Adsorbable organic bromine compounds (AOBr) in aquatic samples: a nematode based

toxicogenomic assessment of the exposure hazard

Nadine Saula,*, Stephen R. Stürzenbaumb, Shumon Chakrabartia, Nora Baberschkea, Thora Liekea, Anke Putschewc, Cindy Kochanc, Ralph Menzela, Christian E. W. Steinberga

a Department of Biology, Freshwater and Stress Ecology, Humboldt-Universität zu Berlin, Späthstr. 80/81, 12437 Berlin, Germanyb School of Biomedical Sciences, Analytical and Environmental Sciences Division, King's College London, 150 Stamford Street, London, SE1 9NH, UKc Chair of Water Quality Control, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany

* Corresponding author:Telephone: 0049 (0)30 6397 4444Fax: 0049 (0)30 636 9446Email address: nadines1976@aol.com

Abstract

Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in

German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of

microcystins, are one of the main producers of natural organobromines. However, very little

is known about how environmental realistic concentrations of organobromines impact

invertebrates. Here, the nematode C. elegans was exposed to AOBr-containing surface water

samples and to a Microcystis aeruginosa enriched batch culture (MC-BA) and compared to

single organobromines and microcystin-LR exposures. Stimulatory effects were observed in

certain life trait variables, which were particularly pronounced in nematodes exposed to MC-

BA. A whole genome DNA-microarray revealed that MC-BA led to the differential

expression of more than 2000 genes, many of which are known to be involved in metabolic,

neurologic, and morphologic processes. Moreover, the up-regulation of cyp- and the down-

regulation of abu-genes suggested the presence of chronic stress. However, the nematodes

were not marked by negative phenotypic responses. The observed difference in MC-BA and

microcystin-LR (which impacted lifespan, growth and reproduction) exposed nematodes was

hypothesized to be likely due to other compounds within the batch culture. Most likely the

exposure to low concentrations of organobromines appears to buffer the effects of toxic

substances, like microcystin-LR.

Keywords: Organobromines; C. elegans; Microcystis aeruginosa; Microcystin; Microarray;

Gene expression 1

1. Introduction

Although more than 5,000 different natural halogenated organic compounds have been

described, most attention has been devoted to marine chlorine compounds (Gribble 2009,

2012). The occurrence of organobromine compounds in freshwaters was highlighted by

Hütteroth et al. (2007) who uncovered that freshwater lakes in Berlin, Germany, can contain

(especially during the late summer months) up to 35 µg/L AOBr (adsorbable organic bromine

compounds). The authors argued that phototrophic organisms, in particular cyanobacteria,

produce AOBr which are released following their death. Moreover, these organisms are also

able to segregate haloperoxidases which catalyze the halogenation of dissolved organic matter

in the presence of halogens and hydrogen peroxide. Organohalogens are considered to exert

endocrine disruptor activities, reproductive and neurological toxicities, and carcinogenic

effects (Shaw et al. 2010) and combined with the fact that lake water itself is a source of

drinking water, may suggest that AOBr in lakes may pose a health risk to wildlife and

humans. However, this notion can be challenged with at least three mutually non-exclusive

counterarguments:

i) The majority of toxicity studies conducted to date have utilized concentrations of AOBr

which are several magnitudes higher than those found naturally in the environment.

Therefore, it is conceivable that lower and ecologically more relevant levels may trigger a bi-

phasic concentration dependent response, a so-called hormesis effect. Indeed, Wang et al.

(2012) observed a hormetic cell proliferation effect in human hepatocytes challenged with

polybrominated diphenyl ethers. Although hormetic effects can be diverse, such as enhancing

growth, increasing brood size or extending lifespan (Calabrese 2010), it is important to

evaluate several life trait variables as, from an ecological point of view, true hormesis applies

only when overall Darwinian fitness is increased.

ii) Some halogenated organic compounds, for example the polyhalogenated monoterpene

halomon produced by the marine red alga Portieria hornemannii, can offer anti-tumor

properties (Fuller et al. 1992; Andrianasolo et al. 2006). Similarly, signs of anti-tumor

activities were reported for natural organohalogens such as bromotyrosines (Simmons et al.

2005), symplocamide A (Linington et al. 2008), chlorosulfolipid (Ciminiello et al. 2001),

brominated oxylipins (Rezanka & Dembitsky 2003), and chlorinated indigoglycosides

(Maskey et al. 2002). Moreover, anti-bacterial and anti-inflammatory properties were also

ascribed to organohalogens (Kuniyoshi et al. 1985; Gribble 2004).

iii) Most toxicity studies have tested single substances in isolation, however cyanobacteria,

one of the main producers of natural organobromines, synthesise several biological active

2

peptides in parallel. These cyanotoxins, like microcystins, are known for their potent

hepatotoxicity (Stewart et al. 2008; Svircev et al. 2010) and their neurotoxicity (Pouria et al.

1998). It is conceivable that hormetic and health promoting activities of organobromines may

attenuate the toxicity of cyanotoxins, at least if an organism is exposed to a mixture of both

compound classes. Cedergreen (2010) provided first tantalizing clues that hormesis may occur

within a mixture scenario as exemplified by root length growth of Lactuca sativa. Indeed,

lakes are composed of numerous other dissolved organic matters, including the humic

substances (HSs). HSs trigger oxidative stress, estrogenic action, as well as the induction of

biotransformation and stress-responsive genes (Steinberg et al. 2006, 2008; Meinelt et al.

2008). Thus, any organismal response should also take the biogeochemical background of

freshwater bodies into account.

Based on these arguments we hypothesize that organobromines in low and environmentally

realistic concentrations are non-hazardous to fauna. Moreover, we hypothesize that

organobromines buffer the toxic effects of cyanotoxins when they occur in parallel. The goal

of this study is to evaluate these hypotheses by testing water samples and a batch culture in

nematodes by using biomolecular and phenotypic methods.

Nematodes are known to be important players in energy flows and nutrient cycling in soils

and freshwater sediments (Covich et al. 1999; Mulder et al. 2011). Therefore, this study made

use of the nematode Caenorhabditis elegans, a model organism with a reputation as a good

model in ecotoxicology (Leung et al. 2008). In detail, the toxicogenomic responses and

changes in key life-history traits were investigated in the nematode challenged with two-fold

concentrated organobromine containing water sampled from two German lakes (Stößensee

and Tegeler See) during the late summer months. In addition, a Microcystis aeruginosa batch

culture (MC-BA) was applied in three different concentrations (10, 20 and 40 % of the

original culture filtrate) to simulate the conditions during a cyanobacterial bloom. The results

obtained were finally compared to analogous experiments previously performed with the

organobromines tetrabromobisphenol-A (TBBP) and dibromoacetic acid (DBAA) as well as

the microcystin-LR (MC-LR) which were recently published in parts (Saul et al. 2014a,

2014b).

2. Material and methods

2.1 Strains and conditions

The wild-type Caenorhabditis elegans strain N2 (var. Bristol) was maintained at 20 °C on

nematode growth medium (NGM) seeded with the Escherichia coli feeding strain OP50

3

according to Brenner (1974). The N2 and the OP50 strain were obtained from the

Caenorhabditis Genetics Centre, University of Minnesota.

2.2 Surface water samples, M. aeruginosa batch culture and test substances

Three lake water samples of approximately 10 L were collected from Stößensee (52° 30’

34’’N 13° 12’ 34’’ E) and Tegeler See (52° 34’ 33’’ N, 13° 15’ 49’’ E) in Berlin, Germany.

Samples were filtered (0.45 µm, cellulose nitrate membrane filters; Sartorius, Germany) and

analyzed for AOBr and microcystins. Samples were lyophilized (ABiTEP, Germany) to a

volume of about 1 L and the AOBr content of each sample was re-analyzed. Furthermore, a

filtrate was taken from a M. aeruginosa (Culture Collections of Algae at the University of

Göttingen, SAG) batch culture grown in 20 L pure Z-medium amended with 200 µg/L

bromide. Table 1 shows the AOBr and microcystin concentrations in the filtrates (original,

after lyophilisation and after subsequent dilution) of the water samples and the batch culture.

Furthermore, the final concentrations which were used for the exposure scenarios in C.

elegans are indicated.

Table 1: Overview of water samples

SamplesMicrocystinin the filtrate

(µg/L)

AOBrin the filtrate (µg/L)

AOBr in samplesFinal AOBr

concentrations for exposure

(µg/L)after

lyophil-isation (µg/L)

after dilution (µg/L)

Stößensee 23.08.2010 Nd 18 148 80 36

=200 %

Tegeler See Nd 12 53 53 24Stößensee 14.09.2010 Nd 16 105 71 32Tegeler See Nd 15 119 67 30Stößensee 12.10.2010 Nd 11 94 49 22Tegeler See Nd 13 60 58 26 M. aeruginosa batch culture (MC-BA)

22.02.2011244 (MC-RR)180 (MC-YR)332 (MC-LR)

103 -234692

10.3 (= 10 %)20.6 (= 20 %)41.2 (= 40 %)

AOBr: adsorbable organic bromine compounds; MC: microcystins; nd: not detectable

To expose the nematodes with the filtrates the NGM agar was prepared according to Table 2

and the OP50 bacteria were concentrated via centrifugation and 450 µl of the diluted filtrate

were added to 550 µl bacteria. 100 µL bacteria mixture were spotted on small agar plates (ø =

35 mm) and 1000 µL on big agar plates (ø = 96 mm).

4

Table 2: Preparation of NGM agarIngredients AmountNaCl 1.5 gBacto-Peptone 1.25 gAgar 8.4 g1 M KH2PO4 12.5 mL5 mg/ml Cholesterol solution 500 μLAqua bidest 262 mL

Autoclaving and cooling down to 55°C in an incubator1 M MgSO4 500 μL1 M CaCl2 500 μLFiltrate after dilution 225 µL

Distribution on petri dishes

Moreover, previously published data (Saul et al., 2014a, 2014b) from DBAA, TBBP (Sigma-

Aldrich, USA), and MC-LR (Enzo Life Sciences, USA) exposures were used for comparative

purposes. Therefore, agar and bacteria containing 22 µg/L and 11 mg/L DBAA and 54 µg/L

and 27 mg/L TBBP (corresponds to 0.1 and 50 µM, respectively) as well as 50 µg/L, 100

µg/L, and 300 µg/L MC-LR were prepared.

The samples contained equal amounts of solvent (final concentration of 0.3 % [v/v] DMSO;

Applichem, Darmstadt, Germany), Z-Medium, or water, respectively.

2.3 AOBr analysis

The concentration of adsorbable organic bound bromine (AOBr) was determined as described

by Oleksy-Frenzel et al. (2000). A 100 mL sample was acidified to pH 2 with HNO3 (conc.).

Dissolved AOBr was enriched on activated carbon and combusted. The reaction gases were

collected in 5 mL ultrapure water containing 4.5 µM Na2S. The solution was injected into an

ion chromatographic system for bromide analysis.

2.4 Analysis of microcystins

Solid phase extraction (SPE) was used to concentrate dissolved MCs from water samples and

from the batch culture sample prior to HPLC (DIN). 500mL water was passed through

Lichrolute RP 18 (300 mg, 3 cc, Merck Millipore) SPE cartridges, conditioned with 4 mL 5 %

methanol and eluted with 6.5 mL methanol acidified with 0.1 % formic acid (v/v). Organic

solvent was removed by a gentle nitrogen stream. Extracts were dissolved in 0.5 mL ultrapure

water and analyzed via an LC system with Triple-Quad MS TSQ Vantage (Thermo Fisher

Scientific) using an electrospray (ESI) interface. Separation was achieved on a Zorbrax

Eclipse XDB C18-UPLC column at 40 °C with a flow rate of 0.4 mL min-1. The LC/MS-MS

gradient solutions (water (A) and methanol (B)) contained 0.006 % acetic acid (v/v) and 5

5

mM HCOONH4. The gradient for microcystin separation was 0–2 % B, 0–1.5 min; 2–50 % B,

1.5–5 min; 50–70 % B, 5–8 min; 70 % B, 8–13 min; 0 % B for re-equilibration. The ESI+

multiple reaction monitoring transitions were m/z 519.8→135.0 and 239.1 (Microcystin-RR),

m/z 995.6 →135.0 and 861.0 (Microcystin-LR) and 1045.5→ 135 and 174.0 (Microcystin-

YR). The limit of quantification was 10 ng L-1 for each microcystin.

2.5 Lifespan assay

30 L4 larvae were transferred to NGM plates dosed with DBAA, TBBP, MC-LR, two-fold

concentrated water samples, or MC-BA, respectively, according to section 2.2 and table 1. A

total of 200 L4 larvae per treatment of the following generation (F1) were transferred onto ten

treatment plates and surviving and dead animals were counted daily until all individuals had

died. Nematodes that failed to respond to contact stimuli were considered to be dead.

Nematodes suffering from internal hatch and those that escaped from the NGM agar were

censored. Adult nematodes were regularly transferred to new treatment plates. Each lifespan

assay was performed at least two times. Statistical significance for alterations in the mean life

span was calculated using a log-rank test (Bioinformatics at the Walter and Eliza Hall

Institute of Medical Research; http://bioinf.wehi.edu.au/software/ russell/logrank).

2.6 Thermal stress resistance and body length

50 L4 larvae (F1) were randomly selected and transferred daily to fresh treatment plates. At

the sixth day of adulthood, treated and untreated nematodes were moved either to 35 °C for

6.5 h and surviving and dead nematodes were counted thereafter (thermal stress test), or to 45

°C for 2.5 h and subsequently sized by means of a digital Microscope at 100-fold

magnification (body length test). Statistical significance was calculated via the Student’s t-test

(SigmaStat 3.5; SPSS Inc., USA). Each concentration was tested in at least three trials.

2.7 Brood size

L4 larvae (F1) were transferred individually to treatment plates and moved to a fresh plate

each day until reproduction was completed at the fourth day of adulthood. The number of

offspring per individual animal was counted daily. Statistical significance was calculated via

the Student’s t-test (SigmaStat 3.5; SPSS Inc., USA). Three trials were performed with 10

nematodes per concentration in each trial.

6

2.8 Gene expression profiling

2.8.1 RNA preparation

Young adult nematodes were treated with sodium hypochlorite (Sigma, Germany) according

to Strange et al. (2007). The obtained eggs were transferred to plates containing the respective

test substance and incubated at 20 °C. At the first day of adulthood the animals were

harvested by rinsing off with M9 buffer, washed at least three times, shock frozen in liquid

nitrogen and stored at -80 ̊C. For each condition, samples were cultivated in triplicate. The

samples were milled with 0.5 mm glass beads in a homogenizer (SpeedMill Plus, Analytik

Jena, Germany) and thereafter the RNA was extracted using the innuSPEED Tissue RNA kit

(Analytik Jena, Germany). The quality of the extracted RNA was examined by gel

electrophoresis and the quantity with a Spectrophotometer (NanoDrop 2000, Thermo

Scientific, USA). The RNA was stored at -80 °C and was used for the microarray as well as

the qRT-PCR experiments.

2.8.2 DNA Microarray

RNA was amplified with the MessageAMP™Premier RNA Amplification Kit (Ambion,

USA) which relies on the T7 in vitro transcription (IVT) amplification technology. First- and

second-strand cDNA synthesis, cRNA synthesis, labeling, fragmentation, chip hybridization,

and scanning were performed according to the manufacturer’s specifications (Affymetrix,

USA). The whole genome microarray chip (GeneChip C. elegans Genome Array, Affymetrix,

USA) contains more than 22,500 transcripts from C. elegans. Three chips as well as three

RNA samples were used per condition (biological replicates). No technical replicates were

performed; however, each gene is represented by approximately 11 different probe sequences

on the chip. The quality and quantity of the RNA, cDNA, and cRNA were assessed via

capillary electrophoresis (Bioanalyzer 2100, Agilent Technologies, UK) and via

spectrophotometry (NanoDrop 2000, Thermo Scientific, UK).

The transcription expression values were pre-processed with the MAS5 algorithm

(Affymetrix Expression Console 1.2.0.20). After background correction, probe set

summarization and CHP file generation, the expression signals were normalized using the

relative median over all genes algorithm (BRB-ArrayTools 4.3.0). Spots with spot intensities

below the minimum signal value of 10 were excluded. Differentially expressed genes (DEGs)

were identified with a random-variance t-test and a SAM (Significance Analysis of

Microarrays) test. Genes were considered statistically significant if their p-value was less than

0.05, the false discovery rate less than 0.3 and the fold change compared to control at least

≤0.67 or ≥1.5. The cut-offs were chosen according to the previously published microarray

7

data with DBAA, TBBP and MC-LR (Saul et al., 2014a, 2014b) which facilitates the

comparison between the array results. Indeed, Dalman et al. (2012) highlighted that altered

cut-offs can lead to different results and should be uniformly chosen when datasets are

compared. To perform the GO Term analysis, the DEGs were processed with the Database for

Annotation, Visualization and Integrated Discovery (DAVID 6.7) (Huang et al. 2009a,

2009b).

2.8.3 qRT-PCR

The M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (Promega, USA) was

used to synthesize the cDNA at 42 °C for 90 minutes. The quantitative Real-Time Polymerase

Chain Reaction (qRT-PCR) was performed with the BIORAD MyiQ iCycler (BIORAD,

Germany) and the qRT-PCR Green Core Kit (Jena Bioscience, Germany). All expression

levels were normalized with the qRT-PCR values of the reference genes act-1 and cdc-42 and

the quality of the PCR product was evaluated via gel electrophoresis. Significant changes

(compared to the control) were determined by means of a Student’s t-test. Genes were

considered statistically significant if their p-value was less than 0.05 and the fold change

compared to control at least ≤0.67 or ≥1.5. qRT-PCR was performed to verify the quality of

the microarray data. The efficiency of each primer pair, the primer sequences, the annealing

temperatures, and the results of the qRT-PCR trials are shown in Online Resource 1.

3. Results and Discussion

3.1 Exposure to water samples and MC-BA affects only marginally the lifespan and

stress resistance of nematodes

In August, September, and October 2010, water from two lakes (Stößensee and Tegeler See,

both in Berlin, Germany) were sampled, filtered and lyophilized. Nematodes were exposed to

a two-fold concentrated sample of the respective lake waters. The M. aeruginosa batch culture

(MC-BA) was filtered and the nematodes were cultivated in 10 %, 20 %, or 40 % of MC-BA.

Table 1 summarizes the AOBr and microcystin contents in each sample. 

The lifespan was monitored in nematodes exposed to the water samples and to the single

organobromines (DBAA and TBBP) and MC-LR. Neither the surface water samples from

Lakes Stößensee and Tegeler See nor the MC-BA samples triggered a change of mean

lifespan in C. elegans (Fig. 1). The final concentrations of AOBr in the surface water and

MC-BA spiked plates ranged from 10 µg/L to 40 µg/L, thus resembling the concentrated

organobromine plates spiked with 22 µg/L and 54 µg/L DBAA or TBBP, respectively. The

elevated levels of 11 mg/L and 27 mg/L can only be found in extreme environments, such as

8

in industrial or hospital waste waters (Furhacker et al. 2000; Kummerer 2001; Shomar 2007).

Interestingly, low concentrations of TBBP prolonged the lifespan of C. elegans, whereas high

concentrations elicited a decrease in lifespan, which is in line with the hormesis effect. In

contrast, only the elevated concentrations of DBAA prolonged the lifespan of nematodes. The

DBAA and TBBP results were drawn from an extensive concentration-dependent lifespan

analysis (Saul et al., 2014a).

Exposure to 100 and 300 µg/L MC-LR resulted in a reduction of lifespan (partly published in

Saul et al., 2014b). The 40 % MC-BA sample comprises of about 130 µg/L MC-LR and 300

µg/L total measured microcystins, therefore it was surprising that the MC-BA exposure did

not mirror the toxicity observed in MC-LR exposed nematodes. It is conceivable that other

cyanobacterial exudates moderate the harmful effects caused by microcystins. Given that the

two organobromines tested here induce some degree of life-extension, suggests that

organobromines may, at least in part, be responsible for this attenuation. Since DBAA and

TBBP represent only a small fraction of all possible organobromines, it cannot be excluded

that the organobromines in the MC-BA filtrate act in a different way. TBBP is a brominated

flame retardant which occurs in sewage treatment plants and freshwater sediments (Law et al.,

2006), but there is no suggestion that it is also part of the AOBr pool tested here. However,

Hütteroth (2006) analyzed the AOBrs in the Berlin lakes Wannsee and Tegeler See by menas

of GC-MS (gas chromatography-mass spectrometry) and detected, amongst others, DBAA.

The lifespan trials were conducted in laboratory controlled conditions, however in nature

nematodes frequently encounter additional stressors, for example thermal stress. Most

exposure scenarios did not influence the resistance to thermal stress, however one water

sample from Tegeler See enhanced the resistance significantly. Furthermore, though

statistically not significant, exposure to 40 % MC-BA induced a marginal decrease in survival

and the 54 µg/L TBBP exposure group was marked by a slight increase (Fig. 2). The thermal

stress test of TBBP and DBAA was previously published in Saul et al. (2014a).

3.2. Exposure to MC-BA increases nematode growth and brood size

Whilst low concentrations of MC-LR and TBBP and also the DBAA exposures did not

influence the reproductive performance of C. elegans, elevated concentrations of MC-LR and

TBBP suppressed the reproductive output (Fig. 3 and partly presented in Saul et al. 2014a,

2014b). Therefore, it was again surprising to note that the 40 % MC-BA exposure scenario

stimulated reproductive output. The microcystins within the MC-BA sample were seemingly

not able to induce a reproductive toxicity. Which constituents of the batch culture were

responsible for this effect is, at present, not known, but the organobromines tested here did

9

not enhance the reproductive outcome.

The measurement of the body length revealed similar results, namely that the 40 % MC-BA

exposure increased the size of the worms significantly and that MC-LR led to a decrease in

the size. Only the higher DBAA concentration elicited a response which was similar in

magnitude to the MC-BA effect (Fig. 4 and partly presented in Saul et al. 2014a, 2014b).

In previously performed neurotoxicity assays, the impact of MC-LR, DBAA, TBBP, the

Stößensee lake samples, as well as the MC-BA filtrate (40%) on autonomic (locomotion,

pharynx pumping frequency, and defecation) and sensory (thermal, chemical, and mechanical

sensory perception) functions were measured (Ju et al., 2014a, 2014b; Lieke et al., 2015). It

was shown that 50 µM TBBP had an impact on three neurological functions (locomotion,

defecation, and chemical sensory) and thus, can be classified neurotoxic whereas 50 µM

DBAA led to neurostimulation of two autonomic functions (locomotion and pumping

frequency) and only exerted a temporary impact on the defecation interval. Similar

stimulations were also found for 0.1 µM TBBP which suggests the presence of an hormetic

effect. Furthermore, MC-LR impacted locomotion, pumping, and mechanical behaviour

whereas MC-BA displayed a lesser degree of neurotoxic action (only a temporarily impact in

thermotactic behaviour) but rather neurostimulation concerning autonomic functions. These

observations align well with the bioassay results (Figure 1, 3, and 4) which uncovered

stimulating activities for DBAA and MC-BA and adverse effects for MC-LR and TBBP.

Furthermore, the October sample of lake Stößensee increased locomotion and the pharynx

activity (Ju et al., 2014a). However, these stimulating activities are not reflected in the

lifespan, growth, stress resistance or reproduction assay (Figure 1-4). This indicates that the

behavioural assays might be more sensitive compared to the standard bioassays.

3.3 Global transcriptomics (microarrays) highlight metabolic and neurological changes

in MC-BA treated nematodes

Young (treated and untreated) adult nematodes were harvested and the global gene expression

changes were determined by means of a whole genome DNA-microarray. In total, 1266

transcripts were shown to be down-regulated and 761 up-regulated following an exposure to

40 % MC-BA and a GO Term analysis of these DEGs revealed that 45 GO Terms were over-

represented, including several metabolic and morphological processes (Table 3). The affected

GO terms in the “Cellular Component” group are all related to membrane constituents and

they are also overrepresented in nematodes exposed to DBAA, TBBP and MC-LR (Saul et al.

2014a, 2014b). However, there are no further GO term matches between MC-LR and MC-BA

treated nematodes. A closer look reveals that several abu-genes (activated in blocked

10

unfolded protein response) were down-regulated (Table 4). Given that these proteins are

known to be induced during stress (Urano et al. 2002), suggests that the observed down-

regulation may reflect the stress-protective effect of MC-BA. On the other hand, the up-

regulation of several cyp-genes (cytochrome P450 family), known for their role in the

metabolism of xenobiotics (Menzel et al. 2001, 2005; Ioannides 2008), implies an increase in

chemical stress. A similar pattern was recently described by Peltonen et al. (2013) who

describe that chronic alcohol exposure can lead to the repression of several abu-genes and the

induction of cyp-genes in C. elegans. Whether this represents a hallmark expression pattern

characteristic of chronic stress exposure warrants further investigations.

In addition, numerous neurological and signaling processes were modulated (Table 3). The

GO Terms related to the neurotransmitter transport, the phosphorylation/dephosphorylation

processes, the response to temperature stimuli, and the locomotory behaviour are essential for

the process and response to stimuli from the environment. Numerous unc-genes

(UNCoordinated) were shown to be differentially (up and down) regulated in MC-BA

exposed worms (Table 4).

To verify the quality of the microarray data, five genes were also tested in a qRT-PCR assay.

gur-3, hen-1, unc-17, and unc-30 were chosen since they are differentially expressed in MC-

BA as well as in MC-LR (gur-3, hen-1, unc-30; Saul et al., 2014b), TBBP (unc-17, unc-30;

Saul et al., 2014a) and/or DBAA (hen-1, unc-17; Saul et al., 2014a) exposed nematodes. The

microarray data of these genes match the results obtained by qRT-PCR (Online Resource 1).

Moreover, sma-2 was chosen due to its up-regulation by MC-BA and its involvement in body

length regulation (Morita et al., 1999). Since MC-BA (40%) treated nematodes displayed an

increase body length, the up-regulation of sma-2 might be linked. Indeed, the up-regulation of

sma-2 was also observed via qRT-PCR, however, the significance limit was not reached

(Online Resource 1).

Interestingly, numerous behaviour, metabolism and transport related genes, which were also

influenced by MC-BA (Table 3), were also differentially regulated when exposing C. elegans

to the organochlorine contaminant octachlorostyrene (Kim and Choung, 2009) or to

organochlorine-rich river sediments (Menzel et al., 2009). However, only 12 of the 45 MC-

BA-affected GO Terms and only a few abu and cyp genes were affected by these river

sediments. Comparisons of the GO-Terms influenced by 50 µM TBBP and DBAA and the

Elbe river sediments revealed similar results (Saul et al., 2014a, 2014b). However, since

studies with organochlorines in environmental realistic concentrations are rare in C. elegans,

it is challenging to compare these results.

The transcriptome analysis of nematodes exposed to the Stößensee derived surface water

11

sample identified only few DEGs. The water sample taken in August repressed 13 genes and

induced 128 genes, including three cyp-genes. Likewise, only one transcript was down-

regulated and 33 genes up-regulated by the October sample (Online Resource 2). The water

sample taken in August only affected GO Terms linked to cell signaling processes (Table 5),

which were all also significantly affected in nematodes challenged with MC-LR, DBAA and

TBBP (Saul et al. 2014a, 2014b), but not by MC-BA.

Table 3: GO Terms of MC-BA influenced DEGs

12

GO Term DEGs (n)

% of total DEGs P-Value

13

Bio

logi

cal p

roce

ss

GO:0043687 post-translational protein modification 109 5.43 7.20E-13GO:0006464 protein modification process 114 5.68 3.13E-11GO:0043412 biopolymer modification 116 5.78 8.85E-11GO:0006793 phosphorus metabolic process 107 5.33 1.21E-10GO:0006796 phosphate metabolic process 107 5.33 1.21E-10GO:0006468 protein amino acid phosphorylation 74 3.69 1.74E-08GO:0006470 protein amino acid dephosphorylation 29 1.44 1.92E-06GO:0016311 dephosphorylation 30 1.49 2.65E-06GO:0016310 phosphorylation 76 3.79 2.91E-06GO:0019538 protein metabolic process 169 8.42 3.24E-04GO:0040002 collagen and cuticulin-based cuticle development 19 0.95 4.97E-04GO:0007592 protein-based cuticle development 19 0.95 6.58E-04GO:0042335 cuticle development 19 0.95 6.58E-04GO:0044267 cellular protein metabolic process 125 6.23 0.00646GO:0009266 response to temperature stimulus 10 0.50 0.01147GO:0010171 body morphogenesis 66 3.29 0.01677GO:0006836 neurotransmitter transport 9 0.45 0.01908GO:0040032 post-embryonic body morphogenesis 4 0.20 0.03624GO:0007626 locomotory behavior 9 0.45 0.03663GO:0009409 response to cold 3 0.15 0.03968GO:0006631 fatty acid metabolic process 9 0.45 0.04597GO:0005976 polysaccharide metabolic process 9 0.45 0.04597GO:0044036 cell wall macromolecule metabolic process 4 0.20 0.04853GO:0016998 cell wall macromolecule catabolic process 4 0.20 0.04853

Cel

lula

r co

mpo

nent GO:0016021 integral to membrane 721 35.92 7.42E-13

GO:0031224 intrinsic to membrane 722 35.97 7.53E-13GO:0044425 membrane part 731 36.42 4.84E-12GO:0016020 membrane 740 36.87 8.06E-11

Mol

ecul

ar fu

nctio

n

GO:0042302 structural constituent of cuticle 64 3.19 8.56E-25GO:0005198 structural molecule activity 102 5.08 4.02E-15GO:0004674 protein serine/threonine kinase activity 73 3.64 9.17E-07GO:0004721 phosphoprotein phosphatase activity 38 1.89 3.27E-06GO:0004672 protein kinase activity 74 3.69 6.27E-06GO:0004725 protein tyrosine phosphatase activity 28 1.40 1.38E-05GO:0042578 phosphoric ester hydrolase activity 48 2.39 1.40E-05GO:0016773 phosphotransferase activity, alcohol group as acceptor 78 3.89 5.55E-05GO:0016791 phosphatase activity 42 2.09 5.84E-05GO:0004713 protein tyrosine kinase activity 56 2.79 2.49E-04GO:0016301 kinase activity 79 3.94 3.87E-04GO:0016772 transferase activity, transferring phosphorus-containing groups 84 4.19 0.00356GO:0016788 hydrolase activity, acting on ester bonds 64 3.19 0.00456GO:0015294 solute:cation symporter activity 9 0.45 0.03207GO:0005326 neurotransmitter transporter activity 6 0.30 0.03933GO:0015370 solute:sodium symporter activity 7 0.35 0.04964GO:0016290 palmitoyl-CoA hydrolase activity 3 0.15 0.04999

Table 4: Selected DEGs in MC-BA treated nematodesDown-regulated gene-classes and genesabu (Activated in Blocked Unfolded protein response):

14

abu-1, abu-6, abu-7, abu-8, abu-9, abu-10, abu-11, abu-13, abu-14, abu-15clec (C-type LECtin):clec-2, clec-3, clec-4, clec-47, clec-57, clec-62, clec-72, clec-73, clec-79, clec-92, clec-95clec-123, clec-138, clec-156, clec-158, clec-175, clec-180, clec-193, clec-203, clec-214, clec-216, clec-228col (COLlagen):col-38, col-39, col-41, col-48, col-49, col-60, col-62, col-63, col-65, col-71, col-73, col-77, col-79, col-84, col-88, col-90, col-91, col-97, col-102, col-104, col-107, col-109, col-111, col-113, col-118, col-120, col-126, col-130, col-137, col-138, col-139, col-14, col-141, col-145, col-147, col-148, col-149, col-150, col-156, col-158, col-161, col-166, col-167, col-175, col-180, col-186dpy (DumPY: shorter than wild- type): dpy-2, dpy-3, dpy-5, dpy-8, dpy-9grl (GRound-Like (grd related)): grl-5, grl-7, grl-10, grl-16, grl-21, grl-23, grl-27lgc (Ligand-Gated ion Channel): lgc-21, lgc-27, lgc-33, lgc-51, lgc-54nhr (Nuclear Hormone Receptor family):nhr-8, nhr-45, nhr-50, nhr-51, nhr-106, nhr-115, nhr-123, nhr-156, nhr-188, nhr-195pqn (Prion-like-(Q/N-rich)-domain-bearing protein):pqn-13, pqn-26, pqn-29, pqn-32, pqn-54, pqn-60, pqn-62, pqn-63, pqn-66, pqn-72, pqn-74, pqn-84, pqn-94snf (Sodium: Neurotransmitter symporter Family): snf-2, snf-4, snf-5, snf-9, snf-11srh (Serpentine Receptor, class H):srh-7, srh-49, srh-59, srh-109, srh-182, srh-183, srh-222, srh-239, srh-271, srh-301str (Seven TM Receptor): str-9, str-19, str-109, str-159, str-166ugt (UDP- GlucuronosylTransferase): ugt-2, ugt-19, ugt-21, ugt-50, ugt-62unc (UNCoordinated): unc-11, unc-17, unc-22, unc-31, unc-42, unc-43, unc-129Up-regulated gene-classes and genesclec (C-type LECtin ):clec-21, clec-22, clec-27, clec-82, clec-128, clec-135, clec-190, clec-210, clec-211, clec-241cyp (CYtochrome P450 family):cyp-14A3, cyp-25A1, cyp-33C7, cyp-33D1, cyp-34A10, cyp-34A7, cyp-34A9, cyp-35A3, cyp-35C1fbxa (F-box A protein): fbxa-18, fbxa-103, fbxa-130, fbxa-141, fbxa-213, fbxa-214fbxb (F-box B protein): fbxb-6, fbxb-9, fbxb-20, fbxb-22, fbxb-38, fbxb-47, fbxb-49, fbxb-59, fbxb-74, fbxb-85, fbxb-90, fbxb-98, fbxb-100, fbxb-102, fbxb-105, fbxb-107gcy (Guanylyl CYclase): gcy-2, gcy-4, gcy-19, gcy-25, gcy-33nhr (Nuclear Hormone Receptor family):nhr-2, nhr-15, nhr-25, nhr-82, nhr-118, nhr-126, nhr-162, nhr-185, nhr-211, nhr-221, nhr-236, nhr-255, nhr-281sre (Serpentine Receptor, class E (epsilon)): sre-8, sre-29, sre-32, sre-40, sre-52, sre-53srg (Serpentine Receptor, class G (gamma)): srg-4, srg-37, srg-44, srg-58, srg-61, srg-68srh (Serpentine Receptor, class H): srh-20, srh-32, srh-35, srh-37, srh-55, srh-67, srh-70, srh-73, srh-115, srh-128, srh-136, srh-140, srh-194, srh-200, srh-203, srh-243, srh-247, srh-251, srh-265sri (Serpentine Receptor, class I): sri-4, sri-28, sri-50, sri-54, sri-73srj (Serpentine Receptor, class J): srj-16, srj-19, srj-25, srj-37, srj-42, srj-49srw (Serpentine Receptor, class W): srw-23, srw-33, srw-38, srw-43, srw-55, srw-83, srw-96, srw-97, srw-100, srw-101, srw-112, srw-136srx (Serpentine Receptor, class X):srx-19, srx-21, srx-33, srx-38, srx-51, srx-64, srx-67, srx-85, srx-104, srx-128srz (Serpentine Receptor, class Z): srz-12, srz-20, srz-23, srz-53, srz-58, srz-64, srz-79, srz-103str (Seven TM Receptor):str-4, str-20, str-39, str-45, str-66, str-78, str-89, str-119, str-122, str-130, str-141, str-145, str-169, str-170, str-188, str-193, str-198, str-217, str-236, str-244, str-250, str-255unc (UNCoordinated): unc-2, unc-26, unc-30, unc-43, unc-49, unc-58, unc-61

Table 5: GO Terms of influenced DEGs by Lake Stößensee samples

15

GO Term DEGs (n)

% of total DEGs P-Value

August 2010

MFGO:0004872 receptor activity 16 11.76 0.01632GO:0004871 signal transducer activity 16 11.76 0.02848GO:0060089 molecular transducer activity 16 11.76 0.02848

October 2010MF GO:0016782 transferase activity, transferring sulfur-containing groups 2 5.88 0.03299

MF: molecular function

4. Conclusion

The water samples from the Stößensee contained up to 18 µg/L AOBr. But even two-fold

concentrated samples did not induce a toxic response in nematodes. In contrast, water taken

from the Tegeler See in September was able to enhance survival during thermal stress. At the

molecular level, which is arguably more sensitive than a life-cycle endpoint, only few genes

and GO Terms were affected by the lake water samples. MC-BA samples triggered

‘beneficial’ growth and brood size changes in C. elegans. Several hundred transcripts were

shown to be influenced by the exposure to MC-BA, however the up-regulation of cyp- and the

down-regulation of abu-genes may reflect some level of chronic stress. Moreover, several GO

Terms associated with metabolic and neurological processes were affected by MC-BA, but no

significantly adverse effects were observed in the life traits measured. Surprisingly, the

transcript expression pattern of MC-LR exposed nematodes was not mirrored in the MC-BA

transcripts. It is likely that the effects of MC-LR and MC-BA differed due to hitherto

unidentified compounds in the filtrate. These compounds could be responsible for the up-

regulation of several cyp-genes which could be able to detoxify the microcystin. This

assumption is underlined by Li et al. (2013) who deduced that CYPs are responsible for the

detoxification of microcystins in zebrafishes. Given that the exposure to TBBP induces two

cyp-genes (Saul et al. 2014a), suggests that the organobromines in the filtrate may act as the

cyp-inducers. C. elegans is one of the most popular invertebrate model organism with a vast

availability of biomolecular data. Being native to a compost heap might suggest that it is less

suited for aquatic studies. However, C. elegans occupies the interstitial space between

particles filled with water and can also be found in freshwater habitats (Hirschmann, 1952;

Andrassy, 1984; Zullini, 1988). Indeed, it was demonstrated that C. elegans is a suitable test

organism for freshwater sediment bioassays (Höss et al., 1999) and for aquatic toxicity tests

(Ura et al., 2002). Moreover, the uptake mechanisms of xenobiotics in C. elegans comprise

three different exposure routes: ingestion, uptake through the skin, and uptake via sensory

16

neuronal endings (Kaletta and Hengartner, 2006) which assures the uptake of the test

substances.

Nevertheless, to underpin the results of this study, more frequent aquatic nematodes such as

members of the Monhysterida and Plectida or of the subclass Chromadoria (Abebe et al.,

2008) or the well-studied cladoceran Daphnia magna (Martins et al., 2007) should be tested

in future studies (if sufficient quantities of test substances are available). Having said that, it is

apparent that the standard C. elegans plate assay appears remains an attractive option when

testing newly emerging contaminants and early evaluation of biological effects.

The results presented here suggest that AOBrs in aquatic samples may not be as toxic as

originally assumed, and that the exposure to low levels of organobromines may buffer the

effect of other toxins, at least within the studied nematode.

5. Acknowledgement

This research was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE

673/18, ME 2056/3 (RM), and PU 199/6-1 and King’s College London (SRS). Furthermore,

we thank the Caenorhabditis Genetics Centre, which is funded by the National Institutes of

Health National Centre for Research Resources, for the supply of the Caenorhabditis elegans

strains and the King’s College London Genomics Centre for their support and access to

microarray facilities. We declare that there is no conflict of interests and that the experiments

comply with the current laws of the countries where the experiments were conducted.

6. References

Abebe E, Decraemer W, De Ley P (2008) Global diversity of nematodes (Nematoda) in

freshwater. Hydrobiologia, 595:67-78. doi: 10.1007/s10750-007-9005-5

Andrassy I (1984) Klasse Nematoda. Gustav Fischer Verlag, Stuttgart, Germany

Andrianasolo EH, France D, Cornell-Kennon S, Gerwick WH (2006) DNA methyl transferase

inhibiting halogenated monoterpenes from the Madagascar red marine alga Portieria

hornemannii. J Nat Prod 69:576–579. doi: 10.1021/np0503956

Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94.

Calabrese EJ (2010) Hormesis is central to toxicology, pharmacology and risk assessment.

Hum Exp Toxicol 29:249-261. doi: 10.1177/0960327109363973

Cedergreen N (2010) Predicting hormesis in mixtures. Integr Environ Assess Manag 6:310-

311. doi: 10.1002/ieam.41

17

Ciminiello P, Fattorusso E, Forino M, Di Rosa M, Ianaro A, Poletti R (2001) Structural

elucidation of a new cytotoxin isolated from mussels of the Adriatic sea. J Org Chem

66:578-582. doi: 10.1021/Jo001437s

Covich AP, Palmer MA, Crowl TA (1999) The role of benthic invertebrate species in

freshwater ecosystems: zoobenthic species influence energy flows and nutrient

cycling. Bioscience 49:119-127. doi: 10.2307/1313537

Dalman MR, Deeter A, Nimishakavi G, Duan ZH (2012) Fold change and p-value cutoffs

significantly alter microarray interpretations. BMC Bioinformatics 13 Suppl 2:S11.

doi: 10.1186/1471-2105-13-S2-S11.

DIN (German Institute for Standardization), 2007. Water quality - Determination of

microcystins. Method using solid phase extraction (SPE) and high performance liquid

chromatography (HPLC) with ultraviolet (UV) detection. ISO 20179:2007-10

Fuller RW, Cardellina JH, Kato Y, Brinen LS, Clardy J, Snader KM, Boyd MR (1992) A

pentahalogenated monoterpene from the red alga Portieria hornemannii produces a

novel cytotoxicity profile against a diverse panel of human tumor cell lines. J Med

Chem 35:3007–3011. doi: 10.1021/jm00094a012

Furhacker M, Scharf S, Weber H (2000) Bisphenol A: emissions from point sources.

Chemosphere 41:751-756. doi: 10.1016/S0045-6535(99)00466-X

Gribble GW (2004) Natural organohalogens: a new frontier for medicinal agents? J Chem

Educ 81:1441-1449. doi: 10.1021/ed081p1441

Gribble GW (2009) Naturally Occurring Organohalogen Compounds: A Comprehensive

Update, vol 91. Springer, Vienna, New York

Gribble GW (2012) Occurrence of halogenated alkaloids. Alkaloids Chem Biol 71:1-165. doi:

10.1016/B978-0-12-398282-7.00001-1

Hirschmann H (1952) Die Nematoden der Wassergrenze mittel-fränkischer Gewässer. Zool Jb

(Syst) 81:313–436.

Höss S, Haitzer M, Traunspurger W, Steinberg CEW (1999) Growth and fertility of

Caenorhabditis elegans (nematoda) in unpolluted freshwater sediments: Response to

particle size distribution and organic content. Environ Toxicol Chem 18:2921–2925.

doi: 10.1002/etc.5620181238

Huang DW, Sherman BT, Lempicki RA (2009a) Systematic and integrative analysis of large

gene lists using DAVID bioinformatics resources. Nat Protoc 4:44-57. doi:

10.1038/nprot.2008.211

18

Huang DW, Sherman BT, Lempicki RA (2009b) Bioinformatics enrichment tools: paths

toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res

37:1-13. doi: 10.1093/Nar/Gkn923

Hütteroth A (2006) Bildung organischer Bromverbindungen in Oberflächengewässern sowie

Studien zum Verhalten während der Uferfiltration. PhD thesis (in German), Doktor

der Naturwissenschaften, Technische Universität Berlin, Berlin

Hütteroth A, Putschew A, Jekel M (2007) Natural production of organic bromine compounds

in Berlin lakes. Environ Sci Technol 41:3607–3612. doi: 10.1021/es062384k

Ioannides C (2008) Cytochromes P450: Role in the Metabolism and Toxicity of Drugs and

other Xenobiotics, Royal Society of Chemistry

Ju J, Lieke T, Saul N, Pu Y, Yin L, Kochan C, Putschew A, Baberschke N, Steinberg CE (2014a)

Neurotoxic evaluation of two organobromine model compounds and natural AOBr-containing

surface water samples by a Caenorhabditis elegans test. Ecotoxicol Environ Saf 104:194-201.

doi: 10.1016/j.ecoenv.2014.03.009

Ju J, Saul N, Steinberg CEW, Kochan C, Putschew A, Pu Y, Yin L (2014b) Cyanobacterial

xenobiotics as evaluated by a novel Caenorhabditis elegans neurotoxicity screening

test. Int J Environ Res Public Health 11:4589–4606. doi: 10.3390/ijerph110504589

Kaletta T, Hengartner MO (2006) Finding function in novel targets: C. elegans as a model

organism. Nat Rev Drug Discov 5:387-398. doi: 10.1038/nrd2031

Kim SJ, Choung SY (2009) Whole genomic expression analysis of octachlorostyrene-induced

chronic toxicity in Caenorhabditis elegans. Arch Pharm Res 32:1585-1592. doi:

10.1007/s12272-009-2111-3.

Kümmerer K (2001) Drugs in the environment: emission of drugs, diagnostic aids and

disinfectants into wastewater by hospitals in relation to other sources - a review.

Chemosphere 45:957-969. doi: 10.1016/S0045-6535(01)00144-8

Kuniyoshi M, Yamada K, Higa T (1985) A Biologically-Active Diphenyl Ether from the

Green-Alga Cladophora-Fascicularis. Experientia 41:523-524. doi:

10.1007/Bf01966182

Law RJ, Allchin CR, de Boer J, Covaci A, Herzke D, Lepom P,Morris S, Tronczynski J, de

Wit CA (2006) Levels and trends of brominated flame retardants in the European

environment. Chemosphere 64:187–208. doi:10.1016/j.chemosphere.2005.12.007

Leung MCK, Williams PL, Benedetto A, Au C, Helmcke KJ, Aschner M, Meyer JN (2008)

Caenorhabditis elegans: An emerging model in biomedical and environmental

toxicology. Toxicol Sci 106:5–28. doi: 10.1093/toxsci/kfn121

19

Li X, Ma J, Fang Q, Li Y (2013) Transcription alterations of microRNAs, cytochrome

P4501A1 and 3A65, and AhR and PXR in the liver of zebrafish exposed to crude

microcystins. Toxicon 73:17-22. doi: 10.1016/j.toxicon.2013.07.002

Lieke T, Steinberg CEW, Ju J, Saul N (2015) Natural marine and synthetic xenobiotics get on

nematode’s nerves: Neuro-stimulating and neurotoxic findings in Caenorhabditis

elegans. Mar Drugs 13. doi:10.3390/md130x000x

Linington RG, Edwards DJ, Shuman CF, McPhail KL, Matainaho T, Gerwick WH (2008)

Symplocamide A, a potent cytotoxin and chymotrypsin inhibitor from the marine

cyanobacterium Symploca sp. J Nat Prod 71:22-27. doi: 10.1021/Np070280x

Martins J, Oliva Teles L, Vasconcelos V (2007) Assays with Daphnia magna and Danio rerio

as alert systems in aquatic toxicology. Environ Int 33:414-425. doi:

10.1016/j.envint.2006.12.006

Maskey RP, Grun-Wollny I, Fiebig HH, Laatsch H (2002) Akashins A, B, and C: Novel

chlorinated indigoglycosides from Streptomyces sp GW 48/1497. Angew Chem Int Ed

Engl 41:597-599. doi: 10.1002/1521-3773(20020215)41:4<597::Aid-

Anie597>3.0.Co;2-Z

Meinelt T, Schreckenbach K, Pietrock M, Heidrich S, Steinberg CEW (2008) Humic

substances (review series). Part 1: Dissolved humic substances (HS) in aquaculture

and ornamental fish breeding. Environ Sci Pollut Res 15:17–22. doi:

10.1065/espr2007.08.448

Menzel R, Bogaert T, Achazi R (2001) A systematic gene expression screen of

Caenorhabditis elegans cytochrome P450 genes reveals CYP35 as strongly xenobiotic

inducible. Arch Biochem Biophys 395:158–168. doi: 10.1006/abbi.2001.2568

Menzel R, Rödel M, Kulas J, Steinberg CEW (2005) CYP35: xenobiotically induced gene

expression in the nematode Caenorhabditis elegans. Arch Biochem Biophys 438:93–

102. doi: 10.1016/j.abb.2005.03.020

Menzel R, Swain SC, Höss S, Claus E, Menzel S, Steinberg CE, Reifferscheid G,

Stürzenbaum SR (2009) Gene expression profiling to characterize sediment toxicity--a

pilot study using Caenorhabditis elegans whole genome microarrays. BMC Genomics

10:160. doi: 10.1186/1471-2164-10-160.

Morita K, Chow KL, Ueno N (1999) Regulation of body length and male tail ray pattern

formation of Caenorhabditis elegans by a member of TGF-beta family. Development

126:1337-1347.

Mulder C, Boit A, Bonkowski M, de Ruiter PC, Mancinelli G, van der Heijden MGA, van

Wijnen HJ, Vonk JA, Rutgers M (2011) A belowground perspective on Dutch

20

agroecosystems: How soil organisms interact to support ecosystem services. in: Guy

W (Ed.). Advances in Ecological Research, Academic Press, pp. 277-357. doi:

10.1016/b978-0-12-374794-5.00005-5

Oleksy-Frenzel J, Wischnack S, Jekel M (2000) Application of ion-chromatography for the

determination of the organic-group parameters AOCl, AOBr and AOI in water.

Fresenius J Anal Chem 366:89–94. doi: 10.1007/s002160050016

Peltonen J, Aarnio V, Heikkinen L, Lakso M, Wong G (2013) Chronic ethanol exposure

increases cytochrome P-450 and decreases activated in blocked unfolded protein

response gene family transcripts in Caenorhabditis elegans. J Biochem Mol Toxicol

27:219-228. doi: 10.1002/jbt.21473

Pouria S, De Andrade A, Barbosa J, Cavalcanti RL, Barreto VTS, Ward CJ, Preiser W, Poon

GK, Neild GH, Codd GA (1998) Fatal microcystin intoxication in haemodialysis unit

in Caruaru, Brazil. Lancet 352:21–26. doi: 10.1016/S0140-6736(97)12285-1

Rezanka T, Dembitsky VM (2003) Brominated oxylipins and oxylipin glycosides from Red

Sea corals. European J Org Chem 2003:309-316. doi: 10.1002/ejoc.200390034

Saul N, Baberschke N, Chakrabarti S, Stürzenbaum SR, Lieke T, Menzel R, Jonas A,

Steinberg CE (2014a) Two organobromines trigger lifespan, growth, reproductive and

transcriptional changes in Caenorhabditis elegans. Environ Sci Pollut Res Int 21:10419-10431. doi: 10.1007/s11356-014-2932-6

Saul N, Chakrabarti S, Stürzenbaum SR, Menzel R, Steinberg CE (2014b) Neurotoxic action

of microcystin-LR is reflected in the transcriptional stress response of Caenorhabditis

elegans. Chem Biol Interact 223C:51-57. doi: 10.1016/j.cbi.2014.09.007

Shaw SD, Blum A, Weber R, Kannan K, Rich D, Lucas D, Koshland CP, Dobraca D, Hanson

S, Birnbaum LS (2010) Halogenated flame retardants: do the fire safety benefits

justify the risks? Rev Environ Health 25:261-305. doi:

10.1515/REVEH.2010.25.4.261

Shomar B (2007) Sources of adsorbable organic halogens (AOX) in sludge of Gaza.

Chemosphere 69:1130-1135. doi: 10.1016/j.chemosphere.2007.03.074

Simmons TL, Andrianasolo E, McPhail K, Flatt P, Gerwick WH (2005) Marine natural

products as anticancer drugs. Mol Cancer Ther 4:333-342.

Steinberg CEW, Kamara S, Prokhotskaya VY, Manusadžianas L, Karasyova TA, Timofeyev

MA, Jie Z, Paul A, Meinelt T, Farjalla VF, Matsuo AYO, Burnison BK, Menzel R

(2006) Dissolved humic substances - ecological driving forces from the individual to

the ecosystem level? Freshw Biol 51:1189-1210. doi: 10.1111/j.1365-

2427.2006.01571.x

21

Steinberg CEW, Meinelt T, Timofeyev MA, Bittner M, Menzel R (2008) Humic substances

(review series). Part 2: Interactions with organisms. Environ Sci Pollut Res 15:128–

135. doi: 10.1065/espr2007.07.434

Stewart I, Seawright AA, Shaw GR (2008) Cyanobacterial poisoning in livestock, wild

mammals and birds - an overview. Adv Exp Med Biol 619:613-637. doi: 10.1007/978-

0-387-75865-7_28

Strange K, Christensen M, Morrison R (2007) Primary culture of Caenorhabditis elegans

developing embryo cells for electrophysiological, cell biological and molecular

studies. Nat Protoc 2:1003–1012. doi: 10.1038/nprot.2007.143

Svircev Z, Baltic V, Gantar M, Jukovic M, Stojanovic D, Baltic M (2010) Molecular aspects

of microcystin-induced hepatotoxicity and hepatocarcinogenesis. J Environ Sci Health

C Environ Carcinog Ecotoxicol Rev 28:39-59. doi: 10.1080/10590500903585382

Ura K, Kai T, Sakata S, Iguchi T, Arizono K (2002) Aquatic acute toxicity testing using the

nematode Caenorhabditis elegans. J Health Science, 48:583–586

Urano F, Calfon M, Yoneda T, Yun C, Kiraly M, Clark SG, Ron D (2002) A survival

pathway for Caenorhabditis elegans with a blocked unfolded protein response. J Cell

Biol 158:639-646. doi: 10.1083/jcb.200203086

Wang L, Zou W, Zhong Y, An J, Zhang X, Wu M, Yu Z (2012) The hormesis effect of BDE-

47 in HepG2 cells and the potential molecular mechanism. Toxicol Lett 209:193-201.

doi: 10.1016/j.toxlet.2011.12.014

Zullini A (1988) The ecology of the Lambro river. Rivista di Idrobiologia 27:39-58.

Figure legends

Fig. 1

The percentage change of mean lifespan compared to the untreated control during exposure to

the lake samples, the M. aeruginosa batch culture, two single organobromines and

microcystin-LR is shown. Each bar represents the mean of two to four trials with a total of at

least 188 nematodes per concentration. The error bars represent the standard error of the mean

and differences are considered significant with ** p < 0.001.

22

Fig. 2

The survival (in percent) following a thermal stress of 35°C for 6.5 hours was monitored.

Each bar represents the mean of three to five trials with a total of at least 124 nematodes per

concentration. The error bars represent the standard error of the mean and differences are

considered significant with * p < 0.05.

Fig. 3

The reproductive output was assessed over three trials with a total of 30 nematodes per

concentration. Each bar represents the mean of the three trials and the error bars represent the

standard error of the mean. Differences are considered significant with * p < 0.05 and ** p <

0.001.

23

Fig. 4

The body length of the nematodes was measured at the sixth day of adulthood in three to four

independent trials with a total of 81-156 nematodes per concentration. Each bar represents the

mean of the trials and the error bars represent the standard error of the mean. Differences are

considered significant with * p < 0.05 and ** p < 0.001.

24