Current issues
in ecotoxicology research
Luděk Bláha (blaha@sci.muni.cz)
RECETOX PřF MU
PHARMACEUTICALS
Example 1 - DICLOFENAC
Unexpected effects at NON-TARGET species
- nephrotoxicity at vultures
- Relevant also in EU
(ESP, EL,CY)
Example 2 – AVERMECTIN-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
?
MIXTURE EFFECTS … VERY LOW CONCENTRATIONS
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
International ring test (2012-13)
Testing comparability of existing and innovative bioassays for water quality assessment
EU WFD
priority
substances
Different
concentrations
EQS
= limit
(Environmental
Quality
Standard)
MIXTURE EFFECTS … VERY LOW CONCENTRATIONS
Example: Effects of mixtures on D. rerio fish embryos
Control
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
MIXTURE EFFECTS … VERY LOW CONCENTRATIONS
Example: Effects of mixtures on X. laevis frog embryos
Controls
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
MIXTURE EFFECTS … VERY LOW CONCENTRATIONS
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
Kde „tradiční“ ekotoxikologie
nestačí
Nano-eco-toxicology
Nanoparticles - examples
Ecotoxicity 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 ??
Mechanistic and Computational
(ECO)TOXICOLOGY
OrganismChemical
Adverse Effects
Death
Altered Reproduction
Inhibition of Growth
Tumorigenicity
Skin irritation
…
+
Traditionally – Evaluation of adverse effects using the whole organism models
Hazard assessment
REGULATORY FOCUS
(APICAL ENDPOINTS)
Newly added aspects
What are the mechanisms (MoA – mode of action)?
Can mechanisms serve for predictions?
Extrémní rozvoj analytických technologií  „OMICS“
A další „ómy“
• Lipidóm
• Mikrobiom …
Sběr omics podporují strategické dokumenty & projekty
Toxicity Testing in the 21st Century: A Vision and a Strategy
US National Academies of Sciences
http://www.nap.edu/catalog/11970.html
OrganismChemical
Adverse Effects (EC50)
Death
Inhibition of Growth
Altered Reproduction
Tumor
Skin irritation
…
+
Traditionally – Evaluation of adverse effects using the whole organism models
+10^4 Chemicals HTS Chemical-biological interactions,
Mechanistic Toxicological Data
Hazard assessment
New – Ex vivo / in vitro / In chemico / In silico Methods
Key task/question:
How to link MECHANISTIC INFORMATION with APICAL ENDPOINTS ?
Kvantitativní mechanistické modelování
PLoS Comput Biol. 2016 Apr 20;12(4):e1004874.
Fig 1. The HPOL signaling network in rainbow
trout as formulated in our model.
Arrows and symbols on graph follow CellDesigner vs. 4.4
notation (www.celldesigner.org). GnRH is secreted from the
hypothalamus into the pituitary stimulating the production of
mFSH and mLH, which then leads to formation of FSH and LH,
respectively. FSH, which is being continuously secreted from the
pituitary, travels to the ovaries to stimulate production of E2. E2
then travels to the liver to bind with E2 receptors (R; translated
from mR) to form ER. ER then stimulates the production of
mVTG, which produces VTGL. Secreted VTG then travels from the
liver to the ovaries via the plasma (VTGP) where it is absorbed by
follicles in stages 3 through 6 (the proportion of follicles in these
stages are denoted by Sj, j = 3, 4, 5, and 6) during vitellogenesis,
the rate of which is affected by FSHP, to promote oocyte growth
(OAvg). Oocyte growth then progresses the oocytes through the
stages using a Weibull distribution created from OAvg together
with OVar. In the later stages LHP stimulates the oocytes to
produce DHP. Finally, oocytes undergo final maturation (SFOM)
and combined with DHP, determine when the fish ovulates
PLoS Comput Biol. 2016 Apr 20;12(4):e1004874.
Kvantitativní mechanistické modelování
Fig 3. HPOL model
predictions for (A)
pituitary levels of FSHβ
subunit mRNA, (B)
pituitary levels of LHβ
subunit mRNA, (C)
Hepatic levels of E2
receptor mRNA and (D)
Hepatic levels of VTG
mRNA
Observed data (dark
grey circles; mean ±TG
mRn = 3)
PLoS Comput Biol. 2016 Apr 20;12(4):e1004874.
Kvantitativní mechanistické modelování