E5080 / E0323
Ecotoxicology
Ecotoxicology in Legislative Praxis
Jakub Hofman
1

Use in praxis
2


Use of bioassays in praxis
§„praxis“ = environmental protection, regulation, legislation ...
§
§use of bioassays depends on: are they officially required by legislation/regulation??
§this is changing in time! – legislation development
§
§bioassays are used in both prospective (MORE) and retrospective (LESS) approaches
§
§in prospective approach, very often in concept of RISK ASSESSMENT
3

Use of bioassays in praxis
§mainly for hazard identification / characterization of chemical substances including important
specific groups of
pesticides, biocides, pharmaceuticals, cosmetics ...
properties of same chemical (unique CAS No.), including ecotox, are constant
including the setting of limits for pollution
use of bioassays in legislation
§
§less (but increasing) for complex samples (materials) in prospective approach (e.g. before their
use) – wastes, sewage sludge, fertilizers etc.
each sample is different (e.g. sewage sludge sampled different days) – new testing needed
legislative use is rare (only national)
§
§less (but increasing) for complex environmental samples (water, soil, sediment ...) in
retrospective approach (diagnostics)
legislative use is rare (only national)
4

Risk assessment concept
§ecotoxicological bioassays are used to quantify HAZARD (i.e. kind of property of chemicals /
materials ...)
§
§RISK is then result of combination of hazard and exposure – probability that hazard will occur
5

Risk assessment concept
HAZARDS
§are also physico-chemical and toxicological and other harmful properties
§ecotoxicological hazards are derived from the results of bioassays:
1.direct use of LCx/ECx, NOEC, LOEC ...
2.tuning these values towards PNEC (Predicted No Effect Concentration), often by the most sensitive
result + using so called uncertainty factors
§
6

§PNEC derivation from ecotoxicity data
§objective is to protect real ecosystems, safe concentration
è need to extrapolate bioassay results to be valid for ecosystems
§how much information we have? à uncertainty factors, 1, 10, 100, 1000
Risk assessment concept
Species sensitivity distribution (SSD)
HC5 = 95% protection level
Extrapolation
PNEC (limits, EQS)
7
ECHA (2008): Guidance on information requirements and chemical safety assessment. Chapter R.10:
Characterisation of dose [concentration]-response for environment.
https://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-
assessment




Risk assessment concept
RISK
§combines by some way
EFFECT data (LCx/ECx, NOEC, NOAEL, PNEC etc.)
with EXPOSURE (Predicted Environmental Concentration – PEC, Measured Environmental Concentration –
MEC, etc.)
§depends on situation (factors)
§can be mitigated and managed
§
9




Risk assessment concept
§human health risk assessment (HHRA) – human health as endpoint
§ecological risk assessment (EcoRA, ERA) – ecosystems, non-human organisms and populations as
endpoints
§environmental risk assessment (ERA) – unclear term, involves both
§
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Battery of bioassays
12


Battery of bioassays
§different organisms give different response to toxicants (sensitivity, bioavailability, exposure,
metabolization ...) and also different conditions and factors in different bioassays
§none single bioassay can give complete response !!
§
§usually it is very good to combine more bioassays together = battery
§more and „better“ (e.g. chronic preferably to acute) bioassays used è lower uncertainty of the
finally derived hazard and risk of the tested chemical substance (lower „uncertainty factors“ in
risk assessment used)
§
§selection should follow defined final goal of the ecotoxicity testing
13

Battery of bioassays
§different approaches (different purposes/legislation needs) how to do it:
1.combine bioassays based on some principle
e.g. golden rule = combine trophic levels: a) producer (plants), b) consumer (invertebrates,
vertebrates), c) destruent (microbes)
+ also combine different routes of exposure, test duration etc.
2.mix scientific principle (1) with practical demands (low number of tests, quick, cheap,
standardized tests ... etc.) - most common batteries in praxis:
oalgae / D. magna / fish for aquatic environment
oearthworm (enchytraeid/springtail) / plant for soil environment
3.tiered approach = based on the results of previous tier its decided if next (and what next)
testing will be done:
fast screening bioassays à standardized acute bioassays à chronic/prolonged studies à field tests
(so called higher tiers)
§
14

Battery of bioassays
Example: ISO guidelines showing how to combine bioassays for testing soils and soil-like materials
15
ISO 15799: Guidance on the ecotoxicological characterization of soils and soil materials
ISO 17616: Guidance on the choice and evaluation of bioassays for ecotoxicological characterization
of soils and soil materials

Battery of bioassays
Example: Battery prepared for testing ecotoxicity of wastes
§current situation:
ohttps://ec.europa.eu/environment/waste/index.htm
oDirective 2008/98/EC on waste (Waste Framework Directive)
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02008L0098-20180705
oH14 Ecotoxicity is one of the hazardous properties of wastes
oit is assessed based on the analysis of wastes and database data
ono real testing with bioassays
16

Battery of bioassays
Example: Battery prepared for testing ecotoxicity of wastes
§there was huge ringtest focused on selection of appropriate bioassays
for testing wastes (13 countries, 59 labs, 500 kg of waste tested ...)
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Ecotoxicological Characterization of Waste Image Image Image

Battery of bioassays
Example: Czech legislation for waste
§H14 Ecotoxicity hazardous property determination (decree 376/2001):
owaste eluate 10 L/kg (EN 12457-4)
oif any of these tests has LC(EC,IC)50 ≤ 10 ml/L, the waste sample is H14
-ISO 6341 Daphnia magna acute
-ISO 8692 Algae growth
-ISO 7346-2 Fish acute test
-mustard Sinapsis alba germination and root growth
o
o
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Battery of bioassays
Example: Czech legislation for waste
§ecotoxicity testing for waste use on land surface or landfilling (decree 294/2005):
owaste eluate 10 L/kg (EN 12457-4)
ouse of waste for for closing landfills, forming protective layer, sealing layer or reclamation
layer of landfill is allowed if:
-Fish acute test: no mortality or behavior change
-Daphnia magna acute test: < 30% imobilization compatred to control
-Algae growth test: < 30% inhibition compared to control
-Sinapis alba germination and root elongation: < 30% inhibition compared to control
19

Battery of bioassays
Example: Czech legislation for dredged sediments
§before application on agricultural land
§limits are given for concentration of pollutants
§ecotoxicity bioassays and criteria are given
o
20
21_%20Excavation_jpg Potworms (Enchytraeids doerjesi) Example of light coloured springtail
(Folsomia candida) Nitrifying bacteria isolated from landfill leakage
ISO 16387
ISO 11267
11269-1
ISO 15685
všechny koncentrace kompost

Examples of ecotoxicity bioassays use in legislation
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Testing of chemical substances
§mainly „chemicals“ – they have CAS No.
§> 100 millions of chemicals known
§> 100 000 we produce and use
§HPVC = high production volume chemicals > 1000 t/y production
§„priority pollutants“ – e.g. Water Framework Directive, Stockholm Convention ...
22
https://echa.europa.eu/cs/information-on-chemicals/ec-inventory

Testing of chemical substances
§developed legislation (EU, US ...) requires determination of ecotoxicity (+ lot of other
properties – phys-chem, toxicity ...) before marketing and use of chemicals
§what does it mean:
oprecise testing using standardized (OECD ...) bioassays in accredited labs with all validity
requirements and quality assurance / quality control (QA/QC) measures
oresults of ecotoxicity clearly expressed avoiding any confusion or missinterpretation:
-what parameter – ECx/LCx, NOEC ...
-which units – mg/L, mol/L, mg/kg, % ...
-specify clearly possible variants in procedure: i.e. D. magna, 24h, LC50 = XY mg/L
-...
oresults used for risk assessment of chemicals, labelling, decision making, authorizations,
restrictions, management etc.
o
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Example 1
Labelling chemicals - GHS
24
Informační centrum OSN

GHS
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Globally Harmonised System of Classification and Labelling of Chemicals
(https://unece.org/about-ghs)
§„hazardous to aquatic environment“
§
§
§
§
§
§
§OECD tests preferred and GLP (good laboratory praxis) should be followed
oacute aquatic toxicity
ochronic aquatic toxicity
opotential for or actual bioaccumulation
odegradation (biotic or abiotic) for organic chemicals
§
§
o
Informační centrum OSN

Acute classification categories 1 to 3
§defined on the basis of the acute toxicity data only (EC50 or LC50)
ofish 96h (OECD 203)
oDaphnia 48h (OECD 202)
oalgae growth inhibition test (OECD 201)
o
GHS
26
United Nations (2019): Globally Harmonized System of Classification and Labelling of Chemicals
(GHS). Eighth revised edition. https://unece.org/ghs-rev8-2019
Informační centrum OSN

Informační centrum OSN
Chronic classification categories 1 to 3
§based on tiered approach è
1.
1.available information on chronic toxicity
-fish early life stage (OECD 210)
-daphnia reproduction (OECD 211)
-algae growth inhibition test (OECD 201)
-NOEC or ECx (e.g. EC10)
2.
2.or acute toxicity data combined with
environmental fate data
o
GHS
27
United Nations (2019): Globally Harmonized System of Classification and Labelling of Chemicals
(GHS). Eighth revised edition. https://unece.org/ghs-rev8-2019

chronic classification
                 data
available                                                                      data unavailable
GHS
28
United Nations (2019): Globally Harmonized System of Classification and Labelling of Chemicals
(GHS). Eighth revised edition. https://unece.org/ghs-rev8-2019
Informační centrum OSN

GHS
29
United Nations (2019): Globally Harmonized System of Classification and Labelling of Chemicals
(GHS). Eighth revised edition. https://unece.org/ghs-rev8-2019
aquatic hazards summary
Informační centrum OSN

Example 2
Chemicals – in EU
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REACH
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§https://ec.europa.eu/environment/chemicals/index_en.htm
§
§Regulation 1907/2006, concerning the Registration, Evaluation, Authorisation and Restriction of
Chemicals (REACH)
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02006R1907-20200824
§
§responsible is European Chemicals Agency (ECHA) https://echa.europa.eu/home
§

REACH
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§REACH allows to produce and use in EU only such chemical substances that are registered, evaluated
and authorized in EU (does not apply to substances with production or import below 1 t/y)
§the risk they may pose to human health and the environment must be characterized and they must be
classified accordingly
§registration is based on technical dossier, which includes data on the properties of the
registered substance determined by defined testing procedures
Regulation No 440/2008, laying down test methods pursuant to Regulation No 1907/2006 (REACH)
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02008R0440-20191016
§for substances with production or import > 10 t/y also Chemical Safety Report (CSR) must be
prepared involving evaluation of hazards and risks related to production and use
§
§concept: with increasing volume of production (1, 10, 100 and 1000 t/y) number of needed data
(methods, tests, including ecotoxicity bioassays) is increasing

REACH
all substances > 1 t/y
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REACH
all substances > 10 t/y
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Obsah obrázku láhev, interiér, několik Popis byl vytvořen automaticky

REACH
all substances > 100 t/y
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REACH
all substances > 100 t/y
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IMG_7022_al 62 Arthrobacter | coryneforms-2

REACH
all substances > 1000 t/y
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62 plant01.jpg (54.086 Byte)
Japanese quail - Wikipedia

REACH
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Regulation No 440/2008, laying down test methods pursuant to Regulation No 1907/2006 (REACH)
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02008R0440-20191016
lists the methods including procedures
often follows OECD guidelines

REACH
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§chemical safety assessment ~ risk assessment principle = combination of HAZARD and EXPOSURE
§detailed guidelines for each phase
https://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-
assessment

REACH
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Environmental hazard assessment
§PNEC (predicted no effect concentration) for each environmental compartment
§concentration below which adverse effects on ecosystems will not occur
§derived on basis of available information on toxicity to species from relevant environments, i.e.
toxicity test endpoints (LC50s or NOECs/EC10s), using appropriate assessment factors (AF) to
extrapolate from single-species laboratory data to a multi-species ecosystem, to address:
ointra- and inter-laboratory variation of toxicity data
ointra- and inter-species variations (biological variance)
oshort-term to long-term toxicity extrapolation
olaboratory data to field impact extrapolation
o...

REACH
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Environmental hazard assessment
§PNEC determination
ECHA (2011): Guidance on information requirements and chemical safety assessment
Part B: Hazard assessment.
https://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-
assessment
ECHA (2008): Guidance on information requirements and chemical safety assessment. Chapter R.10:
Characterisation of dose [concentration]-response for environment.
https://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-
assessment

REACH
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Environmental hazard assessment
§PNEC determination
ECHA (2011): Guidance on information requirements and chemical safety assessment
Part B: Hazard assessment.
https://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-
assessment
What will be PNECwater ???

REACH
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Environmental hazard assessment
§PNEC determination
ECHA (2011): Guidance on information requirements and chemical safety assessment
Part B: Hazard assessment.
https://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-
assessment

REACH
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Exposure assessment
§PEC (predicted environmental concentration) for each environmental compartment
§models based on production of chemicals,
§
§

REACH
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Environmental risk characterization
§
PEC > PNEC
(HQ>1)
PEC < PNEC
(HQ<1)

REACH
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Example CSR report
https://echa.europa.eu/support/practical-examples-of-chemical-safety-reports
from page 53

REACH
§some data available in EC inventory and IUCLID 6
§but not full Chemical Safety Reports
§example: https://echa.europa.eu/substance-information/-/substanceinfo/100.100.840
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https://echa.europa.eu/cs/information-on-chemicals/ec-inventory
Evropská unie – Wikipedie
https://iuclid6.echa.europa.eu/project-iuclid-6

REACH
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§for chemicals, producers and importers must provide information about risks related to substance
use, based on Regulation No 1272/2008, on the classification, labelling and packaging of substances
and mixtures (CLP Regulation)
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02008R1272-20201114
§information on risks is provided in agreement with Globally Harmonised System of Classification
and Labelling of Chemicals (https://unece.org/about-ghs)
§among plenty of other data and properties, ecotoxicity is evaluated
The GHS hazard pictograms for free download
GHS

Example 3
Pesticides - Plant protection products (PPP) – in EU
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Obsah obrázku tráva, exteriér, obloha, nákladní auto Popis byl vytvořen automaticky
Plant protection products (PPP)
§pesticides are very hazardous chemicals!
§Hofman analysis 11/2018: 204 / 441 approved AS have some classification according to EC 1272/2008
(CLP) - majority AS have 3 or more (max 9 – e.g. Spiroxamin), in total there are 723 hazard
statements found for all approved AS
o12 acute toxicity 1
o28 carcinogenicity 2
o153 acute aq. tox. 1
o11 reprod. tox. 1B
Image Image Image Image Image Image Image Image
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Plant protection products (PPP)
§ecotoxicity assessment is related to the expected behavior and impacts after PPP application
§lot of non-target biota under risk of undesired negative impact
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Plant protection products (PPP)
§lot of non-target biota under risk of undesired negative impact, including indirect effects
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Plant protection products (PPP)
§https://ec.europa.eu/food/plant/pesticides_en
§www.efsa.europa.eu/en/topics/topic/pesticides
§
§in EU, EFSA is responsible for the authorization of PPP
§Regulation 1107/2009, concerning the placing of plant protection products on the market
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02009R1107-20191214
§Regulation 546/2011, uniform principles for evaluation and authorisation of PPP
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02011R0546-20180524
§
§overview of whole process:
https://www.efsa.europa.eu/en/interactive-pages/pesticides-authorisation/PesticidesAuthorisation
https://ec.europa.eu/assets/sante/food/plants/pesticides/lop/index.html
§
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Plant protection products (PPP)
§the whole system is divided to:
o
§active substances approval - done for whole EU
a.s. – substance having action against harmful organisms
§
§authorization of PPP – done in member states (zones)
mix of a.s. and other components: safeners (eliminate or reduce phytotoxic effects of PPP),
synergists (enhance a.s. effect), co-formulants (other components), adjuvants (added to PPP by
end-user)
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Approval of a.s.
§Regulation 283/2013, setting out the data requirements for active substances
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02013R0283-20141117
§Regulation 540/2011, list of approved active substances
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02011R0540-20201126
§EU pesticide database
https://ec.europa.eu/food/plant/pesticides/eu-pesticides-db_en
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dossier
DAR/RAR
peer review
conclusion
decision
•its decided if a.s. is approved or not, what conditions and labelling (CLP) and also limits for
residues are set (MRL)
•
•its valid for 10 year and then re-assessment

Approval of a.s.
§Regulation 283/2013 defines what data, including ecotoxicological, are required for approval of
active substance
§ANNEX section 8
§some details provided on the bioassays performance, but more in specific guideline documents
56
8.1.
Effects on birds and other terrestrial vertebrates
8.3.
Effect on arthropods
8.1.1.
Effects on birds
8.3.1.
Effects on bees
8.1.1.1.
Acute oral toxicity to birds
8.3.1.1.
Acute toxicity to bees
8.1.1.2.
Short-term dietary toxicity to birds
8.3.1.1.1.
Acute oral toxicity
8.1.1.3.
Sub-chronic and reproductive toxicity to birds
8.3.1.1.2.
Acute contact toxicity
8.1.2.
Effects on terrestrial vertebrates other than birds
8.3.1.2.
Chronic toxicity to bees
8.1.2.1.
Acute oral toxicity to mammals
8.3.1.3.
Effects on honeybee development and other honeybee life stages
8.1.2.2.
Long-term and reproductive toxicity to mammals
8.3.1.4.
Sub-lethal effects
8.1.3.
Active substance bioconcentration in prey of birds and mammals
8.3.2.
Effects on non-target arthropods other than bees
8.1.4.
Effects on terrestrial vertebrate wildlife (birds, mammals, reptiles and amphibians)
8.3.2.1.
Effects on Aphidius rhopalosiphi
8.1.5.
Endocrine disrupting properties
8.3.2.2.
Effects on Typhlodromus pyri
8.2.
Effects on aquatic organisms
8.4.
Effects on non-target soil meso- and macrofauna
8.2.1.
Acute toxicity to fish
8.4.1.
Earthworm — sub-lethal effects
8.2.2.
Long-term and chronic toxicity to fish
8.4.2.
Effects on non-target soil meso- and macrofauna (other than earthworms)
8.2.2.1.
Fish early life stage toxicity test
8.4.2.1.
Species level testing
8.2.2.2.
Fish full life cycle test
8.5.
Effects on soil nitrogen transformation
8.2.2.3.
Bioconcentration in fish
8.6.
Effects on terrestrial non-target higher plants
8.2.3.
Endocrine disrupting properties
8.6.1.
Summary of screening data
8.2.4.
Acute toxicity to aquatic invertebrates
8.6.2.
Testing on non-target plants
8.2.4.1.
Acute toxicity to Daphnia magna
8.7.
Effects on other terrestrial organisms (flora and fauna)
8.2.4.2.
Acute toxicity to an additional aquatic invertebrate species
8.8.
Effects on biological methods for sewage treatment
8.2.5.
Long-term and chronic toxicity to aquatic invertebrates
8.9.
Monitoring data
8.2.5.1.
Reproductive and development toxicity to Daphnia magna


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


8.2.5.3.
Development and emergence in Chironomus riparius


8.2.5.4.
Sediment dwelling organisms


8.2.6.
Effects on algal growth


8.2.6.1.
Effects on growth of green algae


8.2.6.2.
Effects on growth of an additional algal species


8.2.7.
Effects on aquatic macrophytes


8.2.8.
Further testing on aquatic organisms


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Authorization of PPP
§Regulation 284/2013, setting out the data requirements for PPP
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02013R0284-20150917
§example of PPP database from CR
http://eagri.cz/public/app/eagriapp/POR/Vyhledavani.aspx?type=0&vyhledat=A&stamp=1609840149173
§
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Authorization of PPP
§Regulation 284/2013 defines what data, including ecotoxicological, are required for PPP
authorization
§ANNEX section 10
§some details provided on the bioassays performance, but more in specific guideline documents
§testing necessary where PPP toxicity cannot be predicted on the basis of data on AS
§aim = to demonstrate PPP is more toxic than AS (bridging studies or limit test may be sufficient);
if yes, definitive testing is required
§
58
10.1.
Effects on birds and other terrestrial vertebrates
10.4.
Effects on non-target soil meso- and macrofauna
10.1.1.
Effects on birds
10.4.1.
Earthworms
10.1.1.1.
Acute oral toxicity to birds
10.4.1.1.
Earthworms — sub-lethal effects
10.1.1.2.
Higher tier data on birds
10.4.1.2.
Earthworms — field studies
10.1.2.
Effects on terrestrial vertebrates other than birds
10.4.2.
Effects on non-target soil meso- and macrofauna (other than earthworms)
10.1.2.1.
Acute oral toxicity to mammals
10.4.2.1.
Species level testing
10.1.2.2.
Higher tier data on mammals
10.4.2.2.
Higher tier testing
10.1.3.
Effects on other terrestrial vertebrate wildlife (reptiles and amphibians)
10.5.
Effects on soil nitrogen transformation
10.2.
Effects on aquatic organisms
10.6.
Effects on terrestrial non-target higher plants
10.2.1.
Acute toxicity to fish, aquatic invertebrates, or effects on aquatic algae and macrophytes
10.6.1.
Summary of screening data
10.2.2.
Additional long-term and chronic toxicity studies on fish, aquatic invertebrates and sediment
dwelling organisms
10.6.2.
Testing on non-target plants
10.2.3.
Further testing on aquatic organisms
10.6.3.
Extended laboratory studies on non-target plants
10.3.
Effects on arthropods
10.6.4.
Semi-field and field tests on non-target plants
10.3.1.
Effects on bees
10.7.
Effects on other terrestrial organisms (flora and fauna)
10.3.1.1.
Acute toxicity to bees
10.8.
Monitoring data
10.3.1.1.1.
Acute oral toxicity


10.3.1.1.2.
Acute contact toxicity


10.3.1.2.
Chronic toxicity to bees


10.3.1.3.
Effects on honey bee development and other honey bee life stages


10.3.1.4.
Sub-lethal effects


10.3.1.5.
Cage and tunnel tests


10.3.1.6.
Field tests with honeybees


10.3.2.
Effects on non-target arthropods other than bees


10.3.2.1.
Standard laboratory testing for non-target arthropods


10.3.2.2.
Extended laboratory testing, aged residue studies with non-target arthropods


10.3.2.3.
Semi-field studies with non-target arthropods


10.3.2.4.
Field studies with non-target arthropods


10.3.2.5.
Other routes of exposure for non-target arthropods


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PPP exposure assessment
exposure is expressed by different ways:
§directly as application rate (e.g. g a.s. / ha)
oinitial, maximum or modifications
§as concentrations in various compartments
oPredicted Exposure Concentration – PEC (PECs, PECsw ...)
opredicted by different environmental fate models
e.g. https://esdac.jrc.ec.europa.eu/projects/focus-dg-sante
e.g. https://www.pesticidemodels.eu/swash/home
= models of transport and behavior of PPP in the environment
(drift, sorption, degradation, mobility, accumulation ...) in time
§dietary daily intake (DDD) – for birds and mammals
§
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Example of PECsoil calculation
§The application rate of Roundup Superhero plant protection product to the oilseed-rape field is 4
L per ha. The concentration of glyphosate in the product is 400 g/L. Half of the glyphosate amount
applied is retained by the oilseed-rape plants and never come to the soil. You also know the
following: the layer of soil considered for the calculations is 5 cm (in this layer glyphosate is
homogenously distributed and does not go deeper); density of wet soil is 2 kg/L; the soil has 20%
water by weight basis; DT50 (half-life) of glyphosate in the soil is 15 days.
§What will be the soil concentration (in mg/kg dry weight) after 30 days from the application
(PECsoil,30d)? Explain the reasons for your answer in detail; if any calculations, show them
clearly.
§
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Example of PECsoil calculation
§Application rate is 4 L per ha. Concentration of glyphosate in the product is 400g/L. That means
in 4 L are 1600 g Glyphosate.
§Half of the glyphosate is retained in the oil seed rape plants half goes to the soil. That means
800 g Glyphosate go into the soil 800g to the plant. 1600g:2 = 800g
§The layer of soil considered is 5 cm = 0,05 m
§The plant protection is sprayed on 1 ha = 10 000 m2
§The volume of land where the pesticide is distributed 10 000m2 x 0,05 m (hight) à V = 500 m3
§500 m3 = 500 000 dm3  = 500 000 L
§Density of wet soil is 2 kg/L à 2 kg/L x 500 000 L = 1 000 000 kg soil
§20% is water à 800 000 kg dry soil
§800 g glyphosate goes into 800 000 kg dry soil
§800 000 mg / 800 000 kg dry soil = 1 mg/kg dry soil
§After 15 days the concentration will be 0,5 mg glyphosate per kg dry soil
§After 30 days the concentration will be 0,25 mg glyphosate per kg dry soil
§
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PPP ecotoxicity and risk assessment
§assessment of (eco)toxicity and acute / short-term / long-term risks for:
obirds and other terrestrial vertebrates
oaquatic organisms (fish, inventebrates, algae, plants, sediment organisms)
obees
onon-target arthropods
oearthworms
osoil macro- and mesofauna (other than  earthworms)
osoil microorganisms (C mineralization, N mineralization)
onon target plants
obiological methods of sewage treatment
§
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PPP ecotoxicity and risk assessment
§fundamental principle: risk assessment = combining effects and exposures
omost often as TER = toxicity/exposure ratio = ratio of effect endpoint and estimated exposure
obut for bees or non-target arthtropods, hazard quotient (HQ) - ratio between exposure and toxicity
(usually in units of g a.s. / ha)
§used endpoints (LD50, NOEL, LC50, EC50, NOEC ... acute, short-, or long-term) and units (e.g. in
body or in environment, initial, long- or short-term ...) depend on the specific bioassay and
results
§the most sensitive organism used in the tests has key influence on the final decisions
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PPP ecotoxicity and risk assessment
§TER is compared to trigger (= Assessment Factor = Safety Factor)
§these are laid down in Regulation 546/2011 and Guidance Documents
§example for birds and mammals (tier 1):
§
§
§example for aquatic organisms (tier 1):
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PPP ecotoxicity and risk assessment
Regulation 546/2011: no authorisation shall be granted if:
§birds and other non-target terrestrial vertebrates
oacute and short-term TER < 10 (using LD50) or long-term TER < 5
osecondary poisoning  from food (fish, earthworms)
§aquatic organisms
ofish and Daphnia TER < 100 for acute exposure and < 10 for long-term exposure
oalgal growth inhibition TER < 10
oBCF > 1000 for readily biodegradable or > 100 for not readily biodegradable AS
§bees
ohazard quotient for oral or contact exposure > 50
§beneficial arthropods other than honeybees
o> 30 % of test organisms affected in lethal or sublethal laboratory tests of max application rate
§earthworms
olong-term TER < 5
§soil microorganisms
onitrogen or carbon mineralisation affected > 25 % after 100 days
However, in general more detailed decision is performed based on specific guidelines!
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higher tier „unless“: „unless clearly established through an appropriate risk assessment that under
field conditions no unacceptable impact occurs after PPP use in accordance with the proposed
conditions of use“
Regulation 546/2011, uniform principles for evaluation and authorisation of PPP
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02011R0546-20180524

PPP ecotoxicity and risk assessment
§details for the testing and evaluation (risk assessment) in guideline documents
§for ecotox mainly these 4 are of key importance:
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•EFSA (2009): Risk assessment for birds and mammals: EFSA guidance document. EFSA Journal 7(12):
1438. http://www.efsa.europa.eu/en/efsajournal/pub/1438
•EFSA (2013): Guidance on tiered risk assessment for plant protection products for aquatic
organisms in edge-of-field surface waters. EFSA Journal 11(7): 3290.
https://www.efsa.europa.eu/en/efsajournal/pub/3290
•European Commission (2002): Guidance document on terrestrial ecotoxicology. Draft Working Document
SANCO/10329/2009, rev.2, final.
https://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_ppp_app-proc_guide_ecotox_terrestr
ial.pdf
•Candolfi (2000): ESCORT2 - Guidance document on regulatory testing and risk assessment procedures
for plant protection products with non-target arthropods.
•EFSA (2013): Guidance on the risk assessment of plant protection products on bees (Apis mellifera,
Bombus spp. and solitary bees). EFSA Journal 11(7): 3295.
https://www.efsa.europa.eu/en/efsajournal/pub/3295

Tiered approach - general
§this approach saves time, money, laboratory organisms etc.
§it tries to go narrow pathway „linking questions about risks asked by stakeholder to answers that
can be provided by researchers“
§its always trade-off between price and accuracy of assessment and these are different for each
tier
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Posthuma et al. 2008, Science of the Total Environment, 406:503-517

Tiered approach - general
§this approach saves time, money, laboratory organisms etc.
§
§Tier 1 = starts with default (worst-case) parameters for exposure
o= standard ecotox bioassays as we know them
orough assessment, conservative
oif risk is not acceptable then à
§Tier 2 = refinement, assessment closer to reality (e.g. representative organisms, real conditions,
considering protective distances ...), if risks not acceptable then à
§Tier 3, Tier 4 ... e.g. mesocosm or field studies
§
§based on the results of previous tier, its decided what to do next
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Tiered approach - examples
Aquatic organisms
§Tier 1 - based on standard laboratory studies
§Tier 2 - based on additional laboratory studies:
oTier 2A – based on geomean/AF approach
oTier 2B – based on SSD approach (Species Sensitivity Distribution)
oTier 2C – based on refined exposure laboratory tests/AF approach
§Tier 3 - based on mesocosm/microcosm studies
§
§
§
§at each tier, regulatory acceptable concentrations (RACs) derived
and compared to PEC
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EFSA (2013): Guidance on tiered risk assessment for plant protection products for aquatic organisms
in edge-of-field surface waters. EFSA Journal 11(7): 3290.
https://www.efsa.europa.eu/en/efsajournal/pub/3290
Obsah obrázku rostlina, dlaň, zelenina Popis byl vytvořen automaticky

Tiered approach - examples
Aquatic organisms
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From: Better Training for Safer Food Initiative workshop, 2019

Tiered approach - examples
Aquatic organisms
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From: Better Training for Safer Food Initiative workshop, 2019

Tiered approach - examples
Birds and mammals
§screening – general model, indicator species
§tier 1 – specific model, generic focal species with different feeding preferences and growth
stages
§higher tier – focal species, corrections using measured data (monitoring studies)
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Indicator species – used in the screening step, it is not a real species but, by virtue of its size
and feeding habits is considered to have higher exposure than (i.e. to be protective of) other
species that occur in a particular crop at a particular time.
Generic focal species – used in Tier 1assessment, it is not a real species, however it is
considered to be representative of all those species potentially at risk. Instead of the one single
food item approach of the screening step in this assessment a mixed diet is applied when
appropriate for the generic focal species. In addition, interception of the spray by the crop is
taken into account by calculating the residue level on the several food types for the birds and the
mammals.
Focal species – used in higher tier assessment, it is a real species that actually occurs in the
crop when the pesticide is being used (see section 6.1.3 for identification of focal species.).

Tiered approach - examples
Birds and mammals
§shortcut values for screening and Tier 1 calculations - describe feeding habits and other
ecological needs for indicator and generic focal species
§
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Theoretical dietary exposure routes for birds feeding in crop (e.g., cereals) sprayed with a plant
protection product. Most of the plant protection product will be deposited in the treated crop
area, but some may enter neighboring water bodies. Exposure to birds in the treated area can
therefore occur by feeding on the crop itself (a), ground‐dwelling (b) or foliar‐dwelling (c)
invertebrates, weeds (d), or weed seeds (e). Birds may also feed on earthworms living in the
treated soil (f) or fish living in neighboring contaminated surface waters (g). Exposure may also
occur by drinking from contaminated puddles within the treated crop area (h)

Tiered approach - examples
Birds and mammals
§higher tiers, e.g. field studies
§
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Tiered approach - examples
Soil organisms                                                            Non-target arthropods
(NTA)
§
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Simplified example of evaluation
Soil organisms
§
Exposure estimation - PECsoil
§percentage of applied spray volume reaching soil depending on interception (e.g. 50%)
§even distribution in the top 5 cm of soil
§soil density of 1.5 g/cm3
§calculate PECsoil in mg a.s. / kg soil
§
§example results for PPP „Pest-Killer“
§application dose is 150 g a.s. / ha
§1 ha of 5 cm soil corresponds to 500,000,000 cm3 which is 750,000 kg soil
§PECsoil is 0.2 mg a.s. / ha
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Simplified example of evaluation
Soil organisms
§
TIER 1
§reproduction effects on earthworms - NOEC are derived
§TER > 5 OK, TER < 5 further studies needed
§
§example results for PPP „Pest-Killer“
otested at 0.02, 0.04, 0.08, 0.16, 0.32, 0.64 mg a.s. / kg
oNOEC was 0.32 mg a.s. / kg
§
§TER = NOEC/PECsoil = 0.32 / 0.2 = 1.6 (< 5, not OK and further studies are needed)
§
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http://t3.gstatic.com/images?q=tbn:ANd9GcR7-3EeXeOV0fHO4s2TOGWEnFANnR74gi6jRAso6-WCvT_OWvmIZlw3FsoM
yA

Simplified example of evaluation
Non-target arthropods (NTA)
§
§in-field and off-field assessment
TIER 1
§risk assessment based on two indicator species:
oTyphlodromus pyri
oAphidius rhopalosiphi
§standard toxicity tests to determine LR50 in g a.s. / ha based on mortality
§example results for PPP „Pest-Killer“
otested at 1.25, 2.5, 5, 10, 20, 40, 80 g a.s./ha
oLR50 for both species = 2.5 g a.s./ha
oall in acreditted labs with GLP
§
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rhopalosiphi - BiasLabs

Simplified example of evaluation
Non-target arthropods (NTA)
TIER 1 (cont.)
§now to determine hazards, compare exposure to effect in hazard quotient (HQ) – exposure
(application rate) and LR50 must not differ in units
§if resulting HQ ≥ 2, there is potential hazard to non-target arthropods
§
§
§
§
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In-field HQ =
Application rate x MAF
LR50
Single application in g a.s. / ha
Ratio between rate after max. recommended no. of applications and the initial rate after a single
application
LR50 expressed in g a.s. / ha
Off-field HQ =
Application rate x MAF x (drift / veg distribution factor) x correction factor
LR50
= % drift/100
(90th percentile)
For 2-D exposure (= glass plate) this = 10
To account for uncertainty between species this = 10

Simplified example of evaluation
Non-target arthropods (NTA)
§
TIER 1 (cont.)
§application details:
oapplication rate = 56 g a.s./ha
oNo. of treatments = 3 per crop
oLR50 = 2.5 g a.s. / ha
§assumptions:
oDrift value – 2.01
oMAF – 2.3
oApplication to field crops at tier 1 use the shortest distance – 1 m (3m for orchards, vines etc)
§
§
§
§in-field risk is not acceptable (HQ ≥ 2), off-field is also not acceptable (HQ ≥ 2)
§
§
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In-field effects:
56 g a.s./ha x 2.3
2.5 g a.s./ha
HQ = 51.52
Off-field effects:
56 g a.s./ha x 2.3 x (2.01/100) x 10
2.5 g a.s./ha
HQ = 10.03

Simplified example of evaluation
Non-target arthropods (NTA)
TIER 1 (cont.)
§
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Simplified example of evaluation
Simplified example of evaluation: non-target arthropods (NTA)
TIER 2
§if HQ ≥ 2 for in-field risk assessment, test affected indicator species + 1 additional species
§if HQ ≥ 2 for off-field risk assessment, test affected species + 2 additional species
§preferred species: Orius laevigatus, Chrysoperla carnea, Coccinella septempunctata, Aleochara
bilineata, Poecilus cupreus
§
§
§
§
§
§
§extended laboratory studies; aged residue studies; semi-field studies; field studies
§
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Chrysoperla carnea | NatureSpot Coccinella septempunctata (Seven-spot ladybird): an aphid predator
for biocontrol Aleochara bilineata Chrysoperla carnea - Chrysoperla - BugGuide.Net

Simplified example of evaluation
Non-target arthropods (NTA)
TIER 2
§HQ approach with criteria of ≥ 2 is not applied !
§predicted exposure rates are calculated based on similar equations as in Tier 1
§compared directly to toxicity endpoints LR50 or ER50 (the lower one should be used)
§i.e. trigger is less than 50% negative effects
§
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PPP assessment – results available
ecotoxicological data and risk assessment (i.e. data on tox/ecotox are combined with data on
exposure and env. fate) are available:
§in Draft Assessment Reports or Renewal Assessment Reports (DAR / RAR)
http://registerofquestions.efsa.europa.eu/roqFrontend/wicket/page?5
(generally: https://www.efsa.europa.eu/en/calls/consultations)
Part A: Summary of each section; List of Endpoints
Part B: Detailed evaluation for each area; Ecotox in section B.9
Part C: Confidential information

§in EFSA Conclusions, Peer Reviews and Peer Review Reports; example of Epoxiconazole:
https://www.efsa.europa.eu/en/efsajournal/pub/rn-138
https://www.efsa.europa.eu/en/efsajournal/pub/4123
http://registerofquestions.efsa.europa.eu/roqFrontend/wicket/page?16
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Example 4
Veterinary pharmaceuticals – in EU
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Veterinary pharmaceuticals
§growing problem – increasing release
of bioactive substances to environment
§
§
§
§
§
§
§
§veterinary medicines - more attention is logical
ohuge consumption
otheir entry into the environment (water and soil) is more possible
(use of agricultural waste on soil)
okilling beneficial soil organisms or development of antibiotic resitance
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Veterinary pharmaceuticals
§Regulation 726/2004, authorisation and supervision of medicinal products for human and veterinary
use
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02004R0726-20190330
§Directive 2001/83/EC, medicinal products for human use
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02001L0083-20190726
§Directive 2001/82/EC, veterinary medicinal products
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02001L0082-20090807
§
§pharmacovigilance – careful assessment of all undesired impacts
§European Medicines Agency (EMA) https://www.ema.europa.eu/en
§part of the registration proces is also Environmental Risk Assessment
§for veterinary - detailed evaluation procedure in EMA CVMP (Committee for Medicinal Products for
Veterinary Use)
https://www.ema.europa.eu/en/committees/committee-medicinal-products-veterinary-use-cvmp
§guidelines including assessment of environmental fate, ecotoxicity and risks
https://www.ema.europa.eu/en/veterinary-regulatory/marketing-authorisation/environmental-risk-asses
sment-veterinary-medicines
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Veterinary pharmaceuticals
§guidelines including assessment of environmental fate, ecotoxicity and risks
https://www.ema.europa.eu/en/veterinary-regulatory/marketing-authorisation/environmental-risk-asses
sment-veterinary-medicines
§VICH GL6 Environmental impact assessment (EIAS) for veterinary medicinal products - Phase I
§VICH GL38 Environmental impact assessments for veterinary medicinal products - Phase II
§
§
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Veterinary pharmaceuticals
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