EUROPEAN COMMISSION
HEALTH & CONSUMER PROTECTION DIRECTORATE-
GENERAL
Directorate E – Safety of the food chain
Unit E.3 - Chemicals, contaminants, pesticides
SANCO/12117/2012 –rev. 0
September 2012
Working Document to the Environmental Safety Evaluation
of Microbial Biocontrol Agents
This document has been conceived as a working document of the Commission Services. It does not
represent the official position of the Commission. It does not intend to produce legally binding
effects.
2
Introduction
The current document has been published by the Organisation for Economic Co-operation and
Development (OECD) under the responsibility of the Joint Meeting of the Chemicals Committee
and the Working Party on Chemicals, Pesticides and Biotechnology.
This document has been developed within the OECD-BioPesticides Steering Group.
This document is written for Member States and industry risk assessors and scientists involved
in the authorisation and approval of microbial plant protection products and their active agents.
With the aim of harmonising the environmental safety assessment of micro-organisms a risk
assessment decision scheme has been developed which is detailed in this document. The use of
micro-organisms in this document is restricted to crop protection for outdoor applications. This
document is intended to be used as guidance in the safety assessment of micro-organisms and
microbial plant protection products; it is not a requirement which has to be followed nor does
it provide mandatory guidance.
It is intended that Member States, EFSA and industry start using this document to gain
experience and in this way improve the safety assessment of micro-organisms and microbial
plant protection products.
Implementation schedule
This document has been finalised in the Standing Committee on the Food Chain and Animal
Health on 28 September 2012. It will apply to applications submitted from 1 July 2013 onwards.
3
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Unclassified ENV/JM/MONO(2012)1
Organisation de Coopération et de Développement Économiques
Organisation for Economic Co-operation and Development
17-Feb-2012
___________________________________________________________________________________________
English - Or. English
ENVIRONMENT DIRECTORATE
JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY
OECD GUIDANCE TO THE ENVIRONMENTAL SAFETY EVALUATION OF MICROBIAL
BIOCONTROL AGENTS
Series on Pesticides
No. 67
JT03316239
Complete document available on OLIS in its ori
g
inal format
This document and any map included herein are without prejudice to the status of or sovereignty over any territory, to the delimitation of
international frontiers and boundaries and to the name of any territory, city or area.
ENV/JM/MONO(2012)1
Unclassified
English - Or. English
ENV/JM/MONO(2012)1
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ENV/JM/MONO(2012)1
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OECD Environment, Health and Safety Publications
Series on Pesticides
No. 67
OECD Guidance
to the Environmental Safety Evaluation
of Microbial Biocontrol Agents
Environment Directorate
ORGANISATION FOR ECONOMIC COOPERATION AND DEVELOPMENT
Paris 2012
ENV/JM/MONO(2012)1
7
Also published in the Series on Pesticides
No. 1 Data Requirements for Pesticide Registration in OECD Member Countries:
Survey Results (1993)
No. 2 Final Report on the OECD Pilot Project to Compare Pesticide Data
Reviews (1995)
No. 3 Data Requirements for Biological Pesticides (1996)
No. 4 Activities to Reduce Pesticide Risks in OECD and Selected FAO Countries.
Part I: Summary Report (1996)
No. 5 Activities to Reduce Pesticide Risks in OECD and Selected FAO Countries.
Part II: Survey Responses (1996)
No. 6 OECD Governments’ Approaches to the Protection of Proprietary Rights
and Confidential Business Information in Pesticide Registration (1998)
No. 7 OECD Survey on the Collection and Use of Agricultural Pesticide Sales
Data: Survey Results (1999) [see also No.47]
No. 8 Report of the OECD/FAO Workshop on Integrated Pest Management and
Pesticide Risk Reduction (1999)
No. 9 Report of the Survey of OECD Member Countries’ Approaches to the
Regulation of Biocides (1999)
No. 10 Guidance Notes for Analysis and Evaluation of Repeat-Dose Toxicity
Studies (2000)
No. 11 Survey of Best Practices in the Regulation of Pesticides in Twelve OECD
Countries (2001)
No. 12 Guidance for Registration Requirements for Pheromones and Other
Semiochemicals Used for Arthropod Pest Control (2001)
No. 13 Report of the OECD Workshop on Sharing the Work of Agricultural
Pesticide Reviews (2002)
No. 14 Guidance Notes for Analysis and Evaluation of Chronic Toxicity and
Carcinogenicity Studies (2002).
No. 15 Persistent, Bioaccumulative and Toxic Pesticides in OECD Member
Countries, (2002)
No. 16 OECD Guidance for Industry Data Submissions for Pheromones and Other
Semiochemicals and their Active Substances (Dossier Guidance for
Pheromones and other Semiochemicals) (2003)
ENV/JM/MONO(2012)1
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No. 17 OECD Guidance for Country Data Review Reports for Pheromones and
Other Semiochemicals and their Active Substances (Monograph Guidance
for Pheromones and other Semiochemicals) (2003)
No. 18 Guidance for Registration Requirements for Microbial Pesticides (2003)
No. 19 Registration and Work sharing, Report of the OECD/FAO Zoning Project
(2003)
No. 20 OECD Workshop on Electronic Tools for data submission, evaluation
and exchange for the Regulation of new and existing industrial
chemicals, agricultural pesticides and biocides (2003)
No. 21 Guidance for Regulation of Invertebrates as Biological Control Agents
(IBCAs) (2004)
No. 22 OECD Guidance for Country Data Review Reports on Microbial Pest
Control Products and their Microbial Pest Control Agents (Monograph
Guidance for Microbials) (2004)
No. 23 OECD Guidance for Industry Data Submissions for Microbial Pest Control
Product and their Microbial Pest Control Agents (Dossier Guidance for
Microbials) (2004)
No. 24 Report of the OECD Pesticide Risk Reduction Steering Group Seminar on
Compliance (2004)
No. 25 The Assessment of Persistency and Bioaccumulation in the Pesticide
Registration Frameworks within the OECD Region (2005)
No. 26 Report of the OECD Pesticide Risk Reduction Group Seminar on Minor
Uses and Pesticide Risk Reduction (2005)
No. 27 Summary Report of the OECD Project on Pesticide Terrestrial Risk
Indicators (TERI) (2005)
No. 28 Report of the OECD Pesticide Risk Reduction Steering Group Seminar on
Pesticide Risk Reduction through Good Container Management (2005)
No. 29 Report of the OECD Pesticide Risk Reduction Steering Group Seminar on
Risk Reduction through Good Pesticide Labelling (2006)
No. 30 Report of the OECD Pesticide Risk Reduction Steering Group: The Second
Risk Reduction Survey (2006)
No. 31 Guidance Document on the Definition of Residue [also published in the
series on Testing and Assessment, No. 63] (2006, revised 2009)
No. 32 Guidance Document on Overview of Residue Chemistry Studies [also
published in the series on Testing and Assessment, No. 64] (2006,
revised 2009)
ENV/JM/MONO(2012)1
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No. 33 Overview of Country and Regional Review Procedures for Agricultural
Pesticides and Relevant Documents (2006)
No. 34 Frequently Asked Questions about Work Sharing on Pesticide Registration
Reviews (2007)
No. 35 Report of the OECD Pesticide Risk Reduction Steering Group Seminar on
"Pesticide Risk Reduction through Better Application Technology" (2007)
No. 36 Analysis and Assessment of Current Protocols to Develop Harmonised Test
Methods and Relevant Performance Standards for the Efficacy Testing of
Treated Articles/Treated Materials (2007)
No. 37 Report on the OECD Pesticide Risk Reduction Steering Group Workshop
"Pesticide User Compliance' (2007)
No. 38 Survey of the Pesticide Risk Reduction Steering Group on Minor Uses of
Pesticides (2007)
No. 39 Guidance Document on Pesticide Residue Analytical Methods [also
published in the series on Testing and Assessment, No. 72] (2007)
No. 40 Report of the Joint OECD Pesticide Risk Reduction Steering Group EC-
HAIR Seminar on Harmonised Environmental Indicators for Pesticide
Risk (2007)
No. 41 The Business Case for the Joint Evaluation of Dossiers (Data
Submissions) using Work-sharing Arrangements (2008)
No. 42 Report of the OECD Pesticide Risk Reduction Steering Group Seminar
on Risk Reduction through Better Worker Safety and Training (2008)
No. 43 Working Document on the Evaluation of Microbials for Pest Control
(2008)
.... Guidance Document on Magnitude of Pesticide Residues in Processed
Commodities - only published in the Series on Testing and Assessment,
No. 96 (2008)
No. 44 Report of Workshop on the Regulation of BioPesticides: Registration
and Communication Issues (2009)
No. 45 Report of the Seminar on Pesticide Risk Reduction through Education /
Training the Trainers (2009)
No. 46 Report of the Seminar on Pesticide Risk Reduction through Spray Drift
Reduction Strategies as part of National Risk Management (2009)
No. 47 OECD Survey on Countries’ Approaches to the Collection and Use of
Agricultural Pesticide Sales and Usage Data: Survey Results (2009)
No. 48 OECD Strategic Approach in Pesticide Risk Reduction (2009)
ENV/JM/MONO(2012)1
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No. 49 OECD Guidance Document on Defining Minor Uses of Pesticides
(2009)
No. 50 Report of the OECD Seminar on Pesticide Risk Reduction through
Better National Risk Management Strategies for Aerial Application
(2010)
No. 51 OECD Survey on Pesticide Maximum Residue Limit (MRL) Policies:
Survey Results (2010)
No. 52 OECD Survey of Pollinator Testing, Research, Mitigation and
Information Management: Survey Results (2010)
No.53 Report of the 1
st
OECD BioPesticides Steering Group Seminar on
Identity and Characterisation of Micro-organisms (2010)
No. 54 OECD Survey on Education, Training and Certification of Agricultural
Pesticide Users, Trainers and Advisors, and Other Pesticide
Communicators: Survey Results (2010)
No. 55 OECD Survey on How Pesticide Ingredients Other than the Stated
Pesticide Active Ingredient(s) are Reviewed and Regulated: Survey
Results (2010)
No. 56 OECD MRL Calculator User Guide (2011)
No. 57 OECD MRL Calculator MRL Statistical White Paper (2011)
No. 58 Report of the OECD Seminar on Pesticide Risk Reduction Strategies
Near/in Residential Areas (2011)
No. 59 Report of the OECD Seminar on Risk Reduction through Prevention,
Detection and Control of the Illegal International Trade in Agricultural
Pesticides (2011)
No. 60 Guidance Document on the Planning and Implementation of Joint
Reviews of Pesticides (2011)
No. 61 OECD Survey on Efficacy & Crop Safety Data Requirements &
Guidelines for the Registration of Pesticide Minor Uses: Survey Results
(2011)
No. 62 OECD Survey on Regulatory Incentives for the Registration of Pesticide
Minor Uses: Survey Results (2011)
No. 63 Guidance Document on Regulatory Incentives for the Registration of
Pesticide Minor Uses (2011)
.... Guidance Notes on Dermal Absorption - only published in the Series on
Testing and Assessment, No. 156 (2011)
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No. 64 Report of the Second OECD BioPesticides Steering Group Seminar on
the Fate in the Environment of Microbial Control Agents and their
Effects on Non-Target Organisms (2011)
No. 65 OECD Issue Paper on Microbial Contaminant Limits for Microbial
Pest Control Products (2011)
No. 66 Guidance Document on Crop Field Trials [also published in the Series on
Testing and Assessment, No. 164] (2011)
Published separately
OECD Guidance for Country Data Review Reports on Plant Protection Products
and their Active Substances-Monograph Guidance (1998, revised 2001, 2005,
2006)
OECD Guidance for Industry Data Submissions on Plant Protection Products and their
Active Substances-Dossier Guidance (1998, revised 2001, 2005)
Report of the Pesticide Aquatic Risk Indicators Expert Group (2000)
Report of the OECD Workshop on the Economics of Pesticide Risk Reduction
(2001)
Report of the OECD-FAO-UNEP Workshop on Obsolete Pesticides (2000)
Report of the OECD Pesticide Aquatic Risk Indicators Expert Group (2000)
Report of the 2nd OECD Workshop on Pesticide Risk Indicators (1999)
Guidelines for the Collection of Pesticide Usage Statistics Within Agriculture and
Horticulture (1999)
Report of the [1st] OECD Workshop on Pesticide Risk Indicators (1997)
Report of the OECD/FAO Workshop on Pesticide Risk Reduction (1995)
© OECD 2012
Applications for permission to reproduce or translate all or part of
this material should be made to: Head of Publications Service,
[email protected], OECD, 2 rue André-Pascal, 75775 Paris
Cedex 16, France
ENV/JM/MONO(2012)1
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About the OECD
The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental
organisation in which representatives of 34 industrialised countries in North and South America, Europe
and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise
policies, discuss issues of mutual concern, and work together to respond to international problems. Most of
the OECD’s work is carried out by more than 200 specialised committees and working groups composed
of member country delegates. Observers from several countries with special status at the OECD, and from
interested international organisations, attend many of the OECD’s workshops and other meetings.
Committees and working groups are served by the OECD Secretariat, located in Paris, France, which is
organised into directorates and divisions.
The Environment, Health and Safety Division publishes free-of-charge documents in ten different series:
Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides and Biocides;
Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety of Novel Foods
and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission Scenario
Documents; and Safety of Manufactured Nanomaterials. More information about the Environment,
Health and Safety Programme and EHS publications is available on the OECD’s World Wide Web site
(www.oecd.org/ehs/
).
This publication was developed in the IOMC context. The contents do not necessarily reflect the
views or stated policies of individual IOMC Participating Organizations
The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was
established in 1995 following recommendations made by the 1992 UN Conference on Environment
and Development to strengthen co-operation and increase international co-ordination in the field of
chemical safety. The Participating Organisations are FAO, ILO, UNDP, UNEP, UNIDO, UNITAR,
WHO, World Bank and OECD. The purpose of the IOMC is to promote co-ordination of the policies
and activities pursued by the Participating Organisations, jointly or separately, to achieve the sound
management of chemicals in relation to human health and the environment.
ENV/JM/MONO(2012)1
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This publication is available electronically, at no charge.
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/ehs/)
or contact:
OECD Environment Directorate,
Environment, Health and Safety Division
2 rue André-Pascal
75775 Paris Cedex 16
France
Fax: (33-1) 44 30 61 80
E-mail: ehscont@oecd.org
ENV/JM/MONO(2012)1
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FOREWORD
This document dealing with biological pesticides is intended to provide guidance to both industry and
regulatory authorities, in the context of applications for the approval of microbial biological control agents
(mBCAs), and for the registration of microbial biological control products (mBCPs). This document has
been developed in the framework of the OECD BioPesticides Steering Group (BPSG), a sub-group of the
OECD Working Group on Pesticides (WGP), that helps member countries harmonise the methods and
approaches used to assess biological pesticides and to improve the efficiency of control procedures.
The BPSG regards its work as “dynamic” intended to address scientific issues as they arise and which may
be impediments to harmonisation and work-sharing of microbial dossiers and monographs. Consequently,
the BPSG has endeavoured to address and develop guidance on other issues as needed. The present
document represents one such area, namely guidance on the environmental safety evaluation of microbial
biopesticides.
The Netherlands and Germany served together as lead countries in the preparation of this guidance
document. It was developed with the aim of harmonizing risk assessment of mBCAs. In order to achieve
that objective a risk assessment decision scheme was developed which clarifies all the individual steps to
be made in the risk assessment. The various sections of this guidance describe each individual step of that
scheme in detail, with the knowledge currently available.
The use of mBCAs in this guidance is restricted to crop protection for outdoor applications. Main groups
of mBCAs are bacteria, fungi, viruses, protozoa and microsporidia. The final goal of applying this decision
scheme is to discern whether in view of the intended use of the product, the submitted data, information
and tests, the potential risk to the environment is considered acceptable or not.
This OECD guidance document was prepared in consultation with OECD member countries and the
regulated industry participating in the OECD BPSG. It is consistent with the OECD guidelines and criteria
for the evaluation of dossiers and for the preparation of reports by regulatory authorities (OECD
Monograph and Dossier Guidance for Microbials, published in 2004, and later revised in 2006).
The present guidance document received final approval of the OECD BPSG by written procedure ending
on 28 June 2011 and of the OECD WGP by written procedure ending 17 November 2011.
This document is being published under the responsibility of the Joint Meeting of the Chemicals
Committee and the Working Party on Chemicals, Pesticides and Biotechnology, which has agreed that it be
unclassified and made available to the public.
ENV/JM/MONO(2012)1
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Table of contents
Glossary and Abbreviations....................................................................................................................13
Introduction..............................................................................................................................................15
The risk assessment scheme....................................................................................................................15
1. Characterisation of the mBCA (BOX 1 of decision scheme) .......................................................17
1.1 Possible modes of action...............................................................................................................17
1.2 Host range .....................................................................................................................................19
1.3 The selection of appropriate test species.......................................................................................20
2. Application type and pattern (BOX 2 of decision scheme) ..........................................................21
2.1 Indoor (no/negligible exposure)....................................................................................................21
2.2 Outdoor .........................................................................................................................................22
3. Fate and behaviour (BOX 3 of decision scheme) ..........................................................................25
3.1 Fate and behaviour in the soil compartment .................................................................................25
3.1.1 Fate of inoculum; multiplication, accumulation in soil...........................................................25
3.1.2 Estimation of PEC soil (BOX 4 of decision scheme)...............................................................28
3.2 Fate and behaviour in the aquatic compartment.....................................................................29
3.2.1 Estimation of PECsw, MoS and MHC (BOX 4 of decision scheme) .......................................29
4. Environmental toxicity (BOX 5 of decision scheme) ........................................................................30
4.1 Test Guidelines..............................................................................................................................30
4.1.1 OECD Guidelines ....................................................................................................................30
4.1.2 Data requirements and risk assessment according to OCSPP Guidelines of US EPA ...........31
4.1.3 Data requirements and risk assessment according to Canadian Test Guidelines...................32
4.1.4 Opinion of the BPSG ...............................................................................................................33
4.2 Waiver options ..............................................................................................................................33
4.3 Terrestrial NTOs ...........................................................................................................................33
4.3.1 Birds and mammals .................................................................................................................33
4.3.2 Bees..........................................................................................................................................36
4.3.3 Non-target arthropods other than bees ...................................................................................38
4.3.4 Terrestrial plants .....................................................................................................................42
4.3.5 Earthworms .............................................................................................................................45
4.3.6 Non-target soil microorganisms..............................................................................................48
4.4 Aquatic NTOs ...............................................................................................................................49
4.4.1 Fish ..........................................................................................................................................50
4.4.2 Aquatic invertebrates...............................................................................................................52
4.4.3 Aquatic plants (including algae) .............................................................................................54
5. Refinement options (BOX 6 of decision scheme) ..........................................................................56
6. Mitigation options (BOX 7 of decision scheme)............................................................................57
7. Issues to be solved in the near future.............................................................................................57
Acknowledgements ..................................................................................................................................58
References.................................................................................................................................................59
Appendix: Suggestions for Relevant Databases....................................................................................63
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Glossary and Abbreviations
Accumulation The rate of decline of viable CFUs is lower than possible increases through
reproduction and/or repeated use of the mBCA
BPPD Biopesticides & Pollution Prevention Division (Office of Pesticide Programs,
US Environmental Protection Agency)
BPSG Biopesticides Steering Group
Bt Bacillus thuringiensis
Btk Bacillus thuringiensis subsp. kurstaki
CCA Chemical Control Agent
DAR Draft Assessment Report: Monographs prepared by a rapporteur member state in
the context of inclusion of active substances in Annex I of the Council Directive
91/414/EEC
DNA Deoxyribonucleic acid
CFU Colony Forming Unit (synonym: microbial unit)
EC European Commission
EC
50
Median Effective Concentration
EFSA European Food Safety Authority
EPF Entomopathogenic Fungi
EPPO European and Mediterranean Plant Protection Organization
ER
50
Median Effective Rate
EU European Union
GAP Good Agricultural Practice
GV Granulovirus
HQ Hazard Quotient
IOBC International Organisation for Biological Control (of Noxious Animals and
Plants)
Infectivity The ability of a microorganism to cross or evade natural
host barriers to infection (EPA, 1996)
IPM Integrated Pest Management
LC
50
Median Lethal Concentration
LR
50
Median Lethal Rate
mBCA Microbial Biological Control Agent
MCC Maximum Challenge Concentration
MDD Maximum Daily Dose
MHD Maximum Hazard Dose
MHC Maximum Hazard Concentration
MoS Margin of Safety
MPCA Microbial Pesticide Control Agent (for the sake of consistency the term
“MPCA” is replaced by “mBCA” in the present document)
Multiplication The regeneration of the microorganism
NOEC No Observed Effect Concentration
NOEL No Observed Effect Level
NPV Nucleopolyhedrovirus
NTA Non-Target Arthropod
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NTO Non-Target Organism
OECD Organisation for Economic Co-operation and Development
OCSPP Office of Chemical Safety and Pollution Prevention
OPPTS Office of Prevention, Pesticides and Toxic Substances. On April 22, 2010 this
name was changed to "Office of Chemical Safety and Pollution Prevention"
(OCSPP)
Pathogenicity The ability to inflict injury and damage in the host
after infection, and depends on host resistance or susceptibility (EPA, 1996)
Persistence Survival and/or establishment for longer periods.
PEC Predicted Environmental Concentration
PIEC Predicted Initial Environmental Concentration
PMRA Pest Management Regulatory Agency. Government department in Canada.
Environment Canada is another Government department
PPP Plant Protection Product
PRAPeR Pesticide Risk Assessment Peer Review (EFSAs PRAPeR Unit is responsible for
the risk assessment in the EU peer review programme of active substances)
REBECA Regulation of Biological Control Agents
STP Sewage water Treatment Plant
TER Toxicity/Exposure Ratio
TGAI Technical Grade of Active Ingredient
Toxicity The injury or damage in a host caused by a poison or toxin where infection by
and/or replication or viability of the microorganism
are not necessarily required (EPA, 1996).
US EPA United States Environmental Protection Agency
UV Ultraviolet
wt weight
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Introduction
This guidance to the environmental safety evaluation of microbial biocontrol agents (mBCAs) is the
follow-up of the risk assessment scheme developed by Mensink (2005). This risk assessment scheme was
later published by Mensink and Scheepmaker (2007). In the OECD meeting in Arlington (April 2008) it
was decided that this risk assessment scheme by Mensink (2005) should be adapted into an OECD
guidance document so as to provide an additional tool to risk assessors.
The authors
1
of this current OECD guidance are foremost familiar with the EU regulations but an
attempt has been made to generalize the guidance without referring to a particular regulation. The scheme
used by Mensink (2005) was restructured, following the natural, most logical flow of a risk assessment.
Thus, a harmonised decision scheme is anticipated that can be used by risk assessors of all nationalities.
The use of mBCAs in this guidance is restricted to crop protection. Main groups of mBCAs are
bacteria, fungi, viruses, protozoa and microsporidia.
The final goal is to discern whether in view of the intended use of the product and the submitted data,
information and tests, the potential risk to the environment is considered acceptable or not. The risk
assessment scheme and its guidance comprise the basic information and risk assessment items on which
consensus was shown by the members of the BPSG. This guidance forms the platform for further data
processing and integration. The risk assessment scheme is not intended for the use of genetically modified
mBCAs as, at least in Europe, these are assessed under another legislation (Directive 2001/18/EC). This
mBCA guidance can serve as input for the risk assessment for genetically modified mBCAs.
The risk assessment scheme
The environmental risk assessment terminates in “Risk acceptable” or “Risk not acceptable”.
Should any path in the risk assessment scheme lead to “Risk not acceptable”, the regulatory authority
will consider all available information in order to determine whether registration may still be desirable
under certain conditions (e.g., proposed mBCA will replace a toxic pesticide). The regulatory authority
will only reject proposed uses in the last instance.
It should also be noted that this scheme does not directly lead to a final authorization as input from
other risk assessment areas such as the human risk assessment also needs to be considered.
1
J.W.A. Scheepmaker
a
, B. Karaoglan
b
, S. Bär
b
a
RIVM-SEC, National Institute of Public Health and the Environment-Expertise Centre for
Substances, Bilthoven, The Netherlands.
b
Federal Environment Agency (UBA), Section IV 1.3 - Plant Protection Products, Ecotoxicology /
Environmental Risk Assessment, Dessau-Roßlau, Germany.
ENV/JM/MONO(2012)1
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Start
BOX 1
Are characterization data
satisfactory?
BOX 2
Consider the use pattern
Yes
Seek additional information
No
BOX 3
Fate and Behaviour
Fate of inoculum; persistence,
potential for accumulation
and multiplication in the environment
BOX 4
Estimation of PECsoil / PECsw
and setting the MHC
BOX 5
Environmental Toxicology
Terrestrial organisms
Aquatic organisms
Non-target microorganisms (if necessary)
Were any adverse effects noted in any
of the non-target studies?
BOX 6
Refined risk assessment at 2
nd
and/or 3
rd
Tier level
(considering quantitative risk
assessment if feasible)
Any adverse effects?
Yes
BOX 7
Can risk be mitigated through
various options?
Risk not acceptableRisk acceptable
No
Yes
No
Yes
No
Decision scheme for
outdoor applications
Contamination/exposure
(soil / surface water)
No
ENV/JM/MONO(2012)1
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1. Characterisation of the mBCA (BOX 1 of decision scheme)
Basic knowledge of the specific microorganism is required before starting the risk evaluation. Special
attention should be given to non-indigenous and those indigenous mBCAs that are applied in different
ecological compartments than they naturally occur (e.g. soil organisms applied to blossoms). These require
a case-by-case approach at the strain level. Indigenous isolates though should still require close scrutiny
since these were selected for a specific biological property or function that may not be available in other
isolates. In some cases, the mBCA could, in theory, differ in some aspects from the species.
The following basic characteristics of the microorganisms should be considered
1. taxonomy;
2. the biology of the microorganism;
• origin;
• mode of action;
• host range;
• ability to survive in various environments (fate);
• niche, natural occurrence;
• life cycle including reproduction methods and dispersal mechanisms.
3. methods to identify the mBCA (e.g. molecular techniques, morphology, growth
substrates/conditions)
1.1 Possible modes of action
The effect of a mBCA depends on the mode(s) of action of the microorganism. The effect can be the
combination of several modes of action as given below, with different processes occurring in parallel. Not
all modes of action for each species of microorganism may already be discovered.
Possible modes of action:
1. antibiosis (e.g. production of toxins, fungal bioactives (metabolites), production of cell-wall
degrading enzymes);
2. toxicity (note: antibiosis is a wider term including the action of toxins);
3. pathogenicity (note: antibiosis and pathogenicity might be overlapping terms. Usually
pathogenicity manifests in effects like mortality or obvious sublethal effects. Dose effect relation
may however be unclear);
4. induction of plant resistance;
ENV/JM/MONO(2012)1
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5. interference with the virulence of a pathogenic target organism;
6. endophytic growth;
7. root colonisation;
8. competition for ecological niche (e.g. nutrients, habitats);
9. parasitisation.
Background information on fungal bioactives (metabolites):
According to EU legislation, relevant metabolites need to be identified and further dealt with in a
separate dossier. So far, this has not occurred yet.
Regulation (EU) No 544/2011 (EU, 2011) states that, if the product action is known to be due to the
residual effect of a toxin/metabolite or if significant residues of toxins/metabolites are to be expected not
related to the effect of the active substance, a dossier for the toxin/metabolite has to be submitted in
accordance with the requirements of Annexes IIA and, where specified, the relevant parts of Annex IIIA.
Regulation (EU) No 544/2011 (EU, 2011) further states under the section fate and behaviour in the
environment: Any relevant metabolites (i.e. of concern for human health and/or the environment) formed
by the test organism under any relevant environmental conditions should be characterised. If relevant
metabolites are present in or produced by the micro-organism, data as outlined under Annex II, Part A,
point 7 may be required, if all of the following conditions are met:
• the relevant metabolite is stable outside the microorganism, see point 2.8, and
• a toxic effect of the relevant metabolite is independent of the presence of the micro-organism,
and
• the relevant metabolite is expected to occur in the environment in concentrations considerably
higher than under natural conditions.
Bacteria and fungi may secrete a wide range of metabolites, mostly products of secondary
metabolism. These metabolites, including toxins, serve different functions depending on the ecological
niche of the microbe, and may occur in many environmental compartments (in particular in soil, surface
waters, groundwater and air), in animal feed or in food for human consumers. These substances could vary
in structure, some are simple organic molecules such as antimicrobial agents produced by fungi and others
are peptides or proteins.
A complete identification and characterisation of all metabolites which are produced by bacteria and
fungi under different (environmental) conditions will not be feasible for technical reasons. However, the
potential for the microorganism to produce metabolites that could be harmful to humans and/or the
environment should be assessed, using information on the mode of action, the potential of related species
and strains to produce relevant metabolites/toxins, adverse effects observed in the (eco)toxicity tests, and
all other relevant information in published scientific literature.
The information provided must be sufficient to permit the performance of a risk assessment for man
and/or environment, arising from potential exposure to the microorganism and metabolites (toxins).
ENV/JM/MONO(2012)1
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1.2 Host range
The basic information on the host range should already give some indication on the possibility of
infectivity or pathogenicity to other species than the target organism. For example, baculoviruses have
narrow host ranges usually confined to one or a few species of closely related insects.
However, it is noteworthy to distinguish between physiological and ecological susceptibility. In
general, it is difficult to compare between physiological host range (determined under laboratory
conditions) and ecological host range (determined under field conditions). In a literature review by Roy
and Cottrell (2008), it was concluded that many factors affecting pathogenicity under both laboratory and
field conditions must be taken into account to make sense of how physiological susceptibility relates, if at
all, to ecological susceptibility. Furthermore, it could be concluded that the lack of physiological
susceptibility should be a reliable indicator that a specific strain or isolate of a pathogen will be highly
unlikely to be infective under field conditions. Studies examining pathogen host range generally show that
physiological susceptibility greatly exaggerates ecological susceptibility (Hajek et al., 1995, 1996; Solter
and Maddox, 1998). In general, information on the host range should be provided in an early stage of
assessment based on a transparently conducted literature search using widely accepted databases. If
literature searches do not yield sufficient results for a new isolate, studies (experimental data) on the host
range should be provided.
Host ranges of selected groups of mBCAs consisting of entomopathogenic fungi, entomopathogenic
bacteria and baculoviruses
The examples given below are mainly based on the experience of the assessment of 4
th
list substances
under Commission Regulation (EC) No 2229/2004 (EU, 2004). The examples are therefore not exhaustive.
At first glance, differences in the extent of (physiological) host ranges between EPF, bacteria und viruses
are obvious.
Entomopathogenic fungi (EPF)
The host ranges of the entomopathogenic fungi Beauveria bassiana (different strains) and
Metarhizium anisopliae surpass the borders of subphylum. Beauveria bassiana is able to attack arthropods
of the subphylum Hexopoda (white flies, thrips, aphids), the subphylum Chelicerata (mites), Crustacea
(sowbugs) and Myriapoda (millipedes). The host range of M. anisopliae includes Coleoptera belonging to
the subphylum Hexapoda as well as mites belonging to the subphylum of Chelicerata.
Entomopathogenic bacteria
Entomopathogenic sporeforming bacteria such as Bacillus thuringiensis (subsp. kurstaki, aizawai,
tenebrionis and israelensis) have specific modes of action due to the presence of δ-endotoxins from the
Cry-protein family and other factors that selectively destroy the gut of target insects. Depending on the
pathotype or combination of bioinsecticidal Cry-proteins this leads to different preferential activity against
target pest species within the insect orders Lepidoptera, Coleoptera and Diptera. The range of susceptible
species also covers non-target insects. For instance Bt subsp. kurstaki is primarily active against
Lepidoptera, but has also been recorded as active against other insect orders such as Coleoptera, Diptera,
Hymenoptera, Hemiptera, Isoptera, Phthiraptera, Siphonaptera, Thysanoptera, Neuroptera, Ephemeroptera
(Glare and O’Callaghan, 2000). In contrast to the example on EPF above, affected orders mentioned above
are within the same subphylum.
ENV/JM/MONO(2012)1
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It should be noted that also nematicidal activity has been reported in the open literature for several Bt
isolates (Leyns et al., 1995). Besides the ability to secrete thermostable nucleotide β-exotoxins, some Bt
strains are able to produce thermolabile factors with nematicidal activity (Mozgovaya et al., 2002). Due to
the nonspecific toxicity of β-exotoxins to insects and mammalian cells, β-exotoxins containing Bt products
have been banned from public use and shall be free from β-exotoxins when tested with fly larvae toxicity
tests or an equivalent HPLC method (WHO, 1999). In this context, an improved fly bioassay was recently
developed by Mac Innes and Bouwer (2009) that is suitable for the routine screening of Bt strains for β-
exotoxins.
For the diverse group of bacterial mBCAs other than Bt broad host specificities cannot be excluded
due to their variety and numerous potential modes of action. Nevertheless, bacterial entomopathogens
other than Bt have been developed which seem to indicate a higher degree of host specificity compared to
commercially available Bt-preparations. For example, the activity of the sporeforming bacterium Bacillus
sphaericus is restricted to certain dipteran species (Glare and O’Callaghan, 2000). Another example is the
non-sporeforming bacterium Serratia entomophila with activity against only a limited range of scarab
species (Jackson et al., 1991).
Baculoviruses
Baculoviruses belong to a family of rod-shaped, enveloped viruses with a circular double stranded
DNA and are divided into the genera granuloviruses (GV) and nucleopolyhedroviruses (NPV) on the basis
of occlusion body morphology (OECD, 2002). Because of their specific mode of action baculoviruses have
a very narrow host range and are strictly host-specific to certain arthropod species. In view of the host
specificity, a distinction can be made between the genera GV and NPV.
The host range of NPV is usually restricted to one or a few species of the genus or family. However,
there are NPV that exhibit a larger host range such as Autographa californica NPV infecting more than 30
species from about 10 insect families (OECD, 2002). In contrast to NPV, the host range of GV appears to
be even narrower and mostly restricted to very few species of a single family (e.g. the family Tortricidae
for Cydia pomonella GV). An increasing body of evidence suggests that Baculoviruses such as Cydia
pomonella GV represent the most specific pesticidal agent of all microbials and chemicals (Hauschild,
2011).
Given the varying host specificities among different groups of mBCAs, it should be noted that,
although the risk caused by entomopathogenic fungi seems to be higher than by entomopathogenic bacteria
or viruses, sustainable adverse effects to arthropods under field conditions are hardly observable due to
specific environmental conditions (e.g. microclimate, high humidity) needed for germination and attacking
the host. In general it can be concluded, that risk assessments based on laboratory determined host ranges
often overestimate the risk compared to field conditions. Therefore a distinction between the physiological
and the ecological host range has to be made.
1.3 The selection of appropriate test species
The choice of non-target species for testing as well as the specific test methods (pathogenicity tests vs.
standard toxicity tests) should be related to the mode of action and the proposed use of the microorganism
in the field.
Australia suggests the approach of radial taxonomic testing in risk assessment procedures for mBCAs.
Radial taxonomic testing essentially involves a taxonomic analysis expanding out from the target species
to look at possible effects on related species, genera, families, tribes, orders etc. (for more information
ENV/JM/MONO(2012)1
24
please refer to the study by Weidemann and Tebeest, 1990). The same approach is taken by Environment
Canada and is termed “centrifugal taxonomic approach” (PMRA, 2001).
Even though an applicant may claim a “narrow host range” for a given mBCA, only directly related
species/genera may have been tested to support this claim. Radial taxonomic testing allows for a broader
consideration of NTOs (non-target organisms) both closely and more distantly related to the target
organism, particularly where there is or may be a shared mode of action (e.g. receptor type), a shared
behaviour, a similar or likely exposure scenario, and/or environmental/biodiversity value (e.g. natives) of a
related species.
Radial taxonomic testing is a useful tool for assessors in that it helps to focus the testing of NTOs in
those taxonomic groups that are most likely to be affected by the mBCA. These may be groups that fall
outside the standard test organisms (e.g. rainbow trout, Daphnia) recommended under current OECD
guidelines for the testing of chemicals. At least, if testing is not conducted, it alerts the assessor to those
NTOs most at risk from the mBCA and appropriate responses can be considered (e.g. imposing
management conditions on the release).
2. Application type and pattern (BOX 2 of decision scheme)
The pattern of use is a very important part of the environmental risk assessment, as it primarily
determines the (potential) extent of exposure. It comprises:
• the application rate expressed in CFU/ha (or other relevant units such as granules/ha);
• the frequency;
• the site (crop, bare soil, slope);
• the time (early or late in the crop; early morning or late evening: this informs exposure
assessment depending on NTO activity);
• type of application (spray, drip, aircraft, ground equipment).
It should be noted that mBCAs require very specific (micro-) conditions and without these
requirements the efficacy is limited. In view of these limitations, biopesticides may be applied more
frequently than their chemical counterparts.
In general, only the exposure of NTOs due to outdoor application (e.g. spray application, granules,
seed treatment) should be considered in the risk assessment. Risk assessments should be based on the
specific mBCA considering the intended use (soil or foliar applications), target pest (fungicide, insecticide
etc.) and mode of action (pathogen, competition for space and nutrients etc.).
2.1 Indoor
Currently, there is no agreement on the definitions of individual protected/covered crop systems like a
specific type of glasshouse. In view of this paucity and uncertainty, the EFSA Panel on Plant Protection
ENV/JM/MONO(2012)1
25
Products and their Residues (PPR) held a workshop in Parma (Italy) on November 17-19, 2009 to discuss
the development of a new Guidance Document on emissions of plant protection products from protected
crop systems such as glasshouses and crops grown under cover (EFSA, 2010). For microorganisms,
exposure routes and amounts may be completely different, if relevant at all. However, for the time being,
the outcome of this EFSA Guidance Document may be considered for the risk assessment of mBCAs.
Exempting data requirements is not recommended by Canada. Instead, Canada recommends that sound
scientific rationales be considered in lieu of data to ensure that no potential risks exist for the mBCA in
question.
Types of application and expected exposure of NTOs:
• Indoor use e.g. mushrooms, harvested crop:
no or negligible exposure
• Indoor glasshouses
Exposure:
− Emissions due to spray drift from permanent structures via open windows and openings can be
considered negligible.
− Exposure of birds, mammals, aquatic organisms, earthworms, soil microorganisms may not be
relevant.
− Exposure of pollinators such as bumblebees and beneficial arthropods such as predatory mites and
parasitic wasps need to be considered as they may be used as part of IPM in combination with
mBCAs. Exposure of non-target insects that invade the glasshouse through open windows is
considered not to be relevant.
− Discharge to surface water needs to be taken into consideration.
2.2 Outdoor
At the start of the risk assessment scheme, the NTOs that will likely be exposed in consideration of
the application type, need to be determined.
ENV/JM/MONO(2012)1
26
Types of application and expected exposure of NTOs:
• Spray applications to bare soil
Exposure:
− Birds and mammals
− Non-target soil dwelling arthropods
− Earthworms and microorganisms
− Emerging plants
− Aquatic organisms (fish, algae, crustaceae, aquatic invertebrates, aquatic plants)
− Other NTOs potentially exposed are reptiles and amphibians
• Spray applications to crops (plants and soil)
Exposure:
− Birds and mammals
− Bees; only in flowering crops [or other pollen producing plants (e.g. gymnosperms)]
− Non-target arthropods (soil and plant-dwelling)
− Earthworms and microorganisms
− Plants
− Aquatic organisms (fish, algae, crustaceae, aquatic invertebrates, aquatic plants)
− Other NTOs potentially exposed are reptiles and amphibians
• Seed treatments (also relevant for bulbs and potatoes)
If an mBCA is applied to seeds by drench, an exposure of NTOs might occur in the soil. Initial
PECsoil should be based on the calculation of the number of CFU of the mBCA per seed
multiplied by the number of seeds, bulbs or potatoes per hectare. This will result in local
exposure of the rhizosphere of the roots of the plants emerging from the seeds, bulbs or potatoes.
The mBCA will multiply and grow along with the newly formed hair roots. In arable fields
drilled with treated seeds, a certain percentage of unburied seeds can be expected whereas the
availability of unburied seeds depends on the used drilling-technique and other agricultural
practices such as seeding depth and soil conditions (de Snoo and Luttik, 2004). Depending on the
amount of spillage, risks to seed eating birds and mammals should be taken into account.
ENV/JM/MONO(2012)1
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Exposure
− Soil arthropods in the rhizosphere
− Earthworms and microorganisms in the rhizosphere
− Granivorous birds and mammals (exposure due to ingestion of treated seeds remaining on the soil
surface following drilling or ingestion of spilled seeds).
Situations less likely but to be aware of:
− If the microorganism is able to grow endophytically, an exposure of NTOs might also occur
aboveground on plant-dwelling arthropods.
• Point applications: tree injection or as a rub on trunks
As trunk treatment is a very specific local application to a limited area, exposure of the terrestrial
compartment could be considered minimal or negligible.
Situations to be aware of:
− Tree injection will not lead to further exposure of the environment, unless the mBCA will grow into
the rhizosphere or will sporulate on the leaves. Example: Tree injection of Verticillium albo-atrum
isolate WCS850 used as vaccine in order to prevent Dutch elm disease.
− There are known cases where fungal preparations applied to the surface of cut wood can sporulate and
spread to nearby trees.
− Example: Chondrostereum purpureum on black cherry (De Jong et al., 1996).
• Spraying from aircrafts
Exposure:
− In general, all compartments will be exposed (air, surface water and soil) although some compartments
will not be exposed under specific conditions [e.g. aerial spraying against the desert locust
(Schistocerca gregaria) in the desert is unlikely to expose water compartments]. Mitigation options are
possible (e.g. no-spray zones, crop stages, time of day). The only difference in the risk assessment
compared to land-based applications is that the relative exposure values for the compartments will be
different.
− Other formulation types than spray solutions can be used for aerial spraying (e.g. solid pellets), which
have other consequences for exposure of environmental compartments, i.e. no crop interception, no
exposure of air.
ENV/JM/MONO(2012)1
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3. Fate and behaviour (BOX 3 of decision scheme)
Comparison of data requirements and risk assessment approaches within the OECD
Regulation (EU) No 544/2011 states that experimental data on fate and behaviour are normally
required unless it can be justified that an assessment can be performed with the information already
available from the open literature for the respective environmental compartment. The respective paragraph
7.1 states as follows: “Where relevant, appropriate information on the persistence and multiplication of the
microorganism, in all environmental compartments has to be given, unless it can be justified that exposure
of the particular environmental compartment to the microorganism is unlikely to occur.” (see also
paragraph 1.3.1.1).
The US and Canadian approach, in contrast to that of the EU, does not generally require formal
environmental fate data for the reason that the fate and behaviour of a mBCA is difficult to evaluate due to
the potential for microbial growth under suitable environmental conditions. Instead of requiring
environmental fate data, the US and Canada follow a tiered approach as described in chapters 4.1.2 and
4.1.3.
Accordingly, the need for environmental fate testing depends on the occurrence of detrimental effects
in the first tier. Moreover, the Canadian registration guidelines (PMRA, 2001) state that the extent of
environmental fate testing is mainly based on the nature of the mBCA, i.e., whether it is indigenous or non-
indigenous to the ecozone(s) of intended use.
General options for waiver
In the EU, waivers are accepted if exposure of the NTO can be excluded by the type of application.
Waivers are also accepted when significant information about the mBCA, e.g. in-depth knowledge of the
biology, life-cycle, mode of action, fate and behaviour in the considered environmental compartment is
available. The rationale should include a transparently conducted scientific literature search for published
pathogenic/toxic effects to NTOs of concern. In this context it should be mentioned that EFSA has recently
published a Guidance Document entitled “Submission of scientific peer-reviewed open literature for the
approval of pesticide active substances under Regulation (EC) No 1107/2009” (EFSA, 2011). This
guidance document shall ensure methodological rigour and transparency, and aims to minimise bias in the
identification and selection of scientific information in dossiers. This EFSA guidance is compatible (e.g. in
terms of format) with existing EU and OECD Guidance documents that are widely used to assist the
preparation of dossiers (EC, 2005b; OECD, 2005, 2006).
3.1 Fate and behaviour in the soil compartment
3.1.1 Fate of inoculum; multiplication, accumulation in soil
In Commission Regulation (EU) No 544/2011, annex part B (EU, 2011) the data requirement on fate
and behaviour specifically asks for information on ‘persistence and multiplication’. Ideally, growth of an
"indigenous" mBCA should, after a short growth period, level off, and continue along the line of the
background microorganisms. If the application of a mBCA is not expected to increase the natural
“background” levels of the species or related species, risks may be considered acceptable or “not
deviating” from “normal”.
ENV/JM/MONO(2012)1
29
It should be evaluated on a case-by-case approach whether the mBCA, based on its identity and
characterization, is likely to survive in the soil. This approach is particularly important for mBCAs that
consist of non-indigenous microorganisms, i.e. strains and/or species that are not found in the natural
environment where the mBCA will be applied. For non-indigenous mBCAs fate/survival tests and NTO
testing may be particularly important. However, as stated in the EU Guidance Document
SANCO/10754/2005 (EC, 2005a), due to large possible ranges of the environmental factors, data on fate
and behaviour of the microorganisms will inevitably show large variability. Therefore, this variability
caused by environmental factors could be larger than the possible differences between strains of the same
species. On the other hand, the EU Guidance (EC, 2005a) also proposed that data on strains should be
treated separately if there is sufficient evidence that strains differ in their environmental fate and
behaviour.
For entomopathogenic fungi B. bassiana, B. brongniartii, and M. anisopliae, data on natural
concentrations in the soil and persistence following applications to the soil have been collected in a study
performed by Scheepmaker and Butt (Scheepmaker & Butt, 2010). This review is freely available on the
OECD site. In this review, a methodology was suggested to determine the natural background level.
Methodology on how to determine the natural background level in three steps
Step 1) Determination upper natural background level
Studies on natural background concentrations were collected from the literature for each of the three
species. Those studies were selected that gave at least three data points from one sampling area. The
overall geometric mean was calculated for the selected studies as the average of the individual log
observations. The overall geometric mean was then the exponent of this value. The derived 95
th
percentile of the geometric mean was chosen to represents the upper natural background level. By
choosing the 95
th
percentile some very high peaks were excluded. For M. anisopliae, B. bassiana and
B. brongniartii, the upper natural background level was approximately 1000 CFU/g soil. It was clear
that natural background concentrations are variable and depend on land use, climate, soil and other
possible factors.
Step 2) Collection of fate/survival data of applied inoculum
Studies on survival of applied inoculum were collected from the literature for each of the three
species. Despite the variety in between the experiments (length, number of sampling during the course
of the experiment, soil, crop, etc.) data from the different sources showed a decline in density for the
three fungal species. This decrease was similar for laboratory experiments, small-scale experiments
and field experiments.
Step 3) Determination of the time needed for applied inoculum to decrease to upper natural
background level
It was graphically estimated that the applied inoculum density decreases to upper natural background
levels within 0.5-1.5 year for B. bassiana, after about 4 years for B. brongniartii and >10 years for M.
anisopliae.
ENV/JM/MONO(2012)1
30
Conclusions for the risk assessment:
• The review of Scheepmaker and Butt (2010) (see link on OECD site) can be referenced in a
waiver/statement to fulfill the persistence in soil data requirement for the three EPF species, B.
bassiana, B. brongniartii, M. anisopliae. Since other EPF species are subjected to the same processes
in the soil, it is assumed that a similar decrease of inoculum will occur in other EPF species.
Therefore, the proposed methodology can be used for other mBCAs as well.
• This review showed that applied inoculum of the three EPF species decreases to natural background
levels in time and that increases of the inoculum are only temporary and depend on the presence of a
population of host insects in the field.
• A wide variety of factors explaining the decline of EPF density was described.
Some general situations with a negative impact on the survival and fate of the inoculum:
− the microorganism is subject to competition and parasitism of the autochthon microbial
community.
− the microorganism is subject to predator pressure.
− the microorganism does not germinate and/or proliferate/or multiply in the soil due to very
specific (micro-) conditions.
− it cannot readily gain energy from hardly degradable substances of limited biodegradability
like lignin.
• It is not feasible to collect a set of background studies that are similar regarding soil condition, strain,
country, crop, etc., for the simple reason that the data in the literature are not uniform and may be very
limited. Moreover, in most cases, studies from the literature are not based on the desired strain for
authorization, as these strains often originate from a specific isolate and can therefore only be found in
a certain area. For these reasons, it is not feasible to develop standardised methods specifying the
minimum number of different conditions, soils, application timings and samplings. This approach is
not practicable and too costly.
• Although species potentially differ in toxicity at the strain level, it is recommended to evaluate
persistence at the species level as it was shown by Scheepmaker and Butt (2010) since densities of
individual strains often follow a very similar decline.
• It should be realized that reproduction of an entomopathogenic fungus may occur in the presence of
the host. If occurring, the PEC may increase during a short period of time. After this period, a steady
decline of the inoculum is expected to occur.
• In contrast to the criterion of persistence for chemicals, there is no criterion for persistence of
mBCAs. From this follows that the length of the period that the applied concentration is higher than
the upper background concentration is to be discussed case-by-case. This is clearly the case for B.
brongniartii and M. anisopliae. In general, the persistent mBCA may be present in an inactive state,
probably in a patchy distribution confined to small pockets in the soil. The mBCA may be activated
under very specific conditions.
ENV/JM/MONO(2012)1
31
3.1.2 Estimation of PEC soil (BOX 4 of decision scheme)
PEC-in crop
For the sake of consistency the widely used term “PEC” (predicted environmental concentration) is
considered in the present document to express (quantify) exposure level, although the term “PED”
(predicted environmental density) may be regarded as more appropriate in ecological terms.
In general, it should be taken into consideration that for many biological products based on
microorganisms the active ingredient is very susceptible to UV light, dry conditions etc. For this very
reason many products need to be applied at a regular interval.
It was agreed in the EU Pesticide Risk Assessment Peer Review (PRAPeR) of plant protection
products (PPP) containing microorganisms to estimate the PECsoil by assuming a density of the soil of
1500 kg/m
3
and a distribution in the soil in the top 5 cm. This approach is in line with PECsoil calculation
of chemical substances. This estimate is conservative as the actual concentration in soil after application is
always lower than predicted due to loss in viability of the mBCA (with the exception of a possible short
period of increases (e.g. reproduction of entomopathogenic fungi in hosts)). It is more difficult to
determine the concentration (population density) of a living organism in soil compared to a chemical
substance. An appropriate measure of mBCAs in soil would be to estimate the predicted initial
concentration in soil (PIEC) using the summation of the nominal concentrations used in the repeated
applications. These PIECsoil values should be compared to background concentrations if available. With
molecular techniques it is possible to determine the exact concentrations in the soil. Other approaches are
available: Environment Canada uses a model into which degradation of inoculum is integrated. This model
assumes distribution in the upper 15 cm of the soil.
Crop interception values should be included in the estimation of the PEC values. Care has to be taken
when using the interception values of chemicals as these are not validated for microorganisms.
Nevertheless, the interception of a crop can be included in the calculation when the application technique
(spray equipment) and formulation (additives, spreader etc.) are similar or identical to chemical products.
If interception would not be considered for the calculation of the PEC values then risk evaluation would be
done on full accumulated PEC values on crop and in soil which would not be equivalent to the risk
assessment of chemicals.
PEC-off crop
Canadian drift model(s) employed to determine off-crop exposures needs to calculate deposition from
different spray methods and equipment (e.g. ground boom, airblast and aerial). Calculating a PEC off-crop
value would only be necessary if exposure of NTOs requires refinement due to adverse effects noted at the
Maximum Hazard Concentration (MHC) and Maximum Hazard Dose (MHD), respectively.
Moreover, the expected concentrations are below the levels tested in the worst case scenario in Tier I
studies. Therefore, side effects are not to be expected. mBCAs are sensitive to UV-light, desiccation and
other abiotic factors, therefore any CFU (microbe) deposited by spray drift has hardly a chance to survive.
ENV/JM/MONO(2012)1
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3.2 Fate and behaviour in the aquatic compartment
3.2.1 Estimation of PECsw, MoS and MHC (BOX 4 of decision scheme)
• PECsw
In the EU PRAPeR Expert Meeting M2 held on 16-18 February 2009, it was proposed that, due to
lack of appropriate methods, initial exposure in surface water can be calculated using the Ganzelmeier drift
tables leading to PEC values (Ganzelmeier et al., 1995, updated by Rautmann et al., 2001), whereas entry
paths such as runoff, drainage and aerial deposition require different approaches. It was concluded that
exposure estimation has to be solved on a case-by-case basis. The above mentioned EU PRAPeR proposal
using initial PEC values can be regarded as a conservative approach since the actual concentration
following application is likely to be lower than the predicted concentration as many environmental
parameters cause loss of viability of the mBCA within a relatively short time frame. Also, the mBCA may
(in some cases) quickly precipitate to the sediment in calm water, leading to lower concentrations
distributed throughout the water. In the latter case, effects on sediment organisms may be required
provided that the mBCA has the potential to adversely affect invertebrates. Therefore, for risk assessment
purposes, particular attention should be paid to environmental conditions affecting viability of mBCAs.
Information should be available on the fate of the mBCA in surface waters with various oxygen conditions,
sensitivity to solar radiation and its influence on growth and germination capability. Based on the outcome
of the 4
th
stage EU review programme containing microorganisms, there is an increasing body of evidence
suggesting that most mBCAs are not viable in non-sterile water due to competition with other
microorganisms or due to unfavourable environmental conditions. Therefore, long-term exposure in
surface water is expected to be unlikely while acute and short-term exposure cannot be excluded.
• MoS
Margins of safety (MoS) between the units of microorganisms per ha on the one hand and toxicity
values on the other hand can be derived. As a general conclusion, a rough estimation of the initial
concentrations seems appropriate in the first Tier level.
• MHC
Canada does not routinely apply spray drift models to assess drift and off-target exposure potential of
mBCAs. The Ganzelmeier drift tables have been considered in Canadian spray drift models for ground
application of conventional chemical pesticides (http://www.hc-sc.gc.ca/cps-spc/pest/agri-commerce/drift-
derive/index-eng.php, last accessed, April 27, 2011). For mBCAs, Canada, however, recommends that
each mBCA be considered separately based on its own biological properties as well as its proposed use
pattern. The MHC approach (Tier I), in Canadian opinion, is the simplest approach since it eliminates the
requirement for fate testing and focuses on potential hazards, i.e., assumes that NTOs may be exposed.
According to the US EPA and Canadian approaches a MHC for surface water of 10
6
CFU/mL or 1000
times the expected microbial concentration in water bodies is defined. In order to determine the MHC or
simply to judge whether the MHC is high enough (margin of safety) the setting of the MHC needs to be
verified prior testing.
ENV/JM/MONO(2012)1
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4. Environmental toxicity (BOX 5 of decision scheme)
The established European environmental risk assessment for chemical pesticides is primarily based on
the calculation of the quotient between ecotoxicological endpoints (ER
50
, LD
50
or NOEC) and predicted
environmental concentrations being the TER-value. This approach has also been used by some European
member states compiling draft assessment reports (DARs) for List III and IV mBCAs. These calculations
served mainly as an approximate assumption of the relation between endpoints gained from submitted
toxicity/pathogenicity studies and estimated environmental concentration (or environmental density). The
estimated environmental exposure were mostly calculated by using methods being developed for chemical
pesticides, as no specific exposure models for mBCAs are available so far. These calculations were
accompanied by further qualitative statements leading altogether to a semi-quantitative risk assessment.
In fact, exposure scenarios for chemicals are not fully applicable to mBCAs. The maximum hazard
concentration (MHC) or maximum hazard dose (MHD) [note: the synonym “Maximum Challenge
Concentration (MCC)” is being used in the Canadian Guidelines] circumvents this problem by using the
maximum amount of active ingredient (mBCA or its toxin) in the toxicity/pathogenicity studies. A
definition of the MHC/MHD to be used can be found in each guideline. As the MHC/MHD approach shall
cover anticipated exposure levels including a safety factor, no further risk calculation is necessary in most
cases. It goes without saying that the MHC/MHD needs to be carefully established. For example for EPF
or viruses, the Canadian guidelines (Environment Canada, 2004) propose additional safety factors due to
their potential multiplication: “For mBCAs that are expected to increase significantly in the environment
following an application, e.g. viruses in insects, the oral dose administered should be no less than the
highest concentration possible in field, e.g. equivalent to the numbers in maximally infected insects”.
The amount of studies required for the risk assessment may depend on various factors and properties
of the mBCA. For instance, production of bioactives (toxins, metabolites) might affect data requirements
and should be considered in the following way: If metabolites/toxins are known to be responsible for the
mode of action, and if there is relevant exposure of NTOs then, in that case, toxicity data should be
available and a risk assessment for the metabolites/toxins of ecotoxicological concern should be
performed. Currently, Butt and Scheepmaker are working on a project that categorises metabolites/toxins
and their effects on NTOs using information that is available in scientific literature. Risk strategies will be
proposed. It is anticipated that this work will be transformed into OECD guidelines for assessing the risks
of fungal metabolites.
4.1 Test Guidelines
4.1.1 OECD Guidelines
Within the EU, standard OECD tests are available to determine the potential toxic effects of
(chemical) pesticides. The advantage of studies according to the OECD guidelines is the determination of
LD
50
, ER
50
values due to the usage of numerous test concentrations. These OECD guidelines are
considered to be less appropriate for assessing possible effects of mBCAs to NTOs since the recommended
duration of these tests might not be sufficiently long to allow infection. Furthermore, some expected routes
of exposure are not considered in all cases.
ENV/JM/MONO(2012)1
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4.1.2 Data requirements and risk assessment according to OCSPP Guidelines of US EPA
US EPA does not require dose-response testing in the first Tier level. OCSPP guidelines give the
option of testing a single group of test species at the MHD (maximum hazard dose), thus giving a No
Observable Adverse Effects Limit. Test Guidelines are available on the website:
http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htm (last accessed, April 27,
2011). The MHD for Tier I testing will be based on a safety factor times the maximum amount of active
ingredient (mBCA or its toxin) expected to be available to terrestrial and aquatic plants and animals in the
environment. These Tier I studies are of sufficient duration (i.e., typically 21 to 30 days) to increase the
likelihood of detecting any adverse effects due either to toxicity or infectivity/pathogenicity and allow for
specific routes of exposure. A short summary of the tiered approach is given in Table 1 below.
Table 1: Tiered testing approach by the US EPA
Tier level Explanation
I Tier I consists of maximum dose single species hazard testing on
NTOs
II If adverse effects are observed in Tier I, the potential exposure to the
MPCA is estimated by means of Tier II testing for population
dynamics, fate and expression in the environment
III If Tier II tests show that there may be significant exposure to the
MPCA, Tier III studies to determine a dose response effect or to
examine certain chronic effects will be performed to determine if the
minimum infective dose is less than the exposure or if there are other
considerations that would decrease the observed effects in the
environment.
IV Tier IV tests, under simulated or actual environmental conditions,
are to be designed on a case-by-case basis to evaluate any specific
problem that cannot be resolved by lower tier testing.
According to the OCSPP approach, dose-response tests are only needed if any adverse effects are
observed in Tier I MHD studies. In practice, US EPA never needed microbial pesticide NTO studies
rendering an LD
50
. If unacceptable adverse effects are identified in Tier I tests, Tier II tests are performed
attempting to quantify levels of the mBCA to which the susceptible non-target species may be exposed.
Tier II Environmental expression testing consists of simulated terrestrial and aquatic applications of the
mBCA. Terrestrial and aquatic applications are conducted in a contained environment (greenhouse,
aquaria) to assess survival and growth in soil, vegetation, water and sediment. The contained environment
in the environmental expression tests are generally based on natural materials from the proposed use site
(sediment, soil, plants, and marine/estuarine liquids) which are arranged as naturally as possible, and held
within a plastic, glass or other container to prevent escape of the microbial agent.
As a result, Tier II information indicating that the mBCA will not survive or persist in the
environment to which it is applied, can be submitted as support for a request for waiver of some or all of
Tier I testing requirements. In case of lasting concerns as an outcome of Tier I and Tier II studies, further
Tier III (prolonged Tests) or Tier IV (field testing) studies may be required.
ENV/JM/MONO(2012)1
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4.1.3 Data requirements and risk assessment according to Canadian Test Guidelines
Environment Canada has developed a Guidance Document for testing the pathogenicity and toxicity
of new microbial substances to aquatic and terrestrial organisms (Environment Canada, 2004). This
guidance document is available on:
http://www.ec.gc.ca/Publications/default.asp?lang=En&xml=F9BF9993-4BAC-4215-BD3E-
9B0962980915 (last accessed April 27, 2011).
The Canadian Guidance Document takes into account various sources of information, including
guidance in PMRA
2
s microbial registration guidelines (DIR2001-02), US EPA test guidelines and OECD
test guidelines. PMRA registration guidelines (Regulatory Directive DIR2001-02, Guidelines for the
Registration of Microbial Pest Control Agents and Products) provide general guidance on study design
and reporting and are available on PMRA’s website (http://www.hc-sc.gc.ca/cps-spc/alt_formats/pacrb-
dgapcr/pdf/pubs/pest/pol-guide/dir/dir2001-02-eng.pdf, last accessed, April 27, 2011).
According to Canadian registration guidelines (PMRA, 2001) a four-tiered testing approach is
followed similar to the approach by the US EPA. However, minor deviations are noted regarding the
testing criteria of the NTOs and fate testing procedures (see Table 2 below).
Table 2: Tiered testing approach according to Pest Management Regulatory Agency (2001)
Tier level Explanation
I Test organisms in Tier I are exposed to maximum hazard or
Maximum Challenge Concentration (MCC) of the mBCA.
Criteria for non-target to be tested are as follows:
taxonomically related, infected by mBCA, high exposure potential,
similar physiology, susceptible to related pathogens, representative
species from seven broad taxonomic groups
II Adversely affected species from Tier I toxicology tests are exposed
to Lower Challenge Concentrations (LCC).
Conditionally required fate studies are as follows:
pure culture testing, microcosm testing, small- or large-scale
field studies
III* Adversely affected species from Tier II toxicology tests are exposed
to multiple concentrations (determination of LC50, LD50, EC50
values)
Conditionally required fate studies are as follows:
small- or large-scale field studies
IV Adversely affected species from Tier II are investigated in small-
scale field studies in which the end-use product should be used.
*Tier III testing is not required for indigenous mBCAs
Overall, data requirements for mBCAs are similar between regulations in Canada and the US with
regard to effect analysis, whereas few test systems recommended in the Guidance Document (Environment
Canada, 2004) are not usually required in the US EPA Microbial Pesticide Branch such as the 56-day
2
Note that Environment Canada and PMRA are two different governmental departments in Canada
ENV/JM/MONO(2012)1
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earthworm reproduction study or the 28-day collembolan reproduction study, respectively. In addition,
Canadian guidelines were developed for both single concentration and multi-concentration-tests taking into
account maximum hazard testing approach. The Canadian guidelines are more advantageous because they
contain more specific statements regarding certain test conditions and test criteria.
4.1.4 Opinion of the BPSG
The BPSG had agreed to use the US Microbial Pesticide Testing Guidelines although Canadian test
guidelines may also be used.
4.2 Waiver options
Waivers can be granted in two situations:
1. If exposure of the NTO can be excluded by the way of application.
2. If significant information is available for the mBCA, e.g., in-depth knowledge of the biology,
life-cycle, mode of action, fate and behaviour in the considered environmental compartment.
The rationale should include a transparently conducted literature search on the pathogenic/toxic
effects to NTOs of concern according to EFSA’s Guidance Document on Submission of scientific peer-
reviewed open literature (EFSA, 2011).
4.3 Terrestrial NTOs
4.3.1 Birds and mammals
a) Birds
Available test guidelines
1) US EPA test guidelines OCSPP 885.4050 Avian Oral Tier I.
In this study, a MDD (maximum daily dose) is administered to young bobwhite quail or mallard
ducks for five days with a following observation period of at least 25 days. If any signs of pathogenicity
and toxicity are manifested on the 30
th
day, observation should continue until recovery, mortality or
unequivocal moribundity is established.
The highest oral dosage level tested is defined by the following formula:
MDD (units) = [mBCA] in TGAI × 5 mL/kg BW × weight of test bird (kg)
where
[mBCA] = concentration of mBCA
TGAI = technical grade of active ingredient
BW = body weight
ENV/JM/MONO(2012)1
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The treatment group is accompanied by three different control groups:
• A negative, non-dosed control group.
• An infectivity control group treated with the mBCA inactivated in such a way as to retain the
structural integrity of the cell.
• A control group in which the birds are dosed with sterile filtrate from production cultures.
2) The test guidelines recommended in the Canadian guidance document are consistent with the US
EPA test guidelines. The Canadian proposal for test method provides additional criteria of validity
(invalid if < 90 % survival in negative control at test end), a more detailed description of conducting a
multi-concentration test and the requirement for assessing infectivity at the end of the test (as a
minimum).
b) Mammals
Available test guidelines
1) US EPA provides specific test guidelines for detecting effects in wild mammals (885.4150).
Overall, results of the toxicology studies might be sufficient to address possible adverse effects to
mammals.
Mammals feeding on insects infested by entomopathogenic mBCAs
Animals feeding on insects can be expected to ingest large quantities of actively growing
microorganisms when they feed on diseased insects. Moreover, there is a possibility for exposure to
potential toxic secondary metabolites synthesized during vegetative growth. This issue is addressed in Tier
I freshwater fish testing (OCSPP 885.4200) since there is an option of exposing fish with infected insects.
However, no test guidelines are available to address this type of exposure in insectivorous birds and
mammals.
Risk assessment
If no negative impact to birds and mammals are observed in the study, the risk can be considered to be
acceptable.
If the MHD study shows negative effects, an attempt to classify the type of effect(s) observed in the
study should be made by using observations of pathogenic symptomatology or pathological changes, gross
necropsies and histopathological findings, and by comparing these observations to those made for the
various control group(s) (e.g. non-infectious control and sterile filtrate control).
ENV/JM/MONO(2012)1
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Toxic effects:
If the observed effects are mainly attributed to compounds with a toxic mode of action, i.e. caused by
toxic co-formulants or other components of the technical material (probably secondary metabolites
resulting from the microbial growth in the batch of production), a dose-response study should be conducted
to obtain reliable ecotoxicological endpoints such as LD
50
/LC
50
/EC
50
/ER
50,
NOEC or NOEL values.
Consequently, a standard risk assessment comparable to chemical pesticides including standard safety
factors might be feasible. In accordance with the Regulation (EC) No 1107/2009 of the European
Parliament and of the Council and the Guidance Document on Risk Assessment for Birds & Mammals
(EFSA , 2009), a TER calculation can be conducted taking into account Annex VI TER trigger values of
10 and 5 for acute and chronic exposure, respectively.
As the toxicity causing metabolites are probably unknown, derived ecotoxicological endpoints must
refer to the amount of technical material or to the units of the mBCAs itself.
Pathogenic effects:
If the adverse effects are caused by pathogenicity, a follow-up with dose-response testing might not
always be appropriate since clear dose-response relationships may not necessarily be observed.
Information on growth-temperature-relationship of the mBCA
The ability of an mBCA to grow at body temperatures can be regarded as a crucial factor in the
evaluation of pathogenic effects.
As stated in the Commission Regulation (EU) 544/2011 (EU, 2011) in annex part B point 2.5
(Infectiveness, dispersal and colonisation ability), the temperature range at which the mBCA grows must
be determined, including minimum, maximum and optimum temperatures. This information is of particular
value as a trigger for effect studies on human health. It is also, to some extent, of importance for evaluating
effects on birds and wild mammals. However, it should be emphasised that a single statement “inability of
growth at temperatures of >36°C” should not be accepted as a waiver. Nevertheless, adequate information
on growth-temperature characteristics together with additional arguments might be acceptable. Further,
unnecessary testing of vertebrates should generally be avoided in view of animal welfare.
Incidental remarks on vertebrates other than birds and mammals:
It is obvious that information on the growth-temperature characteristics of mBCAs do not provide a
strong waiver argument for cold-blooded animals such as amphibians and reptiles. Given the lack of
microbial-specific test methods for amphibians/reptiles and because they are not formally required for
conventional pesticides (except the Metamorphosis Assay with the African clawed frog Xenopus laevis in
case of thyroid active substances), effect studies with (amphibians/reptiles) should only be provided on a
case-by-case basis, e.g., if susceptibility is reported in the open literature, or there is strong evidence for
adverse effects based on the biological properties of the mBCA.
Waiver options
A waiver can be submitted:
− If exposure of birds and mammals is expected to be minimal or negligible.
− If significant information is available for the mBCA, e.g., in-depth knowledge of the biology,
information on growth-temperature characteristics, life-cycle, mode of action, fate and behaviour
ENV/JM/MONO(2012)1
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in the considered environmental compartment must be submitted based on a transparently
conducted literature research for published pathogenic/toxic effects to birds/mammals.
A waiver can be granted if sufficient information is available to conduct a qualitative risk assessment
and thus in logical line of argument a potential risk of the mBCA to birds/mammals can be excluded.
4.3.2 Bees
General aspects
The European Regulation (EC) No 1107/2009 requires that the risk to bees be evaluated. The US EPA
as well as Canada only provide guidelines for testing honey bees.
Available test guidelines
1) OCSPP guidelines 885.4380 Honey Bee Testing Tier I
− No MHD is defined.
− On the basis of 885.4340 (Nontarget insect testing) dosage shall be in suitable increments of up
to 100 times the LD
50
or LC
50
of the pathogen in its natural host, or 10–100 times the
recommended field dosage.
− The method of application depends on the expected route of exposure, either oral or contact or
even whole-hive.
− The recommended test duration is ≥ 30 days. When the mBCA may be expected to affect larvae,
honey bee larvae should be included as test organisms.
2) Honeybees Acute Oral/Contact Toxicity Tests (OECD 213/214) (Tier I)
− Test duration is 2 days which is too short to measure pathogenic as well as toxic effects.
Therefore they do not provide an acceptable alternative to OCSPP guidelines 885.4380.
3) A standardised method by Aupinel et al. (2005)
− This method can be recommended for assessing the risks to bee brood.
4) EPPO 170 guidelines (EPPO, 2010)
− Additional long-term (semi)-field testing may also be required if effects are observed in the first
tier level.
Additional information on laboratory test methods is given below:
ENV/JM/MONO(2012)1
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Risk assessment
The calculation of HQ values as used for chemicals (application rate/LD
50
) is generally regarded as
less feasible for risk assessments with mBCAs because dose-response relationships are rarely observed in
cases of pathogenic effects.
If observed effects are caused by toxicity, dose-response testing should be conducted providing
reliable ecotoxicological endpoints (LC
50,
LD
50
, NOEC). These endpoints can be integrated in standard risk
assessments similar to chemical pesticides including the use of established safety factors. Exploring the
origin of effects observed in control groups receiving an attenuated treatment (microbe-free or non-viable
microbe comprising material from the culture system used for propagation) might be helpful, as adverse
effects caused by this treatment are not due to pathogenicity.
According to the US EPA guideline OCSPP 885.4340, the MHD is up to 10–100 times the
recommended field dosage, thus comprising a safety factor of 10–100. According to Environment Canada
(Environment Canada, 2004), “the maximum hazard concentration is to be equivalent to 100 times the
maximum concentration of microorganisms specified by the notifier for the final tank mix of a microbial
According to the Canadian guidance document (Environment Canada, 2004), US EPA is
undertaking research studies with the intent of developing a standardised laboratory test method for
measuring the ecological effects of microbial substances on honey bees. The publication of Hanley et
al. 2003 (cited in Environment Canada, 2004) describes a laboratory test used to demonstrate the
potential adverse effects of dietary pollen contaminated with microbial or chemical pesticides on larval
or pupal life stages of honey bees. Certain aspects of the test design including larval and pupal mortality
rates as well as reduced pupal weights as biological endpoints are being considered by the US EPA for
possible use when developing a standardized protocol.
The Canadian guidance document mentions two other published reports on laboratory tests
performed with groups of adult honey bees. Ball et al. 1994 (cited in Environment Canada, 2004)
acclimated groups of young adult honey bees (25/cage) to laboratory conditions in cages for 1 week,
followed by their exposure to a mycopesticide administered by spray application. The negative control
group showed a mortality rate of only 7 % during a subsequent 12-day period of observation. Butt and
Goettel (2000, cited in Environment Canada, 2004) using a similar experimental design did not report
the mortality rate of control groups, although a 14-day mortality of only 11 % was found for groups of
adult bees subjected to the lowest microbial test concentration tested, with higher mortality rates (up to
87 %) for higher concentrations. Both research studies indicate that acceptably low (e.g. ≤ 10 %)
mortality rates can be achieved for negative control groups of adult honey bees, in 12-14-day laboratory
test using this experimental design. The Canadian guidance document considers both approaches as
promising, but recommends for applying this test design to conduct preliminary tests of 14 day duration
to ensure that an acceptable control mortality rate of ≤ 10 % can be achieved. Additionally alternatives
for dosing the test groups by feeding them a diet containing the microbial substance or by spray
application should be considered and experimented with in preliminary trials.
ENV/JM/MONO(2012)1
41
product, when it is applied at the maximum label rate”. A subsequent risk calculation requiring an adequate
exposure assessment is not necessary provided that no adverse effects are observed.
In instances of pathogenic effects, these observations have to be considered and classified in context
of the overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-
cycle and biology of the mBCA, environmental conditions for survival, germination and infection).
Since bumble bees have a far lower hive temperature compared to honey bees, they might be more
susceptible to mBCAs, particularly to EPFs having mostly lower optimal growth temperatures (Hokkanen
et al., 2003). This should be taken into consideration. Additional study guidelines adapted to bumble bees
could be helpful.
Waiver options
A waiver can be submitted:
- If exposure of bees is negligible or minimal.
- In case of non-entomopathogenic mBCA, if database searches find no reports of detrimental
impacts of the considered microorganisms on bees and other closely related species of the mBCA
that share the same ecological habitat.
4.3.3 Non-target arthropods other than bees
a) Leaf-dwelling arthropods
General aspects
mBCAs can be applied to control fungal or bacterial plant diseases, weeds or pest insects. In the
control of insect pests, non-target arthropods (NTA) are the organisms that are most at risk, being
relatively closely related to the target organism. Many microorganisms exert their effect(s) through
pathogenicity as well as toxicity.
The European regulation does not distinguish between soil- or leaf dwelling arthropods.
Nevertheless, a differentiation between soil and leaf dwelling arthropods is useful as tests with leaf-
dwelling arthropods can be waived if exposure can be excluded due to application techniques (e.g. soil
drench application) and due to formulation types such as granules, seed treatments and pellets (please note:
as pointed out in chapter 1.2.2, exposure of plant-dwelling arthropods, in theory, might also occur
following seed treatment uses if the microorganism is able to grow endophytically.)
In the US, arthropod tests are only required if the mBCA is intended to control target insect pests by a
mechanism of infectivity (e.g., may create an epizootic condition in non-target insects).
Available test guidelines
To date there are no internationally recognized standard test methods for testing the effects of
microbials on non-target arthropods comparable to existing OECD Test Guidelines.
ENV/JM/MONO(2012)1
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Current test guidelines used for evaluating chemical pesticides are only suitable to a limited extent.
1) The IOBC Test Guidelines by Candolfi et al. (2000) are generally used to determine side-effects
of chemical pesticides on a large range of beneficial arthropods (natural enemies) including both
plant-dwelling (e.g. the parasitic Hymenoptera Aphidius rhopalosiphi, the predatory mite
Typhlodromus pyri) and ground/soil-dwelling arthropods (e.g. the carabid beetle Poecilus
cupreus, the rove beetle Aleochara bilineata and the wolf spider Pardosa spp.). However, these
guidelines do not consider relevant routes of exposure for viruses, fungi and bacteria, nor do they
allow for prolonged exposure and observation periods.
2) The OCSPP guidelines 885.4340 (Non-target Insect Testing Tier I) provides guidance on
developing suitable test designs.
− Test species: Three species of arthropods have to be chosen from at least two identified
groups (i.e. parasitic Diptera, predaceous Hemiptera, predaceous Coleoptera, predaceous
mites, predaceous Neuroptera and parasitic Hymenoptera).
− Route of exposure: Route of exposure should be consistent with the most likely route of
exposure under natural environmental conditions. Exposure in the diet is mostly preferred.
Considering viral and bacterial mBCAs, these guidelines recommend that internal parasites
be tested with virus/bacteria-infected hosts or if they can be cultured in vitro, the
virus/bacteria can be added to the diet. External stages of parasites and predators (if they
are obligatory) may be fed virus/bacteria-infested hosts, virus/bacteria-contaminated media,
or virus/bacteria suspended in sugar or honey solutions. The exposure of fungal mBCAs
should simulate field conditions as much as possible. Humidity might be critical during
exposure.
− Test concentration: The test dosage shall be in suitable increments up to 100 times the LD
50
or LC
50
of the pathogen in its natural host, or 10–100 times the recommended field dosage.
− Control: A concurrent control group treated with microbe-free (or non-viable microbe)
material from the culture system used for propagation is recommended.
− Observations and biological endpoints: Mortality and symptoms of pathogenicity are to be
determined
− Test duration: Test duration depends on the type of microorganism under investigation as
well as on the host species and life stage, 8−10 days for fungi, 21−30 days for bacteria and
up to 30 days for viruses or until control mortality rises up to 20%.
3) The Canadian guidance document (Environment Canada, 2004) also gives useful advice on
designing tests. In chapter 13.3.1, several protocols for testing the effects of microbial pathogens
on non-target beneficial insects and mites are mentioned.
1. Methods for testing the pathogenicity and virulence of fungi on:
- the predatory mite Metaseiulus occidentalis (Sewall and Lighthart, 1989, cited in
Environment Canada, 2004)
- the parasitic wasp Trichogramma pretiosum (Sewall and Lighthart, 1990, cited in
Environment Canada, 2004)
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43
- the green lacewing Chrysoperla carnea (Donegan and Lighthart, 1990, cited in
Environment Canada, 2004)
- the lady beetle Hippodamia convergens (James and Lighthart, 1992, cited in Environment
Canada, 2004)
2. Test for pathogenicity and virulence of bacteria on:
- the lady beetle Hippodamia convergens (James and Lighthart, 1990, cited in Environment
Canada, 2004)
Exposure is generally achieved by dipping the test insects in different concentrations of
test material followed by observation periods of 6 to 10 days and, if possible, the
determination of LD
50
values.
The Canadian guidance document (Environment Canada, 2004), as opposed to US EPA
guideline 885.4340, recommends that the biology of the microorganism (e.g. known
pathogens in the same family or genus) be considered during the selection of a suitable test
organism and the associated biological test method. If closely related microorganisms are
pathogenic to any terrestrial invertebrate, this species of invertebrate should be selected as
a test organism, provided that a suitable biological test method is available. The use of
computerized databases with a focus on environmental safety issues of microbial
pathogens is recommended to identify arthropods species that may be susceptible to a
given mBCA. Database results within the genus of the microorganism should be identified
and reviewed.
4) The review by Fisher and Briggs (1992, cited in Environment Canada, 2004) considers a variety
of important parameters when testing the effects of mBCAs on non-target insects in the
laboratory, including choices of test (host) organisms, various routes of exposure, quantifying the
test concentration, test duration and endpoints. Moreover, a brief description of research
approaches and (non-standard) test methods for measuring effects of microorganisms on honey
bees and other non-target insects is included. The Canadian guidance document regards this
publication as helpful when choosing the test method(s) for terrestrial invertebrates to be applied
to mBCA.
Considerations to entomopathogenic mBCAs
The Canadian proposal for selecting a suitable test organism might be adapted for entomopathogenic
microorganisms, because they are intended to control target arthropods. EPF may have broad host ranges,
including non-target arthropods. Thus, a species found to be susceptible in the database might belong to the
known host range. In instances where an entomopathogen has a broad host range, some effects on non-
target arthropods might occur. In such cases, regulators may have to accept some level of pathogenic
effects to non-target arthropods.
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Risk assessment
The calculation of HQ values as used for chemicals (application rate/LR
50
) is generally regarded as
less feasible for risk assessments with mBCAs because dose-response relationships are rarely observed in
cases of pathogenic effects.
If observed effects are caused by toxicity, dose-response testing should be conducted providing
reliable ecotoxicological endpoints (LR
50
, LC
50
, LD
50
, NOEC). These endpoints can be integrated in
standard risk assessments similar to chemical pesticides including the use of established safety factors.
According to the US EPA guideline OCSPP 885.4340, the MHD is up to 10–100 times the recommended
field dosage therefore comprising a safety factor of 10–100. According to Environment Canada
(Environment Canada, 2004) “the maximum hazard concentration is to be equivalent to 100 times the
maximum concentration of microorganisms specified by the notifier for the final tank mix of a microbial
product, when it is applied at the maximum label rate”. A subsequent risk calculation requiring an adequate
exposure assessment is dispensable provided that no adverse effects are observed.
In instances of pathogenic effects, these observations have to be considered and classified in context
of the overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-
cycle and biology of the mBCA, environmental conditions for survival, germination and infection).
It is concluded that detrimental effects to non-target arthropods within the host range (ecological host
range) have to be accepted to some extent.
Waiver options
A waiver can be submitted:
- If exposure of leaf-dwelling arthropods is considered to be minimal in view of certain application
techniques (e.g. soil drench application) and formulation types (e.g. granules, seed treatment,
pellets).
- In case of non-entomopathogenic mBCA, if database searches find no reports of detrimental
impacts of the considered microorganisms together with sufficient information about mode of
action, biology, life cycle and environmental conditions for survival and reproduction.
b) Soil-dwelling arthropods
Available test guidelines
1) The US EPA provides no test guidelines concerning soil-dwelling arthropods.
2) Canadian guidelines are available for a 28-day reproduction test using the springtail Folsomia
candida (Collembola: Isotomidae).
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45
Database searches may lead to other susceptible soil-dwelling arthropods. These should be considered
as test organisms provided that any testing method is available or might be adapted (e.g. IOBC guidelines)
to microbial test substances.
Risk assessment
A risk calculation analogous to chemicals is generally regarded as less feasible for the risk
assessments of mBCAs because dose-response relationships are rarely observed in cases of pathogenic
effects.
If observed effects are caused by toxicity, dose-response testing should be conducted providing
reliable ecotoxicological endpoints (LR
50
, NOEC). The Canadian Guidelines for F. candida provides the
option of multi-concentration tests. These endpoints can be integrated in standard risk assessments similar
to chemical pesticides including the use of established safety factors. Exploring the origin of effects
observed in control groups receiving an attenuated treatment (microbe-free or non-viable microbe
comprising material from the culture system used for propagation) might be helpful, as adverse effects
caused by this treatment are not due to pathogenicity.
According to Canadian guidelines for testing the springtail F. candida, a MHC for soil of 10
6
microbial units/g soil (dry wt) or 1000 times the expected microbial concentration in soil within the
terrestrial environment is defined. A subsequent risk calculation requiring an adequate exposure
assessment is dispensable provided that no adverse effects are observed. When using other guidelines
adapted for microbial test substances, a similar test exposure should be adopted.
In case of pathogenic effects, these observations have to be considered and classified in context of the
overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-cycle
and biology of the mBCA, environmental conditions for survival, germination and infection).
Waiver options
A waiver can be submitted:
- If exposure of soil-dwelling arthropods is negligible or minimal
- In case of non-entomopathogenic mBCA, if database searches find no reports of detrimental
impacts to soil-dwelling arthropods caused by the considered microorganisms in connection with
sufficient information about mode of action, biology, life cycle and environmental conditions for
survival and reproduction.
4.3.4 Terrestrial plants
General aspects
Information and/or testing for plant toxicity/pathogenicity is not required according to Regulation
(EU) No 544/2011 annex part B.
In contrast to the EU, effects on terrestrial plants have to be assessed in the US and in Canada if the
mBCA is closely related to a known plant pathogen.
ENV/JM/MONO(2012)1
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Diseases of commercially important plants have been intensively studied for decades and many plant
pathogens have been identified and subsequently well characterized. Some plant pathogens have a very
narrow host range and may attack only one species of plant, other plant pathogens may attack a wide range
of plant species, and still other microorganisms have never been identified in association with disease in
plants.
Available test guidelines
1) The US EPA guideline OCSPP 850.4300
These guidelines refer not only to terrestrial but also to aquatic plants. As terrestrial plants are
discussed in this chapter, only this part of these guidelines is considered here.
− Test species: Commercial agricultural crops should be considered as test species since these
plants are more susceptible to plant diseases compared to wild plants being genetically
diverse groups. Besides US EPA emphasizes their commercial importance. The number of
species tested depends on the similarity of the mBCA to known plant pathogens. A rationale
for the selection of the species to be tested must be provided. Commercial agricultural crops
are recommended as test species are listed in the OCSPP guidelines 885.4300. However it is
suggested that depending upon the predicted use pattern, certain forest tree species,
ornamental trees and shrubs, and weed species may need testing.
− Test concentration: One single concentration level at the “maximum label rate” is to be
tested, that means the amount of active ingredient in the recommended quantity of carrier.
− Control: Negative (untreated) as well as positive controls have to be included. Positive
controls are to ascertain that environmental conditions are such that penetration, infection,
and disease development are likely to occur in a susceptible host. Therefore the positive
control should be selected to closely resemble the subject mBCA in terms of taxonomy and
optimal conditions for infection and disease development, if known. In the case of a mBCA
not intended for herbicidal use, the positive control may consist of a known plant pathogen,
with taxonomic characteristics similar to the mBCA and its susceptible host. In the case of a
microbial herbicide, however, the positive control should consist of the target pest weed and
the microbial herbicide.
− Test duration: Plants should be observed weekly or more frequently until normal harvest or
death, or, in the case of perennials, at regular intervals for at least 2 years.
2) The Canadian guidelines
− Test species and test method: various monocotyledons and dicotyledons depending on the
intended way of application and the likelihood of exposure (see section 12 of the Canadian
guidance document). In order to find appropriate test organisms, all results of previous
laboratory tests involving terrestrial plants exposed to the mBCA or ones having similar
characteristics should be taken into consideration. All available research findings from
relevant experimental field studies should be reviewed and considered. Computerized
databases should be consulted as a first step in choosing the appropriate test method and test
host species. Besides the mBCA under investigation, all microorganisms within the same
genus should be identified and reviewed in the same way. Plants found to be adversely
affected by the mBCA or by related microorganisms within its genus should be considered as
test species provided that suitable test methods are available.
ENV/JM/MONO(2012)1
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− Route of Exposure: The test plants should be exposed to the mBCA by whatever route of
exposure that is expected by the proposed use pattern, e.g., in test water, in test soil, by
wounding and spraying. Specific susceptibility of a given life stage, possible ways of entry of
the potential pathogen (seed, root, leaf) and controlled climatic conditions are to be taken into
account
− Test concentration: The MHC is defined for soil as 10
6
microbial units/g soil (dry wt) or
1000 times the expected microbial concentration in soil within the terrestrial environment
provided this is readily attainable under laboratory conditions.
− Control: An additional control group using the sterile filtrate is recommended.
− Test duration: Test duration of only 14 and 21 days, respectively depending on the test plant.
Risk assessment
A risk calculation analogous to chemicals is generally regarded as less feasible for the risk
assessments of mBCAs because dose-response relationships are rarely observed in cases of pathogenic
effects.
If observed effects are caused by toxicity, dose-response testing should be conducted providing
reliable ecotoxicological endpoints (EC
50
). The Canadian Guidelines provide the option of multi-
concentration tests. These endpoints can be integrated in standard risk assessments similar to chemical
pesticides including the use of established safety factors. Exploring the origin of effects observed in control
groups receiving an attenuated treatment (microbe-free or nonviable microbe comprising material from the
culture system used for propagation) might be helpful, as adverse effects caused by this treatment are not
due to pathogenicity.
According to the US EPA guidelines OCSPP 885.4300, one concentration level equal to no less than
the maximum label rate shall be tested. The phrase “maximum label rate” means the amount of active
ingredient in the recommended quantity of carrier, such as water to be used per land area or applied
directly to the surface of a 15 cm or 6 inch column of water. According to Canadian guidance, a MHC for
soil of 10
6
microbial units/g soil (dry wt) or 1000 times the expected microbial concentration in soil within
the terrestrial environment is defined. A subsequent risk calculation requiring an adequate exposure
assessment is dispensable provided that no adverse effects are observed.
In case of pathogenic effects, these observations have to be considered and classified in context of the
overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-cycle
and biology of the mBCA, environmental conditions for survival, germination and infection).
Waiver options
A waiver can be submitted:
- If exposure of terrestrial plants is negligible or minimal.
- In case of mBCAs with no intended use as a herbicide, if database searches find no reports of
detrimental impacts to plants by the considered microorganisms and relative species within the
ENV/JM/MONO(2012)1
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same genus in connection with sufficient information about mode of action, biology, life cycle
and environmental conditions for survival and reproduction.
4.3.5 Earthworms
General aspects
The issue of possible adverse effects (toxicity, infectivity and pathogenicity) to earthworms has to be
addressed according to point 8.5 in part B in the annex to Regulation (EU) No 544/2011 when applying for
the registration of mBCAs.
US EPA has no data requirement assessing the risk to earthworms. REBECA
3
questions the
reasonability of earthworm tests and states “no earthworm pathogens have been reported”. Moreover, it is
argued that earthworms should be adapted to soilborne bacteria and fungi as these are often similar to
mBCAs applied for registration. Indeed, searching for microbial earthworm pathogens is difficult
compared with, e.g., plant or insect pathogens, as there are free databases for diseases of plant or insects
but not for annelids.
Until now, only few studies indicated pathogenic effects to earthworms (see below).
For example, Smirnoff and Heimpel (1961) reported pathogenic effects of B. thuringiensis subsp.
thuringiensis (Thuricide® 30B) in a prolonged study with Lumbricus terrestris. However, further analysis
revealed that observed lethal effects were not attributed to the mBCA. As explained in the review by
Addison J.A. (1993) reported effects were caused by the presence of diatomaceous earth used as carrier in
the formulated product.
Detrimental impacts of Bt-formulations on earthworms and other non-target soil organisms have also
been reported in studies by Addison and Holmes (1995, 1996). The authors examined effects of Bt subsp.
kurstaki on a forest earthworm (Dendrobaena octaedra) and found no effect of unformulated and aqueous
Btk at 1000 times the field concentration, however an oil formulation of Btk reduced survival, growth and
reproduction (Addison and Holmes, 1996).
An earthworm study with B. subtilis QST 713 (Serenade® WP) submitted during the European
process of Annex I inclusion showed sublethal effects such as lethargy and reduced reaction to mechanical
stimuli. Histopathological analysis revealed bacterial colonisation in body tissues. However, it should be
noted that these observations occurred at concentrations that would not be expected under field conditions.
Another example of pathogenic effects to earthworms was published by Vakili (1993). The author
described the isolation of the soilborne fungus Exophiala jeanselmei from cocoons of the earthworm
species Eisenia foetida infected by the fungus. As several attempts to isolate the fungus from the used soil
were not successful, it was concluded that the fungus was carried within the body of the adult earthworms
and disseminated to their cocoons. Adult earthworms were not described as negatively affected.
Deleterious effects were observed only in the reproduction process. The examples above indicate that
standard short-term 14-day earthworm studies focusing on detecting lethal or sublethal effects (weight
loss) are probably not suitable to prove the absence of infectivity or pathogenicity at least by visual
observation.
3
REBECA is an EU policy support action to review possible risks of biocontrol agents, compare regulations in the EU and the US and to propose
alternative, less bureaucratic and more efficient regulation procedures maintaining the same level of safety for human health and the environment
but accelerating market access and lowering registration costs.
ENV/JM/MONO(2012)1
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Earthworms obviously cope with soilborne microorganisms without being infected or negatively
affected. This is due to the long time of evolutionary earthworm-pathogen co-existence, during which
earthworms have developed. Earthworms developed an immune system being native, mainly non-specific,
non-anticipatory and non-clonal. Although there was evidence of specific memory in immune response of
invertebrates under certain conditions, this is limited to the recognition of PAMPs (pathogen-associated
molecular patterns) being present in many microorganisms mediated by pattern recognition receptors like
Toll-like receptors (Rowley and Powell, 2007). Therefore, evidence suggests that the immune strategy of
earthworms is similar to various microorganisms.
Available test guidelines
1) Suitable guidelines determining effects of mBCAs to non-arthropod invertebrates such as
earthworms are considered in the Canadian registration procedure under PMRA Data Code:
M9.6 Non-arthropod invertebrates (PMRA, 2001).
2) The following methodology is recommended according to Section 13 of the Canadian guidance
document (Environment Canada, 2004):
− Test species: Eisenia andrei (also referred to as Eisenia foetida andrei) or the closely related
species Eisenia fetida.
− Testing for infectivity: Infectivity is examined by measuring the concentration of microbial
substance in whole-organism homogenate of earthworms from each treatment during and/or
at the end of the test.
− Route of exposure: The route of exposure is via soil mixed with the test substance or via food.
− Test concentration It is possible to use only one concentration (i.e., MHC) in a single
concentration test or a minimum of seven concentrations including the MHC. The MHC used
for soil mixture is defined as 10
6
microbial units/ g soil (dry wt), or 1000 times the expected
microbial concentration in soil within the terrestrial environment. The definition of the MHC
used in food mixture is 100 times that in the maximum concentration of microorganisms
specified by the notifier for the final tank mix of a microbial product.
− Control: Negative (untreated) as well as positive controls have to be included. The use of a
non-infectious control is strongly recommended. A sterile filtrate control is optional but also
recommended.
− Observations and biological endpoints: The total number of live adult worms on Days 0 and
28, number of live juvenile worms on Day 56, obvious pathological symptoms (e.g. open
wounds) or distinct behavioural abnormalities (e.g. lethargy) have to be recorded. Biological
endpoints are determined for the total number of surviving adult worms on Day 28, total dry
weight and number of surviving juvenile worms on Day 56, number of surviving adult
worms showing atypical appearance and/or behaviour on Day 28 as well as on Day 56.
− Test duration: Effects on survival, reproduction and growth are to be detected within a
timeframe of 56 days.
− Criteria of validity: Tests are considered invalid if mean 28-day survival of adults in negative
control soil < 90 %, if mean reproduction rate for adults in negative control soil < 3 live
ENV/JM/MONO(2012)1
50
juvenile/adult and if mean dry weight of individual live juveniles in negative control soil at
test end < 2 mg.
General remarks regarding the risk assessment for below ground NTOs
The estimation of the PIECsoil might give an overall idea of the exposure of below ground NTO
(earthworms, microorganisms, other NTO). Care has to be taken with the use of the NOEC and LC
50
values which are also calculated with a substantial uncertainty. For most mBCAs, a qualitative risk
assessment based on an overall view of all available information is the most relevant and often the only
possible evaluation. When using OCSPP-tests (first Tier) endpoints are usually based on MHC and
subsequently LC
50
values if effects are observed in an initial stage. LC
50
values are generally not suitable
for assessing risks of pathogenicity since infection/pathogenicity do not necessarily occur in a dose-
response manner. Likewise, an attempt to derive a specific pathogenic threshold level is not deemed
feasible.
The European regulation demands data concerning earthworms and soil-microorganisms that are not
required in the US. However, there are Canadian test guidelines addressing the determination of adverse
effects of mBCAs to earthworms as well as springtails. Therefore, studies might be conducted in
accordance with these guidelines.
Risk assessment
A risk calculation analogous to chemicals is generally regarded as less feasible for the risk
assessments of mBCAs because dose-response relationships are rarely observed in cases of pathogenic
effects.
If observed effects are caused by toxicity, dose-response testing should be conducted providing
reliable ecotoxicological endpoints (LC
50,
LR
50
, NOEC). The Canadian Guidelines provide the option of
multi-concentration tests. These endpoints can be integrated in standard risk assessments similar to
chemical pesticides including the use of established safety factors. Exploring the origin of effects observed
in the control groups receiving an attenuated treatment (microbe-free or nonviable microbe comprising
material from the culture system used for propagation) might be helpful, as adverse effects caused by this
treatment are not due to pathogenicity.
According to Canadian guidelines, a MHC for soil of 10
6
microbial units/g soil (dry wt) or 1000 times
the expected microbial concentration in soil and 100 times the maximum concentration of microorganisms
specified by the notifier for the final tank mix of a microbial product for food mixture is defined. A
subsequent risk calculation requiring an adequate exposure assessment is dispensable provided that no
adverse effects are observed.
In instances of pathogenic effects, these observations have to be considered and classified in context
of the overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-
cycle and biology of the mBCA, environmental conditions for survival, germination and infection).
ENV/JM/MONO(2012)1
51
Waiver options
A waiver can be submitted:
- If exposure of earthworms is negligible or minimal.
- If in-depth knowledge is available on the mode of action, biology, life cycle and environmental
conditions for survival and reproduction of the mBCA excluding any risk to earthworms (e.g.
quality of information known for baculoviruses).
4.3.6 Non-target soil microorganisms
The US EPA BPPD microbiologists do not support testing for effects of microbial pesticides on
microorganisms for the following reasons:
• There may be effects from almost anything added to the soil, but there is no valid way to interpret
any results one might obtain from testing.
• The relative risk from adding microorganisms to the soil microbial community is minimal. Soil
microflora varies immensely spatially and temporally. The natural population has adapted to their
particular environmental niches, and has evolved many defense mechanisms to allow their
survival in those niches.
• Soil microflora is very resilient, e.g. even when the microbial populations are decimated by
methyl bromide, the natural soil populations rebound quickly.
US EPA notes that it is a valid area for research, but is not a very significant risk issue in the big
picture.
In the EU dossiers for the inclusion of mBCAs in Annex I, nitrification and respiration tests were
submitted for Beauveria bassiana strain ATCC 74040, Trichoderma asperellum strain ICC012 (formerly
Trichoderma harzianum Rifai), Trichoderma gamsii strain ICC080 (formerly Trichoderma viride strain
ICC080), Bacillus thuringiensis subsp. aizawai GC-91, B. thuringiensis subsp. tenebrionis NB-176, B.
thuringiensis subsp. kurstaki ABTS-351, PB54, SA-11, SA-12 and EG2348. These functional tests did not
show any adverse effects. This supports the assumption of the resilience of soil-microflora especially
concerning issues of functionality of the microbial community in soil. Therefore, standard nitrification and
respiration tests according OECD Guidelines 216/217 are less suitable to fullfil this data requirement.
Besides, it is worth mentioning that such studies according to OECD 216/217 are designed for chemicals
and are not validated for mBCAs as test substance.
In conclusion, the relevance of carbon mineralization and nitrogen transformation tests seems to be
low as indicated in previous BPSG seminar presentations. However, impacts on microbial community
structures (Pérez-Piqueres et al., 2006; Edel-Hermann et al., 2009) or on symbiotic activity of arbuscular
mycorrhizal fungi may be of relevance in some cases. Studies on these two topics were submitted in the
EU dossiers for the inclusion of Trichoderma atroviride I-1237 and Trichoderma asperellum strain T34 in
Annex I of EU Directive 91/414/EEC (now Regulation (EC) No 1107/2009).
For the EU review process of other mBCAs, literature information, waivers or statements were
submitted. Occasionally, inhibition of microorganisms by an mBCA (e.g. B. thuringiensis
subsp.
tenebrionis NB-176) has been studied using culture techniques (e.g. agar plates), microbial biomass or
ENV/JM/MONO(2012)1
52
enzyme activity (e.g. dehydrogenase). In fact, not one particular test is prescribed and applicants can
choose from a wider range of techniques and also, the non-target group to be tested needs to be chosen
(e.g. fungi, bacteria, actinomycetes or protozoa).
For the sake of increasing knowledge on the non-target effects of mBCAs on non-target soil
microorganisms, a desk study by Scheepmaker and Van de Kassteele (2011) was initiated. In this study,
the effects of chemical pesticides and mBCAs (Azospirillum, Burkholderia, Clonostachys, Pseudomonas,
Streptomyces, Trichoderma, Bacillus, Beauveria, Metarhizium) on non-target microorganisms were
compared. All data derived from published and peer-reviewed studies. Investigated non-target groups were
bacteria, fungi, actinomycetes and protozoa. It was shown that the effects of mBCAs are followed by
recovery within 100 days. Initial effects caused by mBCAs can be either negative or positive. Application
of antagonists, the fungal antagonist in specific, results in initial increases of numbers of bacteria. Most
likely, the antagonists are a rich nutrient source for the resident bacterial population, resulting in rapid
increases of their numbers. The fact that initial effects are short term is in agreement with the current EU
approach that recovery should be observed within an ecological relevant period.
In those cases where actual studies are required to fulfill the data requirement for non-target soil
microorganisms, it is advised to test the most sensitive mBCA/non-target combinations.
This evaluation provides a general picture of expected effect of mBCAs on various groups of soil
microorganisms. Moreover, these analyses can be the basis for a critical discussion of the usefulness of
such information in risk assessments of new mBCAs.
A similar meta regression study is currently performed on the effects of chemical control agents and
microbial biocontrol agents on soil enzyme activities (Scheepmaker et al., in prep.).
Waiver options
A waiver can be submitted:
− If exposure of soil microorganisms is negligible or minimal.
− If a search of published scientific literature finds that effects on the soil microflora are minor and
transient.
4.4 Aquatic NTOs
If significant exposure of aquatic organisms can be expected following application,
toxicity/pathogenicity tests for fish, aquatic invertebrates and algae are required, unless it can be justified
that an assessment of effects on NTOs can be performed with information already available in the open
scientific literature. Testing of aquatic plants may be useful if the mBCA is taxonomically related to a
known plant pathogen.
Since an mBCA that reaches the water surface may tend to precipitate to the sediment it may be
useful to require studies on the effects on sediment-dwelling organisms such as epibenthic chironomid
larvae or endobenthic annelids. However, it should be emphasized that the choice of test organisms should
be verified based on the information on the biological properties (mode of action, host range) as this step is
crucial for the assessment of impact on non-target species.
ENV/JM/MONO(2012)1
53
4.4.1 Fish
General aspects
The possibility of adverse effects to fish has to be addressed adequately if exposure of surface water is
expected following application of the mBCA. This requirement is required by the EU as well as the US and
Canada.
Available test guidelines
1) US EPA provides a test guideline on freshwater fish testing in Tier I (OCSPP 885.4200).
− Test species: In cases of indirect application such as spray drift, one test species is required,
preferably rainbow trout. In cases of direct applications to water, two test species are required,
preferably rainbow trout and bluegill sunfish. Furthermore, the use of young fish is
recommended.
− Route of exposure: Test organisms are to be exposed by two routes, first via mBCA suspended
directly into the water and second via mBCA mixed with food (at least 100 times the
calculated cell density/mL in a 6–inch (15 cm) layer of water immediately following a direct
application to a 6–inch (15 cm) layer of water).
− Test concentrations: Tests are to be conducted with a MHC being 10
6
units/mL or 1000 times
the expected microbial concentration in the aqueous environment.
− Control: A negative, non dosed control group should be run concurrently with the test groups.
A control group in which the fish are exposed to sterile filtrate from production cultures
should be performed concurrently with the test groups.
− Test duration: The test duration is greater than or equal to 30 days.
2) Environment Canada recommends a test method mainly adapted from the OECD guideline No.
215 (“Fish, Juvenile Growth Test), with additional guidance from US EPA test guidelines
OCSPP 885.4200 and the ASTM “Standard guide for conducting Bioconcentration tests with
Fishes and Saltwater Bivalve Molluscs”. This approach is consistent with conditions demanded
in the US EPA guidelines.
− Test species: Test species are comparable to the US EPA guidelines.
− Type of study: Studies are to be conducted as static renewal tests.
− Testing for infectivity: Tests for infectivity are optional.
− Route of exposure: route of exposure is comparable to the US EPA guidelines.
− Test concentration: definitions of the MHC are comparable to the US EPA guidelines.
− Control: Additionally to controls required in the US EPA guidelines, the use of a non-
infectious control is strongly recommended.
ENV/JM/MONO(2012)1
54
− Observations and biological endpoints: Besides observations of fish survival, appearance and
behaviour, necropsy upon death of each fish during test and at test end, histological
investigations of selected tissues and organs have to be conducted.
− Test duration: The test duration is a minimum of 28 days.
Advantages of the Canadian test guidelines are the inclusion of the multi-concentration testing
approach, criteria for test validity and specific recommendations about experimental conditions.
Risk assessment
Risk calculation analogous to chemicals is generally regarded as less feasible for the risk assessments
of mBCAs because dose-response relationships are rarely observed in cases of pathogenic effects. If
observed effects are caused by toxicity, dose-response testing should be conducted providing reliable
ecotoxicological endpoints (LC
50
, EC
50
, NOEC/LOEC). The Canadian guidelines provide the option of
multi-concentration tests. These endpoints can be integrated in standard risk assessments similar to
chemical pesticides including the use of established safety factors. Exploring the origin of effects observed
in control groups receiving an attenuated treatment (microbe-free or non-viable microbe comprising
material from the culture system used for propagation) might be helpful, as adverse effects caused by this
treatment cannot be attributed to pathogenicity.
According to US EPA Test guidelines OCSPP 885.4200 and the Canadian guidelines, a MHC for
water of 10
6
microbial units/mL water or 1000 times the expected microbial concentration in water bodies
is defined. The MHC for exposure via food is at least 100 times the calculated cell density per millilitre in
a 6–inch (15 cm) layer of water immediately following a direct application to a 6–inch (15 cm) layer of
water. A subsequent risk calculation requiring an adequate exposure assessment is dispensable provided
that no adverse effects are observed.
In cases of pathogenic effects, these observations have to be considered and classified in context of
the overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-
cycle and biology of the mBCA, environmental conditions for survival, germination, and infection).
Waiver options
A waiver can be submitted:
- If exposure of fish is negligible or minimal.
- If a microorganism is not able to survive in surface water and sediment.
- If database searches find no reports of detrimental impacts to fish caused by the considered
microorganisms and relative species within the same subfamily or genus in connection with
sufficient information about mode of action, biology, life cycle and environmental conditions for
survival and reproduction.
ENV/JM/MONO(2012)1
55
4.4.2 Aquatic invertebrates
General aspects
The possibility of adverse effects to aquatic invertebrates has to be addressed adequately if exposure
of surface water is expected following the intended application of the mBCA. This requirement is required
by the EU as well as the US and Canada.
Available test guidelines
1) US EPA guidelines OCSPP 885.4240
− Test species: One species of benthic invertebrates should be tested in cases of indirect
exposure of surface water, e.g. spray drift of terrestrial spray applications. If the mBCA is
intended to be applied directly into water bodies, a second planktonic invertebrate has to be
tested. The selected test species should be preferably closely-related to the target host or test
organisms should be chosen likely to prey upon or scavenge the diseased target host
organisms (in case of entomopathogenic mBCAs). Larval life stages are preferred.
− Test concentration: The test substance is applied as a MHC being 10
6
units/mL or 1000 times
the expected microbial concentration in the aqueous environment and is suspended directly
into the test water.
− Control: A negative non-dosed control group as well as a control group exposed to sterile
filtrate from production cultures is to be performed concurrently.
− Observations and biological endpoints: Test organisms are examined for any behavioural,
pathogenic, or toxic effects and especially for infection or any microorganism-related effects
periodically throughout the study and at test termination.
− Test duration: The test duration is 21 days.
2) Environment Canada
Two guidelines are recommended for testing aquatic freshwater invertebrates, one using the
freshwater cladoceran Daphnia magna, the other using the larvae of freshwater midges Chironomus
tentans or Chironomus riparius. The study guidelines for D. magna are an appropriately adapted version of
the OECD guidelines 211 “Daphnia magna reproduction test:
− Test concentration: Test substances can be applied as an MHC being similar to MHC defined
in the US EPA guidelines or in a minimum of five concentrations including the MHC.
− Control: Beside negative control and sterile filtrate control (optional) groups, a non-infectious
control is strongly recommended.
ENV/JM/MONO(2012)1
56
− Observations and biological endpoints: Survival of parental daphnid and success of
reproduction is required whereas testing for infection by measurements of the microbial
concentration in whole-body homogenate is optional.
− Test duration: The test duration is 21 days.
Advantages of the OECD 211-adapted test guidelines are the presence of test validity criteria and well
established test conditions. However, it was noted that the taxonomic relatedness to the host species is not
taken into account. Thus possible pathogenic effects to aquatic invertebrates remain unrecognised when
using solely D. magna as test species. Searching appropriate databases (see below) for potentially negative
impacts of mBCAs and species being closely related to the mBCA might help to assess whether
pathogenic/toxic effects to other aquatic invertebrates are probably overlooked.
Tests with the freshwater sediment invertebrate Chironomus tentans or C. riparius may be required
when mBCAs precipitate quickly into sediment and can probably persist in the sediment for a significant
amount of time (preconditions still have to be discussed). Environment Canada provides test guidelines
adapted from EC test guidelines designed for chemical substances “Test for Survival and Growth in
Sediment Using the Larvae of Freshwater Midges (Chironomus tentans or Chironomus riparius)”.
− Test organisms are third instar C. tentans and first instar C. riparius.
− Test substances have to be mixed in both freshwater and sediment.
− A single-concentration test using the MHC defined as above or a multi-concentration test with a
minimum of five concentrations including the MHC is possible.
− A negative control, a positive (chemical) control must be determined whereas the concurrent
performance of a non-infectious control is strongly recommended and a sterile filtrate control is
optional.
− Observations include numbers of midge larvae on sediment surface, and assessing their behaviour,
appearance and survival. The mean dry weight has to be measured. Testing for infection by
measuring the microbial concentration in whole-body homogenate is optional.
− The test guidelines provide specific experimental conditions and validity criteria.
Risk assessment
A risk calculation analogous to chemicals is generally regarded as less feasible for the risk
assessments of mBCAs because dose-response relationships are rarely observed in cases of pathogenic
effects. If observed effects are caused by toxicity, dose-response testing should be conducted providing
reliable ecotoxicological endpoints (LC
50
, EC
50
, NOEC/LOEC). The Canadian guidelines provide the
option of multi-concentration tests. These endpoints can be integrated in standard risk assessments similar
to chemical pesticides including the use of established safety factors. Exploring the origin of effects
observed in control groups receiving an attenuated treatment (microbe-free or non-viable microbe
comprising material from the culture system used for propagation) might be helpful, as adverse effects
caused by this treatment cannot be attributed to pathogenicity. According to US EPA Test guidelines
ENV/JM/MONO(2012)1
57
OCSPP 885.4240 and the Canadian guidelines, a MHC for water of 10
6
microbial units/mL water or 1000
times the expected microbial concentration in water bodies is defined. A subsequent risk calculation
requiring an adequate exposure assessment is dispensable provided that no adverse effects are observed. In
instances of pathogenic effects, these observations have to be considered and classified in context of the
overall-knowledge about the mBCA (physiological and ecological host range, mode of action, life-cycle
and biology of the mBCA, environmental conditions for survival, germination, and infection).
Waiver options
A waiver can be submitted:
− If exposure of aquatic invertebrates is negligible or minimal.
− In case of non-entomopathogenic mBCA, if database searches find no reports of detrimental
impacts of the considered microorganisms and relative species within the same subfamily or
genus to aquatic as well as terrestrial (soil- or leaf-dwelling invertebrates) in connection with
sufficient information about mode of action, biology, life cycle and environmental conditions for
survival and reproduction.
− If a microorganism is not able to survive in surface water and sediment.
4.4.3 Aquatic plants (including algae)
General aspects
The possibility of adverse effects to aquatic plants has to be addressed adequately if exposure of
surface water is expected following the intended application of the mBCA. This requirement is required by
the EU as well as the US and Canada.
Available test guidelines
1) Commission Regulation (EU) No 544/2011
According to this regulation, information on effects on algal growth, growth rate and capacity to
recover must be reported and effects on aquatic plants other than algae must be reported for mBCAs by the
applicant. No definite test species is defined. However, standard test species for testing chemicals
according OECD test guidelines 201/221 are green algae Pseudokirchneriella subcapitata (formerly
known as Selenastrum capricornutum), Desmodesmus subspicatus (formerly known as Selenastrum
subspicatus), Cyanobacteria Anabaena flos-aquae and Synechococcus leopoliensis and the diatom
Navicula pelliculosa. The gibbous duckweed, Lemna gibba, or the common duckweed, L. minor, are used
as a standard test species representative for macrophytic aquatic plants. In rare cases where concern exists
that the fast-growing Lemna species may underestimate risks, other aquatic macrophytes such as the rooted
dicotyle macrophyte Myriophyllum aquaticum (parrot feather watermilfoil) is commonly used as test
species (optionally conducted with or without sediment). However, an internationally recognised test
method for the latter species is currently lacking.
ENV/JM/MONO(2012)1
58
2) US EPA test guidelines OCSPP 885.4300 “Nontarget Plant Studies Tier I”
Within these guidelines, only a short paragraph refers to aquatic plants and it addresses the issue of
appropriate test species. Accordingly, the green algae Pseudokirchneriella subcapitata (formerly known as
Selenastrum capricornutum), the blue-green alga Anabaena flos-aquae and the duckweed L. gibba are to
be examined. Furthermore, the marine diatom Skeletonema costatum (for application near coasts) and
another freshwater diatom (e.g. Navicula pelliculosa) must be tested. According to the US guidelines,
testing is required for aquatic uses or in cases of expected disseminations of the mBCA to aquatic
ecosystems. Another precondition for test requirements is the potential of the mBCA to survive in natural
water bodies. No test-specific biological methods are provided for these species in this or other Series 885
test guidelines.
3) Environment Canada test guidelines
These guidelines prefer the aquatic duckweed Lemna sp. as test species. In the Canadian guidance
document, an adapted version of Environment Canada duckweed growth inhibition test for chemicals.
Lemna minor as test organism is to be exposed to the mBCA for 7 days with a renewal of each test
concentration at least twice, on Days 3 and 5 of the test. Test substances can be applied as an MHC (10
6
units/mL or 1000 times the expected microbial concentration in the aqueous environment) or in a minimum
of five concentrations including the MHC. Growth rates are calculated on the basis of measured number of
fronds and dry weight at test start and test end. Plant appearance has to be observed at start and end of the
test. Testing for infectivity based on measured concentrations of the mBCA in whole-body homogenates of
L. minor is optional. Beside negative control and sterile filtrate control (optional) groups, a non-infectious
control is strongly recommended. Detailed information on test conditions is given as well as criteria for
validity. One disadvantage of this method is the shortness of test duration as possible pathogenic effects
might not manifest during 7 days of exposure. Extending the duration of the test might be a logical solution
but increasing the exposure period requires a change of validity criteria. Alternatively, an adequately
adapted version of the OECD test guidelines No. 221 may also be accepted according to the Canadian
guidance document. With a limited test duration of only 7 days, these guidelines have the same
shortcoming as the Canadian guidelines and possibly require the same determination of new validity
criteria.
In contrast to European data requirements, studies assessing the effects of a mBCA to algal growth are
usually not required in the US and Canada. US EPA Biopesticide Division staff scientists had commented
on February 8, 2011 as follows: “Although the US guidelines 885.4300 mention that aquatic algae may be
studied, we are not aware of any case where we have asked for algae to be tested. It would be very unusual
to find a microbial pesticide that could present a persistent effect on a beneficial alga.”
As stated in the Canadian guidance document, the adoption of established standard tests for algae,
e.g., according to OECD Test guidelines No. 201, is difficult since algal studies are not compatible with
static renewal test methods. When using relatively high maximum hazard doses/concentrations, static
renewals are often required to maintain water quality and constant exposure. Lowering the initial test
concentration may circumvent this problem but this concentration might be regarded as insufficient when
examining possible pathogenic effects to algae.
ENV/JM/MONO(2012)1
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Risk assessment
A risk calculation analogous to chemicals is generally regarded as less feasible for the risk assessment
of mBCAs because dose-response relationships are rarely observed in cases of pathogenic effects. If
observed effects are caused by toxicity, dose-response testing should be conducted providing reliable
ecotoxicological endpoints (LC
50
, EC
50
, NOEC/LOEC). The Canadian guidelines provide the option of
multi-concentration tests. These endpoints can be integrated in standard risk assessments similar to
chemical pesticides including the use of established safety factors. Exploring the origin of effects observed
in control groups receiving an attenuated treatment (microbe-free or non-viable microbe comprising
material from the culture system used for propagation) might be helpful, as adverse effects caused by this
treatment cannot be not due to pathogenicity. According to the Canadian guidelines, a MHC for water of
10
6
microbial units/mL water or 1000 times the expected microbial concentration in water bodies is
defined. A subsequent risk calculation requiring an adequate exposure assessment is dispensable provided
that no adverse effects are observed. In instances of pathogenic effects, these observations have to be
considered and classified in context of overall-knowledge about the mBCA (physiological and ecological
host range, mode of action, life-cycle and biology of the mBCA, environmental conditions for survival,
germination and infection).
Waiver options
A waiver can be submitted:
− If exposure of aquatic plants is negligible or minimal.
− In case of mBCAs with no intended herbicidal uses, if database searches find no reports of
detrimental impacts to plants by the considered microorganisms and relative species within the
same genus in connection with sufficient information about mode of action, biology, life cycle
and environmental conditions for survival and reproduction.
5. Refinement options (BOX 6 of decision scheme)
In case the risks are deemed to be too high, the risks can be refined by performing Tier 2 or even
Tier 3 experiments. This possibility for refinement is common in the risk assessment of chemical
pesticides. For mBCAs, this option will hardly ever be used. US EPA Biopesticides Division staff
scientists had commented on January 29, 2010, that they have never needed microbial pesticide NTO
studies that would give an LD
50
. Tier 2 level data that are required only if toxic or pathogenic effects are
seen in the Tier I tests have also not been required to date. In contrast to the situation in the US, requests of
higher Tier (semi-field) tests may have occurred more frequently in the EU registration process, for
instance a bumble bee study with an mBCA under greenhouse conditions was submitted in the context of
national approval in Germany.
ENV/JM/MONO(2012)1
60
6. Mitigation options (BOX 7 of decision scheme)
Another option to reduce risks is the possibility of mitigation. Well-known possibilities used for
chemical pesticides are
• Reducing application rates. This option seems to be impracticable since the mode of action of
many mBCAs can be pathogenic and reduced application rates may come into conflict with the
efficacy of a product. No dose-effect correlation is available with such mBCAs and thus,
reduction of applied concentrations is not useful.
• Using drift reducing nozzles. This option can be used when risks are expected to be caused by
spray drift to aquatic organisms or to NTO in adjoining off-crop areas.
• Establishing buffer zones along areas of surface water or off-crop areas to reduce exposure of
surface water to spray drift.
• Rejecting an intended use with unacceptable risks. Although this option is actually not considered
a real mitigation option, this step will eventually be taken when no other options are available.
• Preventing applications to flowering crops to avoid exposure of pollinators. This is a specific
measure to avoid risks for bees and other wild pollinators.
7. Issues to be solved in the near future
In order to harmonise risk assessment procedures among different regulatory agencies, the following
issues that need to be solved in the near future, are highlighted:
• Assessment of fungal metabolites. A publication is in preparation by Butt et al. (in prep.) that
categorises metabolites/toxins and their effects on NTOs from the literature. Risk strategies will
be proposed.
• Other options to improve the qualitative risk assessment of mBCAs, such as better insight into
fate and behaviour of mBCAs.
ENV/JM/MONO(2012)1
61
Acknowledgements
The authors would like to thank the experts from several countries/organisations (Australia, Austria,
Canada, Denmark, France, Germany, IBMA
4
, JRC
5
, the Netherlands, Switzerland, Sweden, United
Kingdom, USA) for giving valuable advices and suggestions and for making improvements on the text and
decision scheme.
4
International Biocontrol Manufacturers’ Association
5
European Commission Joint Research Centre, Institute for Health and Consumer Protection, Chemical Assessment
and Testing Unit
ENV/JM/MONO(2012)1
62
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APPENDIX: SUGGESTIONS FOR RELEVANT DATABASES
Non-target organisms (including birds and mammals)
German Institute of Medical Documentation and Information (DIMDI)
http://www.dimdi.de/static/en/index.html (last accessed, April 27, 2011)
Terrestrial plants
University of Bonn provides a Plant Pathology Internet Guidebook (PPIGB)
http://www.pk.uni-bonn.de/ppigb/menu.htm (last accessed, April 27, 2011)
Aquatic organisms
http://www.diplectanum.dsl.pipex.com/purls/host.htm (last accessed, April 27, 2011)
http://www.fao.org/DOCREP/003/X9199E/X9199E03.htm (last accessed, April 27, 2011)
Terrestrial invertebrates
http://cricket.inhs.uiuc.edu/edwipweb/edwipabout.htm (last accessed, April 27, 2011)
http://arthropodenkrankheiten.jki.bund.de/index_e.php (last accessed, April 27, 2011)
