels are likely to be low. However, no data on other toxins/secondary metabolites produced by this strain during manufacturing or following application are available (see data gap in section 1). Pending on the outcome of this data gap, the need for short-term toxicity or further genotoxicity testing will have to be reconsidered. Overall, the deaths and pathological findings observed in the intratracheal and i.p. toxicity/infectivity studies are likely to be due to the methodology used in the studies (i.e. volume and vehicle; as proposed by the RMS) but as the effects are observed at high dose levels a dose-dependent effect could not totally be ruled out (i.e. as proposed by some MSs). In conclusion, the weight of evidence suggests that the adverse effects observed at high doses are likely to be due not only to the high volume administered but also as a result of the route of administration (i.e. intratracheal and i.p.), rather than the inherent toxicity or infectivity of T. asperellum strain T34 (i.e. based on the pattern of clearance and no growth expected at 37 C and above). The derivation of reference values for the microorganism is not considered necessary based on the lack of significant toxicity, infectivity or pathogenicity in the available studies, and is not possible anyway based on the available data. Therefore, operator and worker exposure estimates are not needed for the microorganism although estimates were provided by the RMS. For bystanders, exposure to the MPCA for indoor scenarios is likely to be negligible in any case. However, taking into account the lack of data about the potential toxins/secondary metabolites, the operator and worker risk assessment cannot be concluded. 7

8 3. Residues Peer review of the pesticide risk assessment of the active substance Trichoderma species have been reported to synthesise many toxins/secondary metabolites. It is not known what toxins/secondary metabolites are produced by this strain and if they are taken up by plants. The consumer risk assessment cannot be finalised until the outstanding issues on toxins/secondary metabolites are addressed and it is confirmed by the toxicological assessment that a quantitative consumer risk assessment is not necessary. This is pertinent in relation to edible crops that might be grown in a greenhouse following carnation production. It may also be pertinent in the situation that spent growing media from carnation production is spread on agricultural or horticultural land (see section 4.1). 4. Environmental fate and behaviour 4.1. Fate and behaviour in the environment of the microorganism No information has been provided in relation to potential interference of T. asperellum strain T34 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC 9 (see specific Annex VI decision making criteria in Directive 2005/25/EC). However as these methods require pathogenic bacteria to be identified and confirmed as absent, it is unlikely that filamentous fungi or their conidia would interfere with methodologies used for such determinations. No information has been provided on the potential transfer of genetic material from T. asperellum strain T34 to other organisms. As fungi, Trichoderma sp. are not expected to possess plasmids in their cytoplasm (only mitochondrial plasmids are known). Consequently it might be expected that the potential for transfer of genetic material from Trichoderma sp. to other organisms is unlikely. Studies on persistence and multiplication in soil show that the soil borne fungus T. asperellum strain T34 persists in growth media for at least 183 days. Trichoderma species occur naturally within the soil micro-flora and, in general, multiply within the rhizosphere. Hence, in the absence of growing roots the capacity for multiplication of the fungus is expected to be more limited. However for the similar strain and same species Trichoderma asperellum ICC012 (where the available data demonstrated it persists in a comparable fashion, i.e. levels above untreated controls after 115 days), EFSA identified a data gap, since it was not shown that Trichoderma asperellum ICC012 would not persist in the environment in concentrations higher than natural background levels, taking into account repeated applications over the years. Consequently a data gap is also identified for T. asperellum strain T34. This data gap is still pertinent in respect of the representative use assessed for T. asperellum strain T34 (glasshouse and indoor use), particularly as what happens to the growth medium at the end of the production was not assessed in the dossier. This route of environmental exposure should be taken into account in territories where spent growing media can be spread on agricultural or horticultural land. The data gap is also relevant for production in temporary greenhouses (e.g. polythene tunnels) for the soil in situ. A study on mobility in soil of T. asperellum strain T34 shows that the main concentration of T. asperellum strain T34 in the treated soil is in the upper 20 cm layer. This is due to the proliferation of plant roots, nutrients and water in this soil layer. Limited movement to deeper soil layers was seen in the study. Studies on persistence and multiplication in water of T. asperellum strains T34 were not available. A study on the presence of T. asperellum strain T34 (and other Trichoderma species) in leachates from pots containing two types of growth media (peat and coir fibre) four months after application where two crops of carnations had been grown, was assessed in the DAR. This study indicated that most of the T. asperellum strain T34 stayed in the plant growth media, but some was found in the leachate. Since T. asperellum strain T34 is applied via irrigation water, propagation/seeding or mixing with 9 OJ No L 330, , p. 32 8

9 growing substrate, surface water exposure is expected to be negligible. Consequently the predicted environmental concentration (PEC) in surface water was not calculated. Trichoderma species have occasionally been isolated from the air both indoors and outdoors. The species appears to occur in the air naturally, the concentrations detected were low compared with other fungal genera. Significant dispersal of spores via aerosols is not anticipated due to the nature of the preparation, the method of application as irrigation, propagation/seeding or mixing with growing substrate, and due to the fact that proliferation is limited to non-lignified parts of the root zone. A study on aerial dispersion of T. asperellum strain T34, in a greenhouse where T34 was applied via drip irrigation systems for two years, indicated that the presence in air of T. asperellum T34 was low Fate and behaviour in the environment of any relevant metabolite formed by the microorganism under relevant environmental conditions Certain Trichoderma species are able to produce many different metabolites such as polyketides, sesquiterpenes (including the mycotoxin group of trichothecenes), viridofungins and peptaibols. Some of these are inhibitory to fungi or bacteria, others have proven toxicity to mammals. A data gap is identified since it is not known if T. asperellum strain T34 will produce any relevant metabolites following their application to soil or if they will be present in the product. It is not clear if such metabolites might fulfil the criteria according to the directive 91/414/EEC: - Stable outside the microorganism - Biologically active independently of the presence of the microorganism - Intended to be applied at levels above background levels Therefore data on the potential for T. asperellum strain T34 to produce metabolites in relation to these criteria are necessary to assess if the further data requirements and the corresponding risk assessment according to Directive 91/414/EEC Annex II part A point 7 (standard data requirements and assessment mandatory for chemical plant protection active substances) are triggered. Consequently a data gap was identified. This data gap is pertinent in respect of the representative use assessed for T. asperellum strain T34, both in situ indoors and following disposal of spent growing media, as what happens to the growth medium at the end of the production was not assessed. This route of environmental exposure would need to be taken into account, in territories where spent growing media can be spread on agricultural or horticultural land. 5. Ecotoxicology No strain specific data on were submitted to address the toxicity, infectiveness or pathogenicity to non-target organisms, with the exception of a study on soil microbial communities and plant growth media wastes treated with the T. asperellum strain T34. In this study, there was no indication of significant specific lasting effects attributed to T. asperellum strain T34 in soils amended with T. asperellum strain T34 treated growth media waste. As exposure of natural aquatic systems consequent to the representative use assessed is considered negligible for T. asperellum strain T34 (see section 4.1), the risk to aquatic organisms from T. asperellum strain T34 exposure is concluded to be low. Since T. asperellum strain T34 is applied by irrigation, in propagation/seeding or mixing with growing substrate in greenhouses or for indoor uses, the environmental exposure for the representative uses might be considered negligible and therefore the risk assessment to non-target organisms from exposure to T. asperellum strain T34, can be considered as low in permanent structures. However in the situation that spent growth media disposal practice includes spreading on agricultural or horticultural land, or production is in temporary greenhouses (e.g. polythene tunnels), the ecotoxicological risk assessment remains open consequent to the data gap identified in section 4.1 (regarding demonstration that T. asperellum strain T34 would 9

10 not persist in the environment in concentrations higher than natural background levels, taking into account repeated applications over the years). No data on T. asperellum strain T34 to address the risk to organisms involved in biological methods for sewage treatment were submitted. The exposure to sewage treatment plants from the representative uses of T. asperellum strain T34 cannot be excluded. Therefore, a data gap was identified to address the risk to organisms involved in biological methods for sewage treatment. It is noted that a data gap for information on toxins/secondary metabolites produced by this strain was identified (see sections 1, 2 and 4) and that, pending on these data gaps, the aquatic risk assessment might need to be further considered for the indoor uses (permanent structures). It is pointed out that in territories where the disposal of spent growth media is not carefully managed, or for temporary greenhouses (e.g. polythene tunnels) the ecotoxicological risk assessment for toxins/secondary metabolites might need to be considered. 10

11 6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment of effects data for the environmental compartments 6.1. Soil Compound (name and/or code) Relevant toxins or secondary metabolites including trichothecenes, viridofungins and peptaibols. Persistence, viability and dynamics May persist months to years in soil. No data available. Ecotoxicology No data available. Assessment may be needed pending on the data gap in section 4. No data available. Assessment may be needed pending on the data gap in sections 1, 2 and Ground water Compound (name and/or code) Mobility in soil >0.1 μg/l 1m depth for the representative uses (at least one FOCUS scenario or relevant lysimeter) Pesticidal activity Toxicological relevance Ecotoxicological activity Relevant toxins or secondary metabolites including trichothecenes, viridofungins and peptaibols. No data available. No data available. Data gap. Data gap pending on their identification and quantification. No data available. Assessment may be needed pending on the data gap in sections 1, 2 and 4. 11

12 6.3. Surface water and sediment Compound (name and/or code) Relevant toxins or secondary metabolites including trichothecenes, viridofungins and peptaibols. Ecotoxicology No data available and not needed for the representative use. No data available. Assessment may be needed pending on the data gap in sections 1, 2 and Air Compound (name and/or code) Relevant toxins or secondary metabolites including trichothecenes, viridofungins and peptaibols. Toxicology Some deaths at 1x 10 7 CFU/rat likely to be due to methodology and route of administration (intratracheal) Rat LC 50 > 3.65 x 10 6 CFU/animal (nose only) No data available. 12

13 7. List of studies to be generated, still ongoing or available but not peer reviewed This is a complete list of the data gaps identified during the peer review process, including those areas where a study may have been made available during the peer review process but not considered for procedural reasons (without prejudice to the provisions of Article 7 of Directive 91/414/EEC concerning information on potentially harmful effects). GLP batch analysis data using validated methods. The OECD issues paper (OECD, 2011) should be used as the reference (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 1). Identification of all toxins/secondary metabolites produced by this strain during manufacture and after application (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see sections 1, 2 and 3). Additional validation data for the method for the determination of the microorganism in the technical material (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see sections 1). Information demonstrating that will not persist in the environment in concentrations higher than the natural background levels, taking into account repeated applications over the years is outstanding. Pending this being addressed, the ecotoxicological risk assessment might need to be re-considered (relevant for all representative uses evaluated, in territories where spent growing media can be spread on agricultural or horticultural land or when the greenhouse type is temporary (e.g. polythene tunnels); submission date proposed by the notifier: unknown; see section 4). Satisfactory information to demonstrate that, under the conditions of use, any relevant toxins/secondary metabolites produced by T. asperellum strain T34 (which may include trichothecenes, viridofungins and peptaibols) will not occur in the environmental compartments in concentrations considerably higher than under natural conditions. Further data on the persistence, transformation and mobility of these compounds may be needed in order to assess the potential for groundwater contamination or exposure of surface water. Pending this being addressed, ecotoxicological risk assessment might need to be re-considered (relevant for all representative uses evaluated, both as a consequence of possible residues in soil in situ and to cover the situation, in territories where spent growing media can be spread on agricultural or horticultural land; submission date proposed by the notifier: unknown; see section 4). Information to address the risk to organisms involved in biological methods for sewage treatment (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 5). 8. Particular conditions proposed to be taken into account to manage the risk(s) identified None. 9. Concerns 9.1. Issues that could not be finalised An issue is listed as an issue that could not be finalised where there is not enough information available to perform an assessment, even at the lowest tier level, for the representative uses in line 13

14 with the Uniform Principles of Annex VI to Directive 91/414/EEC and where the issue is of such importance that it could, when finalised, become a concern (which would also be listed as a critical area of concern if it is of relevance to all representative uses). 1. The production of relevant toxins/secondary metabolites cannot be excluded and therefore the risk assessment cannot be finalised for humans (including operators, workers and consumers) and the environment including the assessment of potential groundwater contamination. 2. Satisfactory information is not available that will not persist in the environment in concentrations higher than the natural background levels, taking into account repeated applications over the years. Consequently the level of exposure in soil of T. asperellum strain T34 cannot be reliably estimated. Therefore the ecotoxicological risk assessment cannot be finalised in relation to temporary greenhouse systems (e.g. polythene tunnels) or when spent growing media is spread on agricultural or horticultural land. 3. The risk assessment for organisms involved in biological methods for sewage treatment cannot be finalised with the available data Critical areas of concern An issue is listed as a critical area of concern where there is enough information available to perform an assessment for the representative uses in line with the Uniform Principles of Annex VI to Directive 91/414/EEC, and where this assessment does not permit to conclude that for at least one of the representative uses it may be expected that a plant protection product containing the active substance will not have any harmful effect on human or animal health or on groundwater or any unacceptable influence on the environment. An issue is also listed as a critical area of concern where the assessment at a higher tier level could not be finalised due to a lack of information, and where the assessment performed at the lower tier level does not permit to conclude that for at least one of the representative uses it may be expected that a plant protection product containing the active substance will not have any harmful effect on human or animal health or on groundwater or any unacceptable influence on the environment. No critical areas of concern were identified. 14

15 9.3. Overview of the concerns identified for each representative use considered (If a particular condition proposed to be taken into account to manage an identified risk, as listed in section 8, has been evaluated as being effective, then risk identified is not indicated in this table.) Representative use Carnation plants Carnation plants Carnation plants (propagation or seeding) (container grown crops: mixing with growing substrate/root bath) (irrigation) Operator risk Worker risk Bystander risk Consumer risk Risk to wild non target terrestrial vertebrates Risk to wild non target terrestrial organisms other than vertebrates Risk aquatic organisms Groundwater exposure active substance Groundwater exposure metabolites to Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Legal parametric value breached Assessment not finalised Legal parametric value breached Parametric value of 10µg/L (a) breached X 1 X 1 X 1 X 1 X 1 X 1 X 1 X 1 X 1 X 1,2,3 X 1,2,3 X 1,2,3 X 1 X 1 X 1 15

16 Assessment not finalised Comments/Remarks X 1 X 1 X 1 The superscript numbers in this table relate to the numbered points indicated in sections 9.1 and 9.2. Where there is no superscript number see sections 2 to 6 for further information (a): Value for non-relevant metabolites prescribed in SANCO/221/2000-rev 10-final, European Commission,

17 REFERENCES ACD/ChemSketch, Advanced Chemistry Development, Inc., ACD/Labs Release: Product version: (Build 29305, 25 Nov 2008). EFSA (European Food Safety Authority), Peer Review Report to the conclusion regarding the peer review of the pesticide risk assessment of the active substance Trichoderma asperellum strain T34. OECD (Organisation for Economic Co-operation and Development), OECD Issue Paper on Microbial Contaminant Limits for Microbial Pest Control Products, ENV/JM/MONO(2011)43. United Kingdom, Draft Assessment Report (DAR) on the active substance Trichoderma asperellum (Strain T34) prepared by the rapporteur Member State the United Kingdom in the framework of Directive 91/414/EEC, May United Kingdom, Final Addendum to Draft Assessment Report on Trichoderma asperellum strain T34, compiled by EFSA, March

18 APPENDICES APPENDIX A LIST OF END POINTS FOR THE ACTIVE SUBSTANCE AND THE REPRESENTATIVE FORMULATION 1 Identity, Biological properties, Details of Uses, Further Information Active microorganism Trichoderma asperellum (Strain T34) Function (e.g. fungicide) Fungicide Identity of the microorganism (Annex IIM 1) Name of the organism: Taxonomy: First description: Samuels, GJ, Lieckfeldt, E, Nirenberg, HI, Trichoderma asperellum, a new species with warted conidia, and redescription of T. viride. Sydowia 51 (1): Strain: T34 Genus: Trichoderma Family: Not known (most of Trichoderma belong to Hypocreale) Division: Not known (most of Trichoderma belong to Ascomycete) Species, subspecies, strain: Identification: Culture collection: Minimum and maximum concentration of the microorganism used for manufacturing of the formulated product (CFU/g; CFU/L, etc.): Identity and content of relevant impurities in the technical grade microorganism: Is the MPCA genetically modified; if so provide type of modification The strains were identified first based on morphological traits and more recently by molecular tools. The molecular identification allows differentiation between strains, which is not possible using morphological identification methods. Spanish Collection of Type Cultures (CECT No ). The initial population in the MPCA should be 1 x CFU/g to give a guaranteed population in the MPCP of 1.0 x 10 9 CFU/g. Open N/A Biological properties of the microorganism (Annex IIM) 18

19 Origin and natural occurrence, background level: Target organism(s): Mode of action: Host specificity: Life cycle: Infectivity, dispersal and colonisation ability: Trichoderma asperellum (Strain T34) was isolated from a natural suppressive compost-peat mix (Cotxarrera et al. 2002). The compost used was commercially available (Metrocompost S.A., Castelldefels, Barcelona, Spain), and was made from vegetable and animal market wastes, sewage sludge and yard wastes in a tunnel system. No specific information is available on the occurrence of the strain. However, Trichoderma spp. are present in nearly all soils and other diverse habitats such as decaying wood. Trichoderma asperellum occurs in both temperate and tropical regions. Fusarium oxysporum f. sp. dianthi (FOD) which causes Fusarium wilt of carnation plants. The mechanism has not been fully determined. Historically, the mode of action has been thought to include mycoparasitism, antibiosis and competition for space and resources in the growth medium (especially iron). However, there is now evidence that T. asperellum produces changes in plant metabolism, increasing growth and enhancing resistance to biotic and abiotic stresses. In addition, the applicant claims that strain T34 induces systemic acquired resistance (SAR) and induced systemic resistance (ISR) in plants. T. asperellum does not have a specific host. It is a general saprophytic fungus occurring in soil, especially in the rhizosphere, but also on organic material. The life cycle of T. asperellum is typical for imperfect fungi T.asperellum strain (T34) is a saprophytic fungus and as such could be expected to grow in soil provided the correct conditions (temperature, light, moisture) allow. The optimum growth temperature for T. asperellum strain T34 is 36 C. At 37 & 38 C it would be expected that no growth would occur but that the fungus would survive to be reactivated at lower temps. Growth is irreversibly halted at temperatures above 38 C. Relationship to known pathogens: T. asperellum T34 strain is not related to any known plant, animal or human pathogen. 19

20 Genetic stability: Production of relevant metabolites/toxins: Resistance/sensitivity to antibiotics/antimicrobial agents used in human or veterinary medicine: Since the isolation (2001), no mutation of traits related to biocontrol activity are observed. Data on metabolite and toxin production were not conclusive but provided reassurance that the product as sold did not contain the trichothecene toxins T2 or Harzianum A and that it was unlikely to produce relevant levels of Trichodermin. There is also evidence that T-34 is unlikely to produce antibiotic or antifungal compounds. Open for further data on toxin/secondary metabolite production. The risk of resistance to T. asperellum developing as a result of the proposed use of T34 Biocontrol is likely to be low and no specific restrictions on use are currently considered necessary. The applicant has also performed calculations that indicate that the relatively low sensitivity to antifungal agents is not a concern as therapeutic doses would produce systemic levels above those required to control T

21 Summary of representative uses evaluated Crop and Member Product F Pest or /or situation state or name G Group of Country or I pests controlled (a) (b) (c) Type (d-f) Formulation Application Application rate per treatment Conc. of as (i) method kind (f-h) growth stage & season (j) number min -max interval between applications (min) kg as/hl; CFU/hL (l) min max water l/ha min max kg as/ha; CFU/ha (l) min max PHI (days) (l) Remarks (m) Carnation plants growing in the greenhouse UK T34 Biocontrol G I Fusarium oxysporum f.sp. dianthi WP % Propagat ion or seeding to 1.08 g of as /m 3-10g of product /m 3 Or 0.5 g of product /m 2 of surface area immediat ely after sowing seeds or rooting cuttings np (*) the doses have been adapted and expressed as in Biol Asses.Do ssier 21

22 Crop and Member Product F Pest or /or situation state or name G Group of Country or I pests controlled (a) (b) (c) Type (d-f) Formulation Application Application rate per treatment Conc. of as (i) method kind (f-h) growth stage & season (j) number min -max interval between applications (min) kg as/hl; CFU/hL (l) min max water l/ha min max kg as/ha; CFU/ha (l) min max PHI (days) (l) Remarks (m) Carnation plants growing in the greenhouse UK T34 Biocontrol G I Fusarium oxysporum f.sp. dianthi WP % Containe r grown crops: Mixing with growing substrate Before planting : preparation of growth medium g of as /L of water for predilution 16.7 ml of this predilution /L of grow medium x 10 4 CFU/ml plant growth medium. 22

23 Root bath Before introduction of plant: growth medium in the pots g of as /L of water for predilution then 1.0 x 10 4 CFU/ml plant growth medium ml of this predilution /L of grow medium At planting x 10 4 CFU/ml of water 23

24 Crop and Member Product F Pest or /or situation state or name G Group of Country or I pests controlled (a) (b) (c) Type (d-f) Formulation Application Application rate per treatment Conc. of as (i) method kind (f-h) growth stage & season (j) number min -max interval between applications (min) kg as/hl; CFU/hL (l) min max water l/ha min max kg as/ha; CFU/ha (l) min max PHI (days) (l) Remarks (m) Carnation plants growing in the greenhouse UK T34 Biocontrol G I Fusarium oxysporum f.sp. dianthi WP % Irrigatio n All growing period 8-12 weeks g of as /L of water for predilution 0.5 x 10 4 CFU/ml plant growth medium ml of this predilution /L of grow medium 24

25 8-12 weeks g of as /L of water for predilution 22.2 ml of this predilution /L of grow medium 1.0 x 10 4 CFU/ml plant growth medium. For uses where the column "Remarks" is marked in grey further consideration is necessary. Uses should be crossed out when the notifier no longer supports this use(s). (a) For crops, the EU and Codex classifications (both) should be taken into account; where relevant, the use situation should be described (e.g. fumigation of a structure) (b) Outdoor or field use (F), greenhouse application (G) or indoor application (I) (c) e.g. biting and suckling insects, soil born insects, foliar fungi, weeds (d) e.g. wettable powder (WP), emulsifiable concentrate (EC), granule (GR) (e) GCPF Codes - GIFAP Technical Monograph No 2, 1989 (f) All abbreviations used must be explained (g) Method, e.g. high volume spraying, low volume spraying, spreading, dusting, drench (h) Kind, e.g. overall, broadcast, aerial spraying, row, individual plant, between the plant- type of equipment used must be indicated (i) CFU/kg or CFU/l. (j) Growth stage at last treatment (BBCH Monograph, Growth Stages of Plants, 1997, Blackwell, ISBN ), including where relevant, information on season at time of application (k) Indicate the minimum and maximum number of application possible under practical conditions of use (l) The values should be given in g or kg whatever gives the more manageable number (e.g. 200 kg/ha instead of g/ha or 12.5 g/ha instead of kg/ha) as well as in number of CFU (m) PHI - minimum pre-harvest interval 25

26 2 Analytical methods Analytical methods for the microorganism (Annex IIM 4.2, 4.3; IIIM 5.4) Manufactured microorganism (principle of CFU count plating methods method): Open for validation data Impurities and contaminating microorganisms in manufactured material (principle of method): Microbial plant protection product (principle of method): ISO standard plating methods CFU count plating methods Open for validation data Analytical methods for residues (viable and non-viable) (Annex IIM 4.5) of the active microorganism (principle of Not required method): of relevant metabolites (principle of method): Open 26

27 3 Impact on Human and Animal Health (Annex IIM 5; IIIM 7) Medical data, surveillance and observations: No strain specific data. Some T. viride strains have been identified in infections of immunocompromised humans. It cannot be excluded that the current T. asperellum strain T34 could be involved in clinical cases. However, in most of the cases, the route of exposure is not relevant for the use of Trichoderma as plant protection product. No adverse effects reported in monitoring of production plant workers. Sensitisation (experience in humans and study results; type of study): Considering that all microbials should be regarded as potential sensitizers, the agreed warning phrase is: Microorganisms may have the potential to provoke sensitising reactions. Positive result with formulated product in a maximisation assay in guinea pigs. No reactions in manufacturing plant personnel. Toxicity after acute oral exposure: after acute inhalation exposure: after acute intraperitoneal/subcutaneous exposure: Rat: LD 50 > 1 x 10 8 CFU/animal Rat: LD 50 > 1 x 10 9 CFU/kg bw Some deaths at 1x 10 7 CFU/rat (intratracheal) Deaths are likely to be due to high volume and route of administration. Rat LC 50.> 3.65 x 10 6 CFU/animal (nose only) Deaths seen in two studies with 7 x /rat. No deaths in subsequent studies with 1.4x x 10 7 /rat Deaths are likely to be due to high volumes and route of administration. Infectivity after acute oral exposure: after acute inhalation exposure: after acute intraperitoneal/subcutaneous exposure: Not infective rapidly cleared Not infective rapidly cleared Not infective rapidly cleared Pathogenicity after acute oral exposure: Red / pink discolouration of pancreas / adrenal at 1 x 10 8 CFU/rat but not replicated in a second oral toxicity study at 1 x 10 9 CFU/kg 27

28 after acute inhalation exposure: Red / pink discolouration of lung / pancreas / adrenal likely to be due to high volumes and route of administration (intratracheal). bw. after acute intraperitoneal/subcutaneous exposure: Adhesions of abdominal organs; discolouration of organs; peritonitis; likely to be due to high doses, high volumes and route of administration. Genotoxicity: Negative in Ames test (supernatant fraction) Further data may be required pending on the identification/quantification of toxins/secondary metabolites (see data gap in section 1). Cell culture study: Not performed not required in the absence of intracellular replication. Short term toxicity/pathogenicity: Not performed. Data may be required pending on the identification/quantification of toxins/secondary metabolites (see data gap in section 1). Specific toxicity, pathogenicity and infectiveness studies No adverse effects were observed in rats after intraperitoneal exposure to sterile filtrate of the medium where T34 grew. Reference values AOEL: ADI: ARfD Not necessary for the microorganism - Not possible to be derived based on the available data Not necessary for the microorganism - Not possible to be derived based on the available data Not necessary for the microorganism - Not possible to be derived based on the available data. Exposure scenarios (including method of calculation) Application method: Compost admixture, spray, root dip, drip irrigation. 28