1. Pollutants in ponds and biodiversity Robert Mandiki Patrick Kestemont University of Namur, URBO

2. Introduction  Release of pollutants in the pond ecosystem has been pointed out as one of the major contributing factors to the decline in organisms living in surface water areas including ponds.  Indeed, high accumulation of some pesticides may disturb various physiological mechanisms of aquatic organisms, such as: - Inhibition of the metabolic pathways controlling the metamorphosis process: low growth and survival rates, malformations, etc - Endocrine disruption of reproductive function: decrease in reproductive efficiency, male sex reversal, unbalance in population structure, etc - Decrease in immune defence and disease resistance: increase of pathological incidence and sensitivity to parasites and pathogens

3. Objectives  The WP5 of the Pondscape project deals with the anthropogenic pollution aspects, and focus on the following specific objectives: 1. To determine whether there are seasonal peak levels of some pesticides in ponds in relation to the pesticide applications scheduled by farmers 2. To verify the impact of the eventual presence of pollutants on some indicators (aromatase activity and VTG induction) of disruption in reproductive function: as a tool for of disruption in reproductive function: as a tool for confirming the presence or absence of oestrogenic compounds confirming the presence or absence of oestrogenic compounds 3. To study the bio-availability of pollutants (pesticides and heavy metals) in relation to pond management (see WP4) by field and mesocosm ecotoxicological studies using a sedentary amphibian Rana temporaria in the Belgian water ponds.

4. 1. Pesticide loads and seasonal changes 1.1. Materials and methods:  Fifteen ponds were selected in Flanders (six) and Wallonia Region (nine) in areas under intensive or semi-intensive agricultural activities vs natural reserves  Water was sampled once over three seasons: mid-April, beginning of July and mid-October  Analyses of widely used pesticides, namely: (1) herbicides: atrazine and its metabolites, simazine, diuron, isoproturon, glyphosate and its surfactant AMPA (2) insecticides : aldrine, chlorfenvinphos,  -endosulfan, dicofol, benzo(b)fluoranthène and benzo(a)pyrene  After water filtrations and extractions, the pesticides were analysed by GC-MS methods

5. 1. Pesticide loads and seasonal changes 1.2. Results:  Whatever the type of pond and season, no insecticide or herbicide was detectable in the selected ponds, except for trace levels of glyphosates and high peaks of isoproturon  Levels of isoproturon peaked especially in October whatever the type of pond (Fig. 1)  Low levels (  10 ppb or less) of glyphosates and its surfactant were detected over all the seasons in almost all the investigated ponds Isoproturon levels were higher in ponds located in agricultural areas vs those in natural reserves (Fig. 2)

6. 2.Other pollutants and occurrence of disruption of reproductive functions  In 40% of the investigated ponds, the isoproturon levels are comparable to values reported in other European countries in other surface water bodies  High accumulation of isoproturon in surface water may affect the detoxification pathways of tadpoles of amphibians and thereby interfere with their welfare (Greulich et al., 2002). 2.1. Materials and methods:  Apart from insecticides known for inducing estrogenic effects (endosufan and polycyclic-aromatic hydrocarbons - PAHs), phenolic compounds (octylphenol, nonylphenol) were assayed in all the water samples used for the dynamics in pesticides loads.  The selected estrogenic compounds were analysed by CG-MS as for previously for pesticides.

7.  Evaluation of the eventual occurrence of reproductive disturbances was done using juveniles of Carassius auratus from six intensive or semi-intensive ponds.  Ten juvenile fish (15 – 65g) from each of these ponds were sampled for blood, brains and gonads.  For each pond, five females and five males were compared for plasma VTG and gonad or brain aromatase activity. VTG level was determined by ELISA method, while brain or gonad aromatase activity was assayed by radioimmunoassay. 2.2. Results:  No phenolic compounds or HAPs was detectable whatever the type of pond.  No abnormal profile was observed in the in vivo indicators of the occurrence of estrogenicity.  Indeed, VTG levels were 300 times higher in females than in males (Fig. 3),

8.  Moreover, the lower level of aromatase activity in male juveniles indicate no disturbance in sex-steroid production (Fig. 5),  Aromatase activity did not differ among the types of ponds (Fig. 6), confirming the lack of estrogenic compounds anywhere.  A normal ratio of intersex-reversal males was observed: 3.3% The latter result and the normal profiles in VTG and aromatase activity in male juveniles indicate that fish populations in the investigated ponds are not exposed to any release of estrogenic compounds.

9. 3.1. Field survey:  Management of ponds in Tommelen by the WP4  Sampling water and sediments before and after dredging  Heavy metal analyses by mass spectrometry methods; isoproturon by GC-MS. 3. Effects of pond management on bio-availability of pollutants and on biodiversity  A field survey of tadpole growth was conducted between February and May 2009 in Tommelen: - Three dredged ponds vs 3 control non impaired - 60 tadpoles at stage 24 - Growth and survival, developmental stages - Glutathione-S-tranferase (GST) 3.2. Eco-toxicological tests:  Fertilized eggs were collected from Tommelen in early March 2009 and transferred to tank conditions in Namur.

10.  Tadpoles at stage 24 were submitted to isoproturon and cadmium challenge during one month at various concentrations  Experimental design:  ISO = Isoproturon  Cd = cadmium  CT = control  Doses: - Iso 1, 10 and 100 µg/L - Cd 1 and 10 µg /L - IsoCd 1 and 10 µg /L  Variables: - Growth, malformations and developmental stages - Detoxification enzymes (GST) - Stress proteins (ST41)

11. 3.3. Results: 3.3 1. Field survey:  Heavy metal and isoproturon analyses are on going.  In vivo tadpole test: - Survival was lower in tadpoles reared in control ponds vs dredged ponds - Tadpoles in dredged ponds showed higher growth rate than controls but not in ontogeny of developmental stages. Mean growth rate: 348 ±78 mg controls 496 ± 57 mg dredged ponds - GST level was significantly higher in control tadpoles vs dredged ponds (Fig. 7), interest in heavy metal contents

12. 3.3.1. Eco-toxicological study:  Survival and developmental stages did not differ whatever the pollutant or the doses  GST level did not differ among treatments but was high in tadpoles at stage 39 than those at stage 41 at the same age (Fig. 9), indicating that pollutants interfered with the establishment of developmental process.  High doses significantly decreased growth rate (Fig. 8) but the dose effect does not seem linear