Analysis of estrogen receptor gene expression in natural water
snails after EE2 exposure
Emilie Björnsdotter
Abstract
Endocrine disruptive chemicals (EDCs) that are discharged into the environment have been
shown to affect growth and reproduction of mollusks. 17- ethinylestradiol (EE2) is the
active component in human contraceptive pills that can affect growth and also transcription of
the estrogen receptor (er) gene in water snails. Although the exact mechanism of er activation
still is unknown in mollusks and further studies are recommended.
The aim of this study was to investigate the effect of EE2 on er gene transcription in two
gastropod species: the prosobranch Bithynia tentaculata (males), and the pulmonate Radix
balthica (hermaphrodite). These snails have different living and reproduction patterns and so
we hypothesized that they would respond differently to EE2 exposure. All snails were
collected from a natural population in southern Sweden. In our laboratory they were exposed
to 10 ng/L and/or 100 ng/L EE2 during 24h and/or 72h.The gene expression was measured in
individual snails after EE2 exposure.
We found a significant species-specific interaction between the effects of EE2 dose and
exposure duration in B. tentaculata males (not in R. balthica). In addition, when we compared
our analyses of male B. tentaculata with the study by Hultin et al. in 2014 our results suggest
that male and female prosobranch snails can respond differently on transcription level to EE2.
Although more females than males have been analyzed. Few studies have been made on the
possible endocrine differences between prosobranch males and females and if there were a
sex-dependent EE2 response this would be interesting to study further.
A suggested control program for toxicity in the process wastewater at Akzo
Nobel Pulp and Performance Chemicals AB in Bohus
- Changes in toxicity, comparing now and then
Frida Eriksson, 30 hec
ABSTRACT
Akzo Nobel Pulp and Performance Chemicals AB in Bohus is a chemical industry located at
the riverside of Göta Älv, Ale municipal, Västra Götalandsregionen. AkzoNobel PPC conduct
activities that may cause adverse effects in the environment and are due to this forced by law
to monitor these activities. This is done by self-monitoring and done according to an own
developed control program. The control program, today, lack specific strategies for
monitoring toxicity in the process wastewater that is released, after internal cleaning, to the
sewage treatment plant (STP) Ryaverket in Gothenburg. The aim of this project was to
develop a suggestion for a control program covering toxicity, taking into account the
parameters of persistency, bioaccumulation and toxicity (PBT), requests from the regulatory
authority, caretakers of the primary recipient and the company itself. To be able to develop a
control program a characterization of the process wastewater was done, showing negligible
toxicity towards bacteria and algae, no observed adverse effects on Nitocra spinipes in a subchronic test with up to 30 % sample mixture, a fraction of persistent material and the need to
further evaluate potentially bioaccumulative substances. Inhibition of nitrification showed
inhibition below limit of concern. The suggested control program is tailor-made for
AkzoNobel PPC and covers the PBT parameters. The characterization was also used to
evaluate current toxicity status compared to previous toxicity status, in the process
wastewater. This evaluation was motivated by the decrease in suspended material by ~ 90 %
since 2012. The comparison showed an indication that the toxicity has decreased but that
there is a fraction of persistent material in the process wastewater today, that needs to be
further evaluated.
A suggested control program for toxicity in the process wastewater at Akzo Nobel Pulp
and Performance Chemicals AB in Bohus
-
Evaluation of the suitability of using Microtox vs. Respirometry - sensitivity or relevance?
Susanna Eriksson, 30hec
Abstract
According to Miljöbalken (chapter 26, 19§), any activity that will cause effects in the
environment or for the human health must plan and monitor the activities in order to prevent
and mitigate such effects. This self-monitoring should include a control program that is
designed according to the primary recipient; a sewage treatment plant (STP) or a direct
discharge. For industries that lead their process wastewater to an STP, inhibition of
nitrification is a crucial parameter. Biodegradation and bioaccumulation also affect the
toxicity and are also taken into account. The process wastewater from AkzoNobel PPC
undergoes chemical cleaning at the site and is then lead to Ryaverket, the sewage treatment
plant in Gothenburg. The aim of this project was to develop a suggestion for a control
program to monitor the toxicity in the outgoing effluent from AkzoNobel PPC. In order to do
so, a characterization of the outgoing effluent was performed which showed that the inhibition
of nitrification was below the limit of concern and that the toxicity of the outgoing effluent is
negligible towards bacteria and algae. No adverse effects were observed on the larval
development of Nitocra spinipes with sample interferences up to 30 %. The outgoing effluent
showed to contain a fraction of persistent material. The control program was developed based
on literature and the type of primary recipient. To choose a method for monitoring the acute
toxicity towards bacteria an evaluation of the two methods; bioluminescence inhibition assay
with Vibrio fischeri (ISO 11348) and the respirometric test (ISO 8192), was performed. The
results show that the respirometric test is applicable for assessing the toxicity of the outgoing
effluent from the site and that the method is more sensitive to the toxicity of this particular
effluent.
Fish cell lines and their potential uses in ecotoxicology: from cytotoxicity studies and
mixture assessment to a co-culture model and mechanistic analyses
Fabienne Roux, 60 hec
ABSTRACT
The environmental risk assessment of chemicals is mostly based on in vivo single compound
experiments. However, due to the increasingly high amount of chemicals, metabolites and
mixtures to be tested, optimization of this assessment is required. Interest in in vitro methods
has been growing greatly in the recent years for economical, practical and ethical reasons, and
the use of cell lines as alternatives to in vivo testing is being seriously considered. This thesis
focused on two different aspects of methods using fish cell lines. The first was the use of
quick and simple cytotoxicity assays as screening method for assessment of mixture toxicity
in the specific case of Swedish coastal waters. Three compounds, copper (II) ions, metoprolol
and tributylphosphate, were tested on RTgill-W1, singularly and in combination based on
their EC50 ratio. The concentration addition (CA) model was used to predict mixture toxicity
and compare with experimental results. CA underestimated the EC50 by less than a factor of
2. The assay itself showed high variability but could be optimized for further mixture testing.
The second, more complex assay consisted of using two different cell lines (RTgill-W1 and
RTL-W1) separated by a permeable insert to mimic the direct exposure of the gill epithelium
to environmental toxicants and indirect exposure of the liver through uptake and metabolism,
as an attempt to improve basic cell line assays. The effects on gene expression level, EROD
activity and glutathione content of benzylbutylphthalate and bisphenol A were analyzed. The
obtained results indicate indirect exposure of RTL-W1. The properties of this co-culture
model are promising but need to be analyzed more in depth before it can be used in toxicity
tests. The characterization of relevant biomarkers and pathways in these cell lines is also
needed.
Keywords: cell lines, in vitro alternatives, RTgill-W1, RTL-W1, NICE, cytotoxicity, mixture,
concentration addition, co-culture, gene expression