Ecotoxicology – Part 3
Current issues in
Research vs Regulation
Ludek Blaha + ecotox colleagues
the assessment of toxicity is needed
to assess for toxicity
When & where the toxicity assessment is needed?
Anytime!
… depending on
researcher’s
budget
As the law says!
… what are the
law(s)? 
View of the researcher View of the regulator
• Industrial chemicals
• Cosmetics
• PPP (pesticides)
• Biocides
• Human
pharmaceuticals
• Veterinary
pharmaceuticals
Chemical laws („bulk“)
nanonanonanonano
REACH
(ECHA)
PPP
(EFSA)
MPs
(EMA)
§§
§§
WFD – surface w.
GWD – ground w.
Air quality
Food and feed
Soil & Sediments
Wastes
SOIL
AIR
WATER
Two §§ approaches:
 Prospective
(chemicals…)
 Retrospective
(mixtures …)
What to assess for toxicity?
Current research topics As required by law
Individual
chemicals
(prospective)
Engineered nanomaterials/particles
Ecological effects (e.g. of pharmaceuticals)
Endocrine disruption & chronic diseases
Industry & biocides (REACH)
PPPs = pesticides
Pharmaceuticals
Cosmetics
Mixtures
(prospective)
Multistressors
+T°C, salinity, pathogens, irradiation, food
Exposome
Contaminated
samples
(retrospective) Can analyzed chemicals
explain observed effects ?
Chemical analyses & limits
Effect testing rare: Remediation,
dredged sediments (CZ), effluents
(DE)
What to assess for toxicity?
Current research topics As required by law
Individual
chemicals
(prospective)
Engineered nanomaterials/particles
Ecological effects (e.g. of pharmaceuticals)
Endocrine disruption & chronic diseases
Industry & biocides (REACH)
PPPs = pesticides
Pharmaceuticals
Cosmetics
Mixtures
(prospective)
Multistressors
+T°C, salinity, pathogens, irradiation, food
Exposome
Contaminated
samples
(retrospective) Can analyzed chemicals
explain observed effects ?
Chemical analyses & limits
Effect testing rare: Remediation,
dredged sediments (CZ), effluents
(DE)
Nanoparticles - examples
Toxicity of nanoparticles …
(Mostly unknown)
Parameters may
Affect ecotoxicity
Composition (chemical)
Surface (size, area)
Charge
Reactivity
Interactions with ions,
other chemicals…
 Effects on
environmental Fate
and toxicity
Ecotoxicity of nanoparticles – RECETOX example
Comparison of toxicity - 4 „appeared to be the same“ particles
(one producer – 4 different lots)
(zerovalent iron – ZVI – Fe0)
?? Why is H16 so toxic ??
… despite of detailed investigation never revealed
PHARMACEUTICALS
Example 1 - DICLOFENAC
Unexpected effects at NON-TARGET species
- nephrotoxicity at vultures
- Relevant also in EU
(ESP, EL,CY)
Example 2 – AVERMEKTIN-like antiparasitics
Ivermectin – antiparasitics in large herds
 Used 2-times per season per sheep/cow
 Kills 100% parasites in sheep
 Released in dung - kills 80-90% larvae of dung flies
 High concentrations in dung (released 2 days post application)
 Persistent in the soil (half-life 30 days)
 Can be washed into adjacent streams (highly toxic to water insects)
Moxidectin – used e.g. in home
„spot on” products
?
Boyles et al. (2011) Science 332 (60251) 41-42
Stress  multigeneration effects
Epigenetics  DNA methylations
2x difference
International ring test (2012-13)
Testing comparability of existing and innovative bioassays for water quality assessment
Main questions:
Are current limits (for individual compounds) safe?
Relevance of “Something from Nothing” phenomenon ?
3 samples
 12 European laboratories – different bioassays
 ČR – RECETOX: 11 bioassays
Carvalho, R. et al. (2014) Mixtures of
chemical pollutants at European legislation
safety concentrations: how safe are they?
Toxicol Sci 141(1): 218-233
EU WFD
priority
substances
Different
concentrations
EQS
= limit
(Environmental
Quality
Standard)
International ring test (2012-13)
Testing comparability of existing and innovative bioassays for water quality assessment
Example: Effects of mixtures on D. rerio fish embryos
Control
Effects of RM 3 (i.e. safe)
mixtures
International ring test (2012-13)
Testing comparability of existing and innovative bioassays for water quality assessment
Carvalho, R. et al. (2014) Mixtures of
chemical pollutants at European legislation
safety concentrations: how safe are they?
Toxicol Sci 141(1): 218-233
Example: Effects of mixtures on X. laevis frog embryos
Controls
International ring test (2012-13)
Testing comparability of existing and innovative bioassays for water quality assessment
Effects of RM 3 (i.e. safe)
mixtures
Carvalho, R. et al. (2014) Mixtures of
chemical pollutants at European legislation
safety concentrations: how safe are they?
Toxicol Sci 141(1): 218-233
Biotest A B C
Microtox 26and 36% stimulation of
luminescence in 15and 30mins of
exposure, respectively
18and 35% stimulation of
luminescence in 15and 30mins of
exposure, respectively
22and 39% stimulation of
luminescence in 15and 30mins of
exposure, respectively
Algae growth inhibition test 96-h
exposure
31% inhibition of growth compared
to solvent control
20% inhibition of growth compared
to solvent control
16% inhibition of growth compared
to solvent control
Acute immobilization test with
D. magna
90% immobilization after 48hours
of exposure; 25% immobilization
occurred in 50% concentration - not
statistically significant
no effect observed no effect observed
Reproduction test with D.
magna (21-d exposure)
100% mortality after 3days of the
test, no reproduction could be
evaluated
31+/- 37% inhibition of
reproduction, not statistically
significant
23+/- 24% inhibition of
reproduction, not statistically
significant
FETAX (96-h exposure) 62+/- 10% of malformed embryos;
no effect on embryo length
observed
43+/- 12% of malformed embryos;
no effect on embryo length
observed
34+/- 14% of malformed embryos;
no effect on embryo length
observed
FET (120-h exposure) effects observed in number of
defected embryos - absence of gas
bladder, (head) deformities and
underdeveloped embryos were
observed the most often.
no significant effects observed effects observed in number of
defected embryos, number of
underdeveloped embryos and
length
In vitro - cytotoxicity no effect observed compared to
solvent control
no effect observed compared to
solvent control
no effect observed compared to
solvent control
In vitro - estrogenicity effect under LOQ effect under LOQ effect under LOQ
In vitro - dioxin-like toxicity effect under LOQ effect under LOQ effect under LOQ
In vitro - androgenicity effect under LOQ effect under LOQ effect under LOQ
In vitro - antiandrogenicity effect under LOQ effect under LOQ effect under LOQ
What to assess for toxicity?
Current research topics As required by law
Individual
chemicals
(prospective)
Engineered nanomaterials/particles
Ecological effects (e.g. of pharmaceuticals)
Endocrine disruption & chronic diseases
Industry & biocides (REACH)
PPPs = pesticides
Pharmaceuticals
Cosmetics
Mixtures
(prospective)
Multistressors
+T°C, salinity, pathogens, irradiation, food
Exposome
Contaminated
samples
(retrospective) Can analyzed chemicals
explain observed effects ?
Chemical analyses & limits
Effect testing rare: Remediation,
dredged sediments (CZ), effluents
(DE)
What to assess for toxicity?
Current research topics As required by law
Individual
chemicals
(prospective)
Engineered nanomaterials/particles
Ecological effects (e.g. of pharmaceuticals)
Endocrine disruption & chronic diseases
Industry & biocides (REACH)
PPPs = pesticides
Pharmaceuticals
Cosmetics
Mixtures
(prospective)
Multistressors
+T°C, salinity, pathogens, irradiation, food
Exposome
Contaminated
samples
(retrospective) Can analyzed chemicals
explain observed effects ?
Chemical analyses & limits
(see lectures: RISK ASSESSMENT part)
Effect testing rare: Remediation,
dredged sediments (CZ), effluents
(DE)
Active sampling
particles vs gaseous phase
• Reference locality – agriculture
(Košetice observatory)
• Region A – industrial
(historically OCPs production)
• Region B – combined: industry,
agriculture, traffic
Novák et al. (2009) Environment International
Contaminated samples? Case study “air“
Chemical
analyses
PCBs
0.001
0.01
0.1
1
REF 1 2 3 4 5 6 1 2 3 4 5 6
A B
ng/m3
log10
particle phase
gas phase
OCPs
0.001
0.01
0.1
1
10
100
1000
REF 1 2 3 4 5 6 1 2 3 4 5 6
A B
ng/m3
log10
particle phase
gas phase
PAHs
0.1
1
10
100
1000
REF 1 2 3 4 5 6 1 2 3 4 5 6
A B
ng/m3
log10
particle phase
gas phase
dioxin-like toxicity
0.1
1
10
100
REF 1 2 3 4 5 6 1 2 3 4 5 6
A B
TEQ(ng/m
3
log10)
particle phase
gas phase
Dioxin-like effects
o Difference B>A
o Difference B vs A – particles vs gas
Antiandrogenic effects
o Quantitative – comparable
o Clear differences in patterns … no effects on particles in „B“ (?)
antiandrogenicity
0.1
1
10
100
1000
REF 1 2 3 4 5 6 1 2 3 4 5 6
A B
1/IC25(1/(m3
/ml)log10)
particle phase
gas phase
Summary on When, Where, What
• Regulatory world
– Assessment of „chemicals“!
• Contaminated samples
- effects rarely tested
– Great value of bioassays
in assessment of contaminated
samples
– Effects observed (!)
– How to set the „limits“?
• Research issues and questions
– Nanomaterials, Pharmaceuticals, EDCs
– Mixtures!
– Exposome