DOES ASELLUS AQUATICUS AVOID SEDIMENT KOEN CONTAMINATED BY THE INSECTICIDE WORKEL LUFENURON?
Aquatic Ecology and Water Quality Management Department
Alterra – Environmental Risk Assessment
Does Asellus aquaticus avoid sediment contaminated by the insecticide lufenuron?
Master thesis for the attainment of the academic degree of MSc. Hydrology and Water Quality - Aquatic ecology
Rapportnr. 010/2011
Koen Workel
September 2011, Wageningen
Supervisors: Dr. ir. Ivo Roessink Dr. Ir. Edwin Peeters MSc Jacqueline Augusiak Dr. Theo Brock
Aquatic Ecology and Water Quality Management Department
Alterra – Environmental Risk Assessment
Niets uit deze uitgave mag worden verveelvoudigd en/of openbaar gemaakt door middel van druk, fotokopie, microfilm of op welke andere wijze ook zonder voorafgaande schriftelijke toestemming van Alterra Wageningen UR.
Alterra B.V. aanvaardt geen aansprakelijkheid voor eventuele schade voortvloeiend uit het gebruik van de resultaten van dit onderzoek of de toepassing van de adviezen. Acknowledgment ‘Doing a thesis should be fun’, said Ivo in our first conversation. Giving the final touch to this report I realize that it was indeed fun! Did very nice experiments, learned many things about research and worked with pleasant people which that made me feel comfortable that really have great knowhow about aquatic ecology and risk assessment. In the first place many thanks to the ERA team of Alterra and Aquatic Ecology and Water Quality Management Department giving me the opportunity to do my master thesis under their supervision. Special thanks go to Ivo Roessink for his enthusiasm, always good mood and supervision. Many thanks to Jacqueline Augusiak for the nice practical cooperation and supervision and Theo Brock for his critical view and good ideas. Thanks to Edwin Peeters for his good supervision from the WU. Could not have done it without help from Hans Zweers, Arrienne Matser, Rene van Wijngaarden, Marie-Claire Boerwinkel, Laura Buijse and others from the ERA team. Thanks to John Beijer and Wendy Beekman for their support in the AEW labs. Summary In 2009 Brock and co-workers , conducted a study where in artificial ditch mesocosms the impact of a benzoylurea (lufenuron) on macroinvertebrates with sprayed and non-sprayed sections was assessed. Lufenuron inhibits the synthesis of chitin and affects the moulting and metamorphosis of arthropods. It was hypothesized that sensitive populations would recover faster from insecticide stress when non-contaminated refuges would be in close proximity. This was confirmed for macroinvertebrates related to the water column, however, sediment dwelling organisms were absent for a long time predominantly on the sprayed sections. It was reasoned that this could be due to (i) the poor ability of sediment bound organisms to migrate, (ii) the long lasting toxicity of lufenuron or (iii) avoidance behaviour. This research aimed to unravel whether one of these possible explanations caused the phenomenon. It was chosen to do a toxicity experiment with Asellus aquaticus, a sediment dwelling organism that has the ability to migrate, to investigate its sensitivity to lufenuron. Sediment was spiked with different concentrations and the specimens were exposed for 21 days. Due to turbid conditions caused by suspension of the sediment, 3 out of 5 replicates were sacrificed at day 5 and the remaining 2 at day 21. No treatment related effects were observed at any of the concentrations at day 5. At the end of the experiment however, pronounced effects were observed where all specimens died at the highest concentration. With the confirmation that A. aquaticus was affected by lufenuron, their avoidance behaviour was tested. In total 16 aquaria (50 x 30 x 30cm) were used divided into 8 controls and 8 treatments. Sediment was divided into a contaminated part, spiked with lufenuron and an uncontaminated part. 10 specimens of A. aquaticus were released in the middle and their positions were manually scored on hourly basis. The hypothesis that they would be more often observed on the uncontaminated part was rejected by the results. There was variation in between the aquaria but there was no avoidance observed. To test the general concept and the used methodology, also the species Anisoptera (Odonata), Zygoptera (Odonata) and Agrypnia sp. (Trichoptera) were tested on avoidance behaviour. For each species 4 aquaria were used each containing 5 specimens. For these species was no avoidance behaviour observed as well. Zygoptera, however, showed in all 4 aquaria a significant preference for one side of the aquaria which probably is caused by the fact that an uneven number of specimens were used (5) and ones they were distributed, their natural ‘sit and wait’ ambush behaviour made that they hardly moved causing a preference for one side. To address both avoidance behaviour as toxicity in a field environment, sediment spiked with different concentrations lufenuron placed in small containers were inserted in an artificial mesocosm ditch. It was expected that colonization by macroinvertebrates would be negatively related to an increasing lufenuron concentration. After an incubation period of 55 days, the containers were removed from the ditch. The found macroinvertebrates were sorted on taxa per container and their dry weight was determined. The results show that there was no significant treatment related response which indicate that there was again no avoidance behaviour observed. Since A. aquaticus has abilities to migrate under water and midges are able to recolonize through the air via egg deposition by adults, it is reasonable that a poor ability to migrate did not caused the phenomenon. Although food avoidance could possibly have taken place, no avoidance behaviour has been observed. Toxicity has been observed by Brock and co- workers and in the toxicity experiment but not in the colonization experiment which might be caused by a constant inflow of new macroinvertebrates or external input of clean food source. Taking into account that there is no avoidance behaviour observed and a poor ability to migrate is probably not an issue as well, it is reasonable that the toxicity of lufenuron possibly caused the long lasting absence of sediment dwelling organisms.
Table of Contents 1 Introduction ...... 2 1.1 Research aim ...... 3 1.2 Research question and hypotheses ...... 4 2 Method ...... 6 2.1 Toxicity experiment ...... 6 2.2 Avoidance behaviour experiment ...... 12 2.3 Colonization experiment ...... 17 3 Results ...... 19 3.1 Toxicity experiment ...... 19 3.2 Avoidance behaviour experiment ...... 20 3.3 Colonization experiment ...... 31 4 Discussion and conclusion ...... 35 4.1 Discussion ...... 35 4.2 Conclusion ...... 37 5 References ...... 38 6 Appendix ...... 40
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1 Introduction
The yield of agricultural farmers has increased worldwide since the use of pesticides, new seed varieties and fertilizers. However, pesticides do not only affect the target organisms but other exposed species as well. This in turn negatively affects biodiversity (McLaughlin and Mineau 1995; Geiger, Bengtsson et al. 2010). Agricultural land in the Netherlands is commonly boarded by small ditches for drainage and with the application of pesticides on agricultural fields, pesticide fractions enter adjacent water bodies through spray drift, runoff, and drainage. Depending on the hydrophobicity of the pesticide, the water column and/or the sediment compartment in the drainage ditch may become contaminated. To assess potential negative effects of pesticides on the environment, experiments are performed on a set of aquatic standard test species such as Oncorhynchus mykiss (rainbouw trout) (Li, Zlabek et al. 2010), Daphnia magna (water flea), Lemna minor L (duckweed) (Singh, Chandra et al. 2008) and Pseudokirchneriella subcapitata (algae) (Perez, Loureiro et al. 2010). Most of such experiments are performed on organisms that live in the water column like fish, Lemna and algae. Due to the high hydrophobicity of some pesticides (e.g. pyrethroids and benzoylurea compounds) there is a transfer from the water column to organic matter in the sediment and research on the potential negative effects on benthic communities is necessary (Amweg, Weston et al. 2006). An overview of frequently used benthic organisms in risk assessments are the midge Chironomus riparius, oligochaetes like Lumbriculus variegates and Tubifex tubifex (Bettinetti, Croce et al. 2005; De Lange, De Haas et al. 2005; Lagauzere, Terrail et al. 2009; Vogt, Hess et al. 2010). Other test organisms are Asellus aquaticus and Ephoron virgo in freshwater environments. In estuarine and marine environments frequently amphipods are used such as Corophium mullisetosum and Ampelisca abdita (Menchaca, Belzunce et al. 2010). Assessments of the potential negative effects of contaminated sediment to benthic organisms demonstrated reduced reproduction of C. riparius when exposed to cadmium via the sediment (Vogt, Hess et al. 2010). Furthermore the oligochaete T. tubifex showed reduced reproduction in relation to DDT contaminated sediment (Bettinetti, Croce et al. 2005). Behavioural responses were tested with A. aquaticus with sediment contaminated with polycyclic aromatic hydrocarbons (PAH) and showed avoidance response towards contaminated sediment (De Lange, Sperber et al. 2006).
Brock and co-workers (2009;2010) performed a study in artificial ditch mesocosms where the impact of a benzoylurea insecticide (lufenuron) on aquatic macroinvertebrates with sprayed and non-sprayed sections was assessed (see figure 1). It was hypothesized that sensitive populations would recover faster from insecticide stress when non-contaminated refuges would be in close proximity. Lufenuron was sprayed to achieve a concentration of 3 µg/L in the water column but due to its hydrophobic properties it adsorbed fast to organic matter and the sediment compartment. This fast dissipation from the water column resulted in a measured maximum concentration of 0.1 µg/L lufenuron one day after application.
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figure 1. Overview of the experimental design of the ditch experiment with the insecticide lufenuron that included both sprayed and unsprayed ditches and ditch sections (Brock, Roessink et al. 2009)
In the ditch experiment with lufenuron, the maximum effect on arthropod populations (the sensitive taxonomic group) appeared 3 to 6 weeks after application. Lufenuron inhibits the synthesis of chitin which affects the moulting and metamorphosis of arthropods and explaining the latency of effects. The hypothesis that populations would recover faster with adjacent non-sprayed sections was proven during the experiment for water column and vegetation related species such as Chaoborus, Cloeon and Zygoptera which suggests that there is migration between the non-sprayed and sprayed sections. Sediment dwelling organisms however, such as larvae from the taxa Caenis, Polycentropidae, Chironomus, Tanypodia and Isopoda showed relatively long lasting effects that were predominantly observed in the sprayed ditch sections. The effect on these taxa at the non-sprayed sections within a treated ditch were absent or relatively minor. Hence, the effects were relatively long lasting but local for the mentioned sediment dwelling organisms. (Brock, Belgers et al. 2010) argues that this phenomenon might be explained by (i) the local long lasting toxicity of the compound, (ii) the poor ability of these organisms to migrate between sprayed and non-sprayed sections or (iii) due to avoidance behaviour.
1.1 Research aim This research aims to investigate which processes (migration, avoidance, or toxicity) could be responsible for the observed phenomena of long-term effects and poor ecological recovery of sediment-associated arthropods in the ditch sections treated with lufenuron. The possible lack of migration can be tackled by selecting a sediment dwelling arthropod that showed the long lasting local response in the treated ditch sections of the experiment of Brock et al. (2009; 2010). By selecting such a mobile arthropod, the main issues that remain as possible mechanism are toxicity and avoidance behaviour. Avoidance of predators, but also for chemical hazards such as pesticide-contaminated sediment by potentially sensitive arthropods, is important for survival and a known response behaviour (Dicke and Grostal 2001). An experiment with six different (terrestrial) spider mite populations with 4 different pesticide residues showed significant avoidance behaviour (Suiter and Gould 1994) and Lumbriculus variegatus actively avoided sediment contaminated with the pesticide Imidacloprid (Sardo and Soares 2010). We selected the isopod A. aquaticus since it is one of the species that showed long term local responses to lufenuron exposure in the ditch experiment. In addition, the species is known for behavioural responses in the sense that sediments contaminated with PAH are avoided (De Lange, Sperber et al. 2006). Behavioural responses of A. aquaticus however are not known for persistent sediment bound insecticides such as lufenuron. Known is that A.
3 aquaticus is a mobile organism with a daily migration pattern allowing the species to migrate between the sprayed and non-sprayed sections (Andrikovics 1981; Marklund, Blindow et al. 2001). This makes A. aquaticus a relevant test organism for a behavioural response experiment. In this Master thesis a 21-d toxicity experiment with A. aquaticus is performed as well, with a focus on lufenuron exposure via the sediment. The results of this toxicity test are used to properly design the avoidance experiment and to evaluate whether local sediment toxicity, or the combination of avoidance and toxicity, might explain the long-term effect without recovery observed in the lufenuron-treated ditch sections.
The lufenuron concentration in the sediment phase has unfortunately not been measured by Brock and co-workers (2009) during their study but was calculated to be between 15 and 325 µg/kg using the TOXSWA exposure model. It should be noted that in all treated ditch sections the same lufenuron dose was used (resulting a nominal concentration of 3 µg lufenuron/L in the water column). For adult specimens of Chironomus riparius a 28 day NOEC of 40 µg/kg was reported. For larval Chironomus riparius it was observed that a concentration above 60 µg/kg caused a serious decline of the survival rate (Hooper, Sibly et al. 2005). There are however some differences between A. aquaticus and C. riparius; the main difference being that C. riparius larvae live in the sediment and A. aquaticus lives on the sediment. Secondly, A. aquaticus is bigger and possibly more robust. Hence, it was expected that effects would occur at higher concentrations than for C. riparius. Therefore a laboratory toxicity test is conducted with a concentration range based on found data. This toxicity experiment was performed with sediment spiked with different concentrations lufenuron. To examine the selectivity of A. aquaticus for lufenuron the present study involves a choice experiment where sediment in an aquarium is divided in an uncontaminated and contaminated part. Hypothesized is that A. aquaticus will avoid the contaminated section and consequently will spend more time on the clean sediment section. Similar experiments were conducted with three other species (Anisoptera (Odonata), Zygoptera (Odonata) and Agrypnia sp. (Trichoptera). These species are not strictly sediment bound what makes them less relevant for testing contaminated sediment avoidance in relation to the previous experiment. Nevertheless, it was of interest to test whether the chosen experimental setup was suitable for other species as well. Additionally, a sediment-container experiment was conducted in an outdoor experimental ditch. These containers contained sediment spiked with different concentrations lufenuron and were used to study colonization by aquatic invertebrates. This was done to address avoidance and toxicity. It was hypothesized is that a negative relation exists between an increasing lufenuron concentration in the sediment and the abundance of aquatic arthropods that colonised the sediment-containers.
Box 1 Introduction to lufenuron The insecticide lufenuron belongs to the chemical group of benzoylureas and is used to control insect pests. Lufenuron causes inhibition of chitin synthesis, a compound which is necessary for the build-up of exoskeletons. It is a hydrophobic, persistent chemical with an octanol-water partition coefficient of 5.12. It is used for the control of several pest species such as Coleoptera, some Thysanoptera, Lepidoptera, and rust mites of the family Eriophiidae (http://sitem.herts.ac.uk/aeru/footprint/en/Reports/420.htm). It is applied for the protection of a large variety of crops such as cotton, maize, potatoes, sugar beets and fruits such as grapes and citrus. Risk assessments indicate that lufenuron has “low acute, and short- and long term toxicity to birds, low acute and chronic toxicity to fish and algae and high acute and chronic toxicity to aquatic macroinvertebrates”It is not harmful to humans (http://www.fao.org).
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1.2 Research question and hypotheses The main research question is: Did a poor migration ability, toxicity or avoidance behaviour cause the long term local effect for sediment dwelling organisms in the mesocosm experiment of Brock et al. (2009;2010)?
The main research questions contains basically three sub-questions. The question whether a poor migration ability by sediment dwelling organisms caused the long-term local effect is basically covered with the choice for A. aquaticus because it can walk several meters per day. The next question is whether A. aquaticus is sensitive to lufenuron exposure via the sediment and the third question whether it actively avoids sediment contaminated with lufenuron.
Three different experiments are conducted in order to answer these question. A toxicity experiment will give insight in the sensitivity of A. aquaticus to lufenuron exposure via the sediment, the avoidance experiment addresses avoidance behaviour and the colonization experiment both toxicity and avoidance of arthropods. Therefore the following hypotheses have been composed.
The hypothesis for the toxicity experiment is: Asellus aquaticus is sensitive to lufenuron exposure via the sediment.
The hypothesis for the avoidance behaviour experiment is: Asellus aquaticus, Anisoptera (Odonata), Zygoptera (Odonata) and Agrypnia sp. will actively avoid sediment contaminated with the pesticide lufenuron.
The hypothesis for the colonization experiment: There will be a negative correlation between the abundance of specimens and taxa that will colonize the containers with sediment and an increasing concentration of lufenuron in the sediment.
In the second chapter, a description is given of the methods used for the toxicity , avoidance behaviour and field experiment. The results of the three experiments are presented in the third chapter followed by a discussion and conclusion in the fourth chapter. The references are listed in chapter 5 and finally, in the appendix additional background information is included.
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2 Method This chapter describes the methods used for the toxicity, avoidance behaviour and the colonization experiment. For all experiments, it was chosen to use a sediment composed according to OECD (Organisation for Economic Co-operation and Development) guidelines. A reason for this choice was that the sediment was in any case free of contaminants. Furthermore, we could ensure a constant sediment composition and easy reproducibility.
2.1 Toxicity experiment Available literature did not provide information concerning Lethal Concentration 50 (LC50) and No Observed Effect Concentrations (NOEC) of lufenuron for Asellus aquaticus. Due to the mode of action of lufenuron (see Box 1) it was expected that it would affect A. aquaticus which corresponds with the results of (Brock, Roessink et al. 2009). When effects would become visible in relation to the concentration was however unknown. For the avoidance test it was necessary to use a concentration by which the animals would be affected but was not lethal during the 5 day avoidance behaviour experiment. Therefore a toxicity experiment was conducted with sediment, spiked with different concentrations of lufenuron. This section describes the methodology of this toxicity experiment pointing out the experimental setup, spiking procedure, the origin of the test species A. aquaticus and sampling.
Experimental setup The toxicity test was conducted in 2.5 L (Weck, Rundrand-Glas 100) beakers situated in a water bath to ensure a constant temperature and light regime. Since A. aquaticus is more active at night (Andrikovics 1981) or under dark conditions, it was decided to create permanent dark conditions during the test. During sampling, a red light was turned on to provide light for the researchers and to maintain dark conditions for the animals (see figure 2). A total of 30 jars with lids to prevent evaporation was placed in the water bath, all containing a layer of approximately 1 cm of sediment, 10 specimens of A. aquaticus, 2 litres of aerated groundwater, that was collected at the Sinderhoeve (www.sinderhoeve.org) and a 150 mm glass pasteur-pipette (VWR International) for aeration of the test vessels (see figure 2). Sediment was added first into test jars, followed by addition of a small water layer and allowed to settle for 2 days. After this period, the jars were carefully filled up with more water. This procedure helped to reduce sediment perturbation to a minimum. This latter step was performed 3 days before the start of the experiment so that any suspended particles had time to settle.
figure 2. Experimental setup of the sediment toxicity test with Asellus aquaticus under light and dark(red) conditions
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The test room had two entrances and was disconnected from neighbouring space by curtains on two sides. It was expected that if any external (demonic) factor would influence our results, they would occur from one or both entrances (see figure 3). The pictured setup allowed for equal distances of the test setup to the entrances. It was decided not to further randomize the setup in order to minimize the chances of human error and consequently avoid cross contamination.
Closed curtain 0A 0B 0C 0D 0E Entrance 20A 20B 20C 20D 20E Closed 40A 40B 40C 40D 40E Entrance Curtain 80A 80B 80C 80D 80E Door 160A 160B 160C 160D 160E 320A 320B 320C 320D 320E Wall figure 3. Overview of the location of the test vessels of the sediment toxicity test with lufenuron and A. aquaticus in the water bath
Sediment Our method for mixing OECD standard sediment was mainly based on OECD Test No. 218: “Sediment-Water Chironomid Toxicity Using Spiked Sediment” and minor practical adjustments have been made based on the method of ISO 16191 norm (OECD 2004). The sediment consisted of a mixture of quartz sand (Geba 0.06-0.25 mm from Eurogrid Dordrecht, The Netherlands), kaolinite clay (Sigma Aldrich), peat (Klasmann Deilmann Benelux), and calcium carbonate (Sigma Aldrich, 99% pure) (see figure 4). The respective dry weight fractions that we applied are given in 0. The OECD guideline prescribes 75-76% quartz sand, 20% kaolinite clay, 4-5% peat and another 0.05-0.1% of calcium carbonate (see figure 4). It was decided to include 5% of peat since A. aquaticus feeds on organic matter and adding additional food to the test would complicate the interpretation of results. We expected no starvation due to food limitation with 5% of peat in the sediment. After mixing all ingredients, a pH adjustment with calcium carbonate was required to achieve a pH of 6.5-7.5 as recommended by the guideline. We found that more calcium carbonate was required than the suggested 0.1 % to raise the pH to recommended levels. ISO 16191 norm also mentions the use of 1% of calcium carbonate instead of 0.1% according OECD. Hence, we decided to lower the use of kaolinite clay to 19% of sediment dry weight. Kaolinite clay came as a very fine, light weighted powder that easily gets suspended in water. This effect could disturb the visibility of test animals, another reason to lower this fraction instead of other ingredients. OECD 218 also recommends the use of peat with a particle size of ≤ 1 mm. For this, it was necessary to sieve the available peat. Grinding demanded too much time. It was also found that sieving required less time using 2 day air dried peat. After mixing all ingredients and adjusting the pH the sediment was wetted to precondition the peat particles and prevent them from floating in the final test setup.
7 table 1. Ingredients OECD standard sediment Constituent % dry weight Quartz sand 75% Kaolinite clay 19% Peat 5% Calcium carbonate 1%
figure 4. Peat (left) and Quartz sand (right)
Spiking The sediment was spiked with 5 different concentrations of lufenuron plus controls (see table 2). For each concentration level 5 replicates were used. table 2. Nominal spiking concentrations (in µg lufenuron/kg dry weight sediment) and number of test vessels used in the laboratory sediment toxicity test with A. aquaticus. Concentration Replicates 320 µg/kg dw 5 160 µg/kg dw 5 80 µg/kg dw 5 40 µg/kg dw 5 20 µg/kg dw 5 0 µg/kg dw 5 Total 30
Brock and co-workers (2009) spiked the sediment via the water phase by spraying. Since we did not want it to be advantageous for A. aquaticus to dig in the sediment to avoid exposure, it was decided to spike the whole sediment instead of only the top layer. A second reason was that the completely spiked sediment was easier to use when setting up the experiment (e.g. less influence of small variations in thickness of the sediment layer in test vessels). The sediment was spiked in 6 portions of 750 gram dry weight in a 2.5 L glass jar in a fume hood. By adding demi water, a slurry was created that was kept homogenous with a mixer. The slurry was needed because lufenuron has strong hydrophobic properties (octanol-water partition coefficient at pH 7, 20˚C of 5.12) and having a non-homogeneous substance would cause heavy loaded organic particles which can be homogeneously mixed. However, it was preferred that all organic matter got evenly loaded. The FMI Lab Pump QG6 inserted the spiking solutions in portions of 100 ml with a speed of ±1.5 ml/h. After a period
8 of settling, the water layer on top of the sediment was removed. However, the water content of the sediment was too high for immediate use and needed to be removed by centrifugation (see figure 5). First analysis of the concentration lufenuron in the sediment showed that the found concentrations were about 40% less than intended. The final analysis however was conducted after finishing this report so the final results could not be incorporated. Consequently, intended concentrations are used in this report.
figure 5. Sediment spiking in the fume hood (left) and settling of spiked sediment (right)
Asellus aquaticus The isopod Asellus aquaticus is a freshwater sediment dwelling organisms that feeds on organic matter and detritus (see figure 6). It is found in all types of waters including rivers, lakes, and ditches and often used as water quality indicator (Aston and Milner 1980; Rask and Hiisivuori 1985; Maltby 1991; Whitehurst 1991).
figure 6. Asellus aquaticus (www.naturfoto.cz)
A. aquaticus was collected in the vicinity of Wageningen in an area called ‘t Binnenveld (see figure 7). Approximately 400 specimens were caught in a small drainage ditch of approximately 1.5 m wide using a fine meshed net. The collected specimens were kept in an aquarium that was aerated. Leaves from Populus canadensis were provided as food. The animals were acclimatized with stepwise replacement of their natural water by groundwater from the Sinderhoeve.
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figure 7. Sampling location of A. aquaticus used in the toxicity experiment (left) and procedure of random selection of test individuals to obtain equal sets of test individuals for each test vessel (right)
Random insertion of A. aquaticus to the test vessels was realized by filling plastic cups with water and adding individuals one by one till all cups looked as equal as possible (see figure 7). Another 20 specimens were used for length and weight characterization of the population (see table 3). The length was measured via pictures taken with a film microscope (magnification: 0.5 x 0.5 x 1) and the computer software Axiovison. Next, the wet weight and dry weight was determined. For drying, the animals were kept at 105˚C for 24 h (see annex A-1). table 3. Overview of length-weight values of A. aquaticus used in the toxicity experiment (n=20) (see annex A-1) Description Average SD SD % Length [mm] 7.205 1.186 16.5 Wet weight [g] 0.044 0.021 47.6 Wet weight/length [g/mm] 0.006 0.002 36.1 Dry weight [g] 0.007 0.003 43.8 Dry weight/length [g/mm] 0.001 0.000 31.5
Measurement endpoints for the sediment toxicity test Since the effect of lufenuron on A. aquaticus is investigated it is important to check water quality variables that could possibly affect the animals. It was decided to measure oxygen, pH, electrical conductivity and temperature (see annex A-2). The experiment started with measuring water quality parameters and adding specimens of A. aquaticus to the jars and turning the aeration on. The equipment used are WTW pH 323 for pH measurements, Eijkelkamp multimeter 18.28 for electrical conductivity and the WTW Oxi 330 for oxygen and temperature measurements. The equipment was always calibrated before use. The aeration was very sensitive and therefore difficult to mitigate. Consequently the jars became turbid in a short period of time (see figure 8). The intention was to regularly observe A. aquaticus and to score their status (dead/alive/affected). However, since the water became turbid the test animals were not visible anymore. By stopping aeration and adding water it was atemped to improve visibility but this failed. Since the necessity is to know the effect after 5 days (duration of avoidance experiment), it was decided to sacrifice three jars per concentration at day 5. For the scoring of A. aquaticus, a bucket was placed in the sink with a fine mesh sized sieve. The water got poured over the sieve leaving the animals behind which were scored dead or alive. Samples of suspended solids and water to determine the lufenuron concentration were taken of each test vessel with an electrical pump (accu-boy) and a pipette of 50 ml. About 40 ml from
10 the water got filtered with a Whatmann GF/F filter and stored at -30˚C and a small volume of the filtered water was put in a WISP vial and stored in a fridge for analysis. Next a sediment sample from each test vessel was taken for lufenuron concentration determination. After 21 days the two remaining jars underwent the same procedure which was within the range (two to six weeks) wherein the maximum treatment-related effects were observed in (Brock, Roessink et al. 2009).
figure 8. Overview of the test vessels of the sediment toxicity test in the water bath. Note the turbidity of the water
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2.2 Avoidance behaviour experiment This second experiment is conducted to test the hypothesis that Asellus aquaticus, larvae from Zygoptera (Damselfly), Anisoptera (Dragonfly) and Agrypnia sp. (Caddisfly) actively avoid sediment contaminated with lufenuron. This chapter describes the methodology of this avoidance behaviour experiment pointing out the experimental setup, manual scoring and test species.
Experimental setup The avoidance behaviour experiment was conducted for 5 days in 16 aquaria (50 x 30 x 30 cm) situated in a water bath. The aquaria’s are divided in 8 controls with clean sediment only and 8 aquaria with both clean (50% of surface area) and lufenuron-spiked sediment (50% of surface area). Each aquarium contained approximately 8 cm of water and approximately 1 cm of sediment. Again OECD sediment was used and treated according to the procedure used in the toxicity experiment except that the sediment needed more CaCO3 to raise the pH to 6.4. The sediment was spiked according the above described procedure with the highest concentration used in the toxicity experiment because this concentration was found to be lethal at the end of the 21-d toxicity experiment but not after 5 days of sediment exposure. Consequently, exposure might give the animals a good reason to avoid it. The controls were filled with uncontaminated sediment while the treatments received half uncontaminated and half contaminated sediment (see figure 9). The first analysis of the concentration in the sediment showed that there was no cross contamination of lufenuron and that the animals had a clear choice between clean and contaminated sediment. The aquaria were stepwise filled with Sinderhoeve groundwater by first adding a layer of 2 cm, a few days later 4 cm and finally 2 cm of copper free tap water because we ran out of groundwater.
figure 9. Constructing the sediment layer of the “treated” aquaria for the avoidance experiment. 50% of the sediment surface received clean and the other 50% sediment spiked with lufenuron.
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Test species avoidance behaviour experiment
Asellus aquaticus The specimens were collected in ‘t Binnenveld, the same area as where the specimens for the toxicity experiment originated from. A characterization of the A. aquaticus’ is shown in table 4. The characterization was performed according to the same procedure as in the toxicity test. table 4. Characterization of Asellus aquaticus used in the avoidance experiment (n=20) (see annex B-1) Asellus aquaticus Description Average SD SD % Length [mm] 8.45 1.31 15.4 Wet weight [g] 0.023 0.009 43.0 Wet weight/length [g/mm] 0.003 0.001 32.6 Dry weight [g] 0.004 0.001 29.8 Dry weight/length [g/mm] 0.001 0.000 17.7
Zygoptera, Anisoptera and Agrypnia sp. After the experiments with A. aquaticus it was decided to make use of the existing experimental setup and test three other species for method development purposes. The actual choice for the species was dependent on the availability of suitable species. While searching for species in ditches at the Sinderhoeve, it appeared that the only species with a by eye visible size and that could be caught in suitable numbers within a reasonable time, were Zygoptera (Damselfly) (see figure 10 and table 5), Anisoptera (Dragonfly) (figure 11 and table 6) and the Trichoptera Agrypnia sp. (figure 12 and table 7).
Zygoptera (Odonata)
figure 10. Zygoptera (Odonata) (www.nwnature.net) Zygoptera (Odonata) (Adult) (www.richard-seaman.com ) table 5. Characterization of Zygoptera (Odonata) used in the avoidance experiment (n=7)(annex B-4) Zygoptera (Odonata) Description Average SD SD % Length [mm] 15.04 1.18 7.9 Wet weight [g] 0.029 0.005 17.4 Wet weight/length [g/mm] 0.002 0.001 11.3 Dry weight [g] 0.006 0.003 40.2 Dry weight/length [g/mm] 0.001 0.001 34.9
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Anisoptera (Odonata)
figure 11. Anisoptera (Odonata) (www.nwnature.net) Anisoptera (Odonata) (Adult) table 6. Characterization of Anisoptera (Odonata) used in the avoidance experiment (n=6) (annex B-4) Anisoptera (Odonata) Description Average SD SD % Length [mm] 14.54 0.96 6.6 Wet weight [g] 0.158 0.026 16.5 Wet weight/length [g/mm] 0.011 0.001 10.0 Dry weight [g] 0.031 0.006 19.7 Dry weight/length [g/mm] 0.002 0.001 14.2
Agrypnia sp.
figure 12. Larvae Agrypnia sp. (www.biologie.swordfish-diving.nl) and adult Agrypnia sp. (www.biopix.com) table 7. Characterization of Agrypnia sp. used in the avoidance experiment (n=7)(annex B-4) Agrypnia sp. Description Average SD SD % Length [mm] 18.46 2.88 15.6 Wet weight [g] 0.096 0.041 42.5 Wet weight/length [g/mm] 0.005 0.003 45.7 Dry weight [g] 0.023 0.010 45.1 Dry weight/length [g/mm] 0.001 0.001 48.6
Due to calibration problems in Axiovison, the software used to measure the length of Anisoptera, Zygoptera and Agrypnia sp., photos were made with a grid in the background so the length could be measured manually.
14
Start experiment In the first run, A. aquaticus was tested on possible avoidance behaviour with the use of all 16 random placed aquaria (see figure 13). The aquaria were labelled C1-C8 for controls and 320A-320H for the treatments. Each aquarium received 10 specimens which were released in the middle. In total 4 cameras were placed in the experimental room each covering 4 aquaria. The cameras video recorded the behaviour of the four different species. Due to the time consuming process of video analyses and a tight manual scoring schedule, only the results of the manual scoring will be discussed in this report.
figure 13. Location of the 8 aquaria with clean sediment and the 8 aquaria with both clean and spiked sediment in the water bath of the experimental room used to test behaviour of Asellus aquaticus
During the second run Zygoptera, Anisoptera and Agrypnia sp. were tested. For every species, four aquaria were used of whereby two comprised controls and two treatments (see figure 14). Of each species, 5 specimens were placed in the middle of the aquaria. The lower numbers compared to avoidance test with A. aquaticus are due to the increased probability of cannibalism (for Anisoptera and Zygoptera) which was observed during the acclimatization period.
figure 14. Location of the 6 aquaria with clean sediment and the 6 aquaria with both clean and spiked sediment in the water bath of the experimental room used for Anisoptera (Odonata) (left), Agrypnia sp. (middle) and Zygoptera (Odonata) (right).
15
Manual scoring Manual scoring took place for 4 days, every hour from 9.00 to 17.00. Each aquaria was divided into 6 different sections. The outer sections could be scored as either completely clean of contaminated and the middle section as an intermediate zone (see figure 15). Since the division between clean and spiked sediment was not completely sharp it was decided to leave this zone out of the results. In practise not all the animals that were inserted in the aquaria were actually observed. Some of the animals must have hidden in the sediment or under peat particles. In that case only the number of the animals actually observed were scored. 21 cm 8 cm 21 cm
figure 15. Overview of the sections (light areas) in the aquaria that were used to score the presence of A. aquaticus
16
2.3 Colonization experiment To test toxicity and avoidance behaviour of sediment in a natural environment, portions of sediment spiked with varying lufenuron concentrations were placed in ditch mesocosms. The main hypothesis being, that higher concentrations would lead to reduced colonization by aquatic macroinvertebrates.
Experimental setup The sediment remaining from the toxicity experiment was mixed per concentration level into an homogeneous substance. Small containers (14.5 x 11 x 5 cm) were used where ropes with corks were attached to track the containers back ones incubated (see figure 16). A total of 21 containers, 3 per concentration, were filled with sediment. 18 containers contained spiked sediment, which was used in the toxicity test, and another 3 contained un- spiked standard OECD sediment. A layer of 4 cm clay (same substrate as ditches) was inserted as underground into the containers because there was not enough spiked sediment to fill it all up (see figure 16). The sediment was carefully divided over the clay surface and had an average thickness of 0.38 cm SD± 13.75%.
figure 16. Containers with clay and attachment of ropes with corks to the containers.
Next, the containers were randomly inserted in the ditch mesocosm (length: 40 m; width: 3.4m at water surface; width 1.6m at the sediment surface; water depth 0.5; volume: 60m³). The containers were placed on one side of the ditch (seen from the width) having one meter in between every box (see figure 17)
17
figure 17. Overview shortly after incubating the containers
The containers were left undisturbed for 55 days allow sufficient colonization time for macroinvertebrates. While using a mobile bridge, the containers were carefully lifted up and placed in a bucket for transport until analysis. The content of the containers was sieved, using different mesh sizes (smallest was 0.212 mm). The macroinvertebrates were identified on species level, abundance and dry weight was measured per container. A distinction between the leaches and snails and other invertebrates has been made because leaches and snails were found in a few samples and would cover over 50% of the total weight. For this reason it was decided to weigh these separately. To test treatments related effects in the abundance and/or biomass of macroinvertebrates, a statistical Williams test was conducted.
18
3 Results This chapter describes the results of the toxicity, the avoidance behaviour experiments and the colonization experiment.
3.1 Toxicity experiment
Water quality variables The average and standard deviation for the different variables temperature, pH, oxygen and electrical conductivity are presented in table 8 below. table 8. Water quality variables measured during the 21 day toxicity experiment (n=156) Variable Average SD SD % Temp. [˚C] 20.8 0.1 0.7 pH 8.1 0.1 1.6 O2 [mg/l] 9.2 1.6 17.5 EC [µS/cm] 194.3 30.5 15.7
Toxicity A. aquaticus The graph below shows the numbers of alive specimens found at day 5 and day 21 (figure 18). At day 5 there were no distinct lethal effects seen that can be related to the concentration lufenuron. At day 21 however, pronounced effects were seen in the test vessels that received sediment spiked with the highest concentration. At this treatment- level all the specimens of A. aquaticus died. In addition, about 50% of the specimens died in the test vessels that received sediment spiked with 160 µg/kg. Test vessels of the 40 and 80 µg/kg treatments both had a replicate where no mortality was observed while in the other replicate respectively 6 and 5 individuals died. The controls showed low mortality (see annex A-4). With (GenStat Release 13.3) a LC50 was calculated for the intended lufenuron concentration at day 5 and day 21. The software works with non-linear regression and due to the non-distinct values at day 5, no fitting trend line was found. Consequently no accurate LC50 value was observed since after 5 days no effect has been observed. At day 21 a more distinct pattern is visible for the intended concentrations and a LC50 was calculated of 164.8 µg/kg with a range between 154 µg/kg and 176.5 µg/kg.
Mean alive A. aquaticus 12 S Control 10 u 20 ug/kg r 8 40 ug/kg v 6 i 80 ug/kg 4 v 160 ug/kg a 2 320 ug/kg l 0 Day 0 Day 5 Day 21 figure 18. Survival of A. aquaticus over time in toxicity experiment.
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3.2 Avoidance behaviour experiment In the first part of this paragraph results of the behaviour experiment are presented for A. aquaticus. In the second part results of behaviour experiments with for the other three taxa are presented Zygoptera (Odonata), Anisoptera (Odonata) and Agrypnia sp.
Asellus aquaticus
Water quality variables The highest variation was found at the electrical conductivity values. The average electrical conductivity changed from the first measurements with 307 to 320 µS/cm on the second and last measurement. Other variables showed minor variation (see annex B-2). table 9. Overview water quality variables(n=32) Variables Average SD SD % Temp. [˚C] 19.5 0.1 0.7 pH 8.1 0.1 0.8 O2 [mg/l] 8.3 0.3 3.2 EC [µS/cm] 313.4 20.2 6.4
Statistical analysis of spatial distribution of A. aquaticus A paired T-test with SPSS (version 17) was conducted to find out whether there were differences in presence scores of A. aquaticus between the left and right sections of the test aquaria. From the data it appears that of the 16 aquaria used in the avoidance experiment, 6 showed a significant difference in presence scores between left and right (see annex B-3). From the 6 aquaria, 3 were used for 3 control and 3 for treatments. The controls had 2 aquaria with A. aquaticus significantly more observed on the right side and 1 aquarium where A. aquaticus was more frequently scored in the left section. In 2 aquaria that contained a section with spiked sediment, significantly more individuals of A. aquaticus were scored on the left and in 1 treated aquarium had a higher score of A. aquaticus individuals on the right. Surprisingly, aquaria that showed significant differences appeared to have a higher score of A. aquaticus on the contaminated side (see table 10).
20 table 10. Statistical evaluation (paired T-test) of the presence of A. aquaticus individuals in the left or right section of the aquaria used for the avoidance behaviour experiment. ID_aquaria t df Sig. (2-tailed) Side C1 4.806 32 .000 Left C2 .651 31 .520 No C3 -.664 30 .512 No C4 -2.749 31 .010 Right C5 -2.995 29 .006 Right C6 -1.625 31 .114 No C7 1.719 31 .096 No C8 -1.792 30 .083 No 320 A 2.467 31 .019 Left-Lufenuron 320 B 2.926 31 .006 Left-Lufenuron 320 C .583 30 .564 No 320 D -2.971 31 .006 Right-Lufenuron 320 E 1.130 31 .267 No 320 F .029 31 .977 No 320 G -.689 31 .496 No 320 H .273 31 .787 No
The average distribution of A. aquaticus between the left and right sections in the aquaria on basis of statistical evaluation with the paired T-test, showed that there is no significant difference between left and right for the controls and the treatments. Furthermore, the paired T-test did not reveal statistically significant differences in presence of A. aquaticus between sediment sections with and without lufenuron. This accounts per individual day and over the whole period (table 11). The results indicate that there is variability in presence scores on left and right sections between the aquaria.
21 table 11. Statistical evaluation (paired T-test) of the presence of A. aquaticus individuals in the left or right section of the aquaria used for the avoidance behaviour experiment from 31-5-2011 to 3-6-2011 and comprised for the whole period. Averaged per treatment on 31-5 ID t df Sig. (2- tailed Controls -0.441 7 0.672 Treatments (L-R) 0.78 7 0.61 Treatments (Luf-Cl) 2.19 7 0.65
Averaged per treatment on 1-6 Controls -1.583 7 0.158 Treatments (L-R) -0.324 7 0.755 Treatments (Luf-Cl) 0.28 7 0.788
Averaged per treatment on 2-6 Controls -0.885 7 0.416 Treatments (L-R) 2.136 7 0.07 Treatments (Luf-Cl) 0.466 7 0.655
Averaged per treatment on 3-6 Controls 0.369 7 0.704 Treatments (L-R) -0.214 7 0.836 Treatments (Luf-Cl) 1.113 7 0.362
Averaged per treatment total period Controls -0.840 28 0.407 Treatments (L-R) 1.038 28 0.307 Treatments (Luf-Cl) 1.620 28 0.115
Distribution A. aquaticus The data for the individual aquaria that showed significant differences in presence scores of A. aquaticus between treated and untreated aquaria sections is shown in distribution graphs 19-20. Given is the percentage of the total observed specimens at a particular side (left or right). Only the left and right part are displayed so the total percentages can be lower than 100% due to individuals that were in the middle section. The displayed minus signs as used in the graphs are used to give the data this comparable expression. Every time point on the y-axis expresses the distribution of individuals divided over left and right at the moment of scoring. That A. aquaticus has been more often observed on one side is visible in the distribution graph for the controls. That side has an overall larger ratio of abundance than the non-preferred side but shows that A. aquaticus have been frequently on the other side as well. For the treatments the similar pattern is observed, so the overall abundance on the lufenuron side was higher but the specimens have been frequently observed on both sides of the aquaria.
22
Distribution A. aquaticus Control 1 31
26 21 16
11 Timepoints 6 1 -100 -50 0 50 100 % per section Left Right figure 19. Distribution of A. aquaticus in Control aquarium 1. Significantly more observed at (.000) left side.
Distribution A. aquaticus Control 4 31
26
21 16
Timepoints 11 6 1 -100 -50 0 50 100 Left Right % per section figure 20. Distribution of A. aquaticus in Control aquarium 4. Significantly (.010) more observed at right side.
Distribution A. aquaticus Control 5 31
26
21 16
11 Time pointsTime 6 1 -100 -50 0 50 100 Left Right % per section figure 21. Distribution of A. aquaticus in Control aquarium 4. Significantly (.006) more observed at right side.
23
Distribution A. aquaticus 320 A 31
26
21 16
Timepoints 11 6 1 -100 -50 0 50 100 % per section Left - Lufenuron Right - Clean
figure 22. Distribution of A. aquaticus in treatment aquarium 320 A. Significantly (.019) more observed at left (lufenuron) side.
Distribution A. aquaticus 320 B 31
26
21 16
Timepoints 11 6 1 -100 -50 0 50 100 % per section Left - Lufenuron Right - Clean figure 23. Distribution of A. aquaticus in treatment aquarium 320 B. Significantly (.006) more observed at left (lufenuron) side.
Distribution A. aquaticus 320 D
29
25
21 17 13
Time points Time 9 5 1 -100 -50 0 50 100 Left - Clean % per section Right - Lufenuron figure 24. Distribution of A. aquaticus in treatment aquarium 320 D. Significantly (.006) more observed at right (lufenuron) side.
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Anisoptera (Odonata), Agrypnia sp. and Zygoptera (Odonata) This paragraph describes the results of the avoidance test conducted with the taxa Anisoptera, Agrypnia sp. and Zygoptera. First the water quality parameters are presented for the aquaria used in the avoidance behaviour experiment, followed by a statistical analysis of the presence scores in the left and right sections of the aquaria.
Water quality variables The measured variables temperature, pH, oxygen and electrical conductivity showed little variation during the behaviour experiment (see table 12 and annex B-6). table 12. Water quality variables (n=24) Variables Average SD SD % Temp. [˚C] 20.1 0.2 1.2 pH 8.0 0.1 1.7 O2 [mg/l] 8.6 0.2 2.5 EC [µS/cm] 343.8 22.3 6.5
Statistical analysis Anisoptera: The paired T-test shows that there is no significant difference between presence scores at the left and right sections of the four aquaria (see table 13 and annex B-7). table 13. T- Statistical evaluation (paired T-test) of the presence of Anisoptera individuals in the left or right section of the aquaria used for the avoidance behaviour experiment (mean over 4 days). ID_aqua t df Sig. (2-tailed) Side C5 -.294 23 .771 No C7 .216 23 .831 No 320 G 1.209 23 .239 No 320 H -.184 23 .856 No
Agrypnia sp.: The paired T-test (table 14), shows that there is no significant difference between presence scores at the left and right sections for three out of 4 aquaria. Treatment aquarium 320 C revealed a significant preference for the side with lufenuron spiked sediment. table 14. Statistical evaluation (paired T-test) of the presence of Phryganea individuals in the left or right section of the aquaria used for the avoidance behaviour experiment (mean over 4 days)
ID_aqua t df Sig. (2-tailed) Side
C1 -1.091 23 .286 No
C4 .604 23 .552 No
320 B .241 23 .812 No
320 C -3.436 23 .002 Right-Lufenuron
Zygoptera: The paired T-test shows in all aquaria significant differences in presence scores between left and right sections of the aquaria (both treatments as controls) (see table 15).
25
Clear avoidance behaviour of lufenuron spikes sediment, however, could not be demonstrated. table 15. Statistical evaluation (paired T-test) of the presence of Zygoptera individuals in the left or right section of the aquaria used for the avoidance behaviour experiment (mean over 4 days). Aqua-ID t df Sig. (2-tailed) Side C2 2.896 23 .008 Left C6 -5.899 23 .000 Right 320 D 12.263 23 .000 Left-Lufenuron 320 E 2.138 23 .043 Left-Clean
Distribution Anisoptera Distribution Anisoptera Control 5
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left % per section Right figure 25. Distribution Anisoptera in control aquarium 5.
Distribution Anisoptera Control 7
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left % per section Right figure 26. Distribution Anisoptera in control aquarium 7.
26
Distribution Anisoptera 320 G
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left - Lufenuron% per section Right - Clean figure 27. Distribution Anisoptera in treatment aquarium 320 G.
Distribution Anisoptera 320 H
21
16
11 Timepoints 6
1 -100 -50 0 50 100 % per section Left - Clean Right - Lufenuron figure 28. Distribution Anisoptera in treatment aquarium 320 H.
The distribution figure 25-28 shows that Anisoptera was observed on both sides without being significantly more on one of the sides.
27
Distribution Agrypnia sp. Distribution Agrypnia sp. Control 1
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left % per section Right figure 29. Distribution Agrypnia sp. in control aquarium 1.
Distribution Agrypnia sp. Control 4
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left Right % per section figure 30. Distribution Agrypnia sp. in control aquarium 4.
Distribution Agrypnia sp. 320 B
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left - Lufenuron% per section Right - Clean figure 31. Distribution Agrypnia sp. in treatment aquarium 320 B.
28
Distribution Agrypnia sp. 320 C
21
16
11 Timepoints 6
1 -100 -50 0 50 100 % per section Left - Clean Right - Lufenuron figure 32. Distribution Agrypnia sp. in treatment aquarium 320 C.
Agrypnia sp. shows a variable distribution over the left and right side from the control aquaria and treatment aquarium 320 B (see fig. 29-32). For treatment aquarium 320 C it is observed that the right side was most frequently visited by the Agrypnia sp. compared to the left side. However, the left side was visited as well.
Distribution Zygoptera Distribution Zygoptera - Control 2
21
16
11 Timepoints 6
1 -100 -50 0 50 100 Left % per section Right figure 33. Distribution Zygoptera control 2.
29
Distribution Zygoptera - Control 6
21
16
11
Timepoints 6
1 -100 -50 0 50 100 % per section Left Right figure 34. Distribution Zygoptera control 6.
Distribution Zygoptera - 320 D
21
16
11
Timepoints 6
1 -100 -50 0 50 100 % per section Left - Lufenuron Right - Clean figure 35. Distribution Zygoptera treatment 320 D.
Distribution Zygoptera - 320 E
21
16
11
Timepoints 6
1 -100 -50 0 50 100 % per section Left - Clean Right - Lufenuron figure 36. Distribution Zygoptera treatment 320 E.
The distribution graphs (fig. 33-36) suggesting that there have been minor movement by Zygoptera. Distributions are sometimes exactly the same for hours.
30
3.3 Colonization experiment The results are presented in graphs giving the mean abundance per taxon per treatment, expressed in nominal lufenuron concentration of the spiked sediment in the containers that were incubated in the experimental ditch. In the second part the biomass (in terms of dry weight) of these taxa are shown per treatment.
Macroinvertebrate abundance A William test has been conducted to discover treatment related effects in the abundance of macroinvertebrates. Juvenile Erpobdella showed an increasing NOEC concentration of 160 µg/kg. It seems that effects are decreasing at a higher concentration which is probably caused by an indirect effect instead a treatment related effect. For all other taxa no NOEC lower than the maximum concentration has been observed (see annex C-1).
Chironomus spec. was found in the colonization containers of all treatments. The lowest numbers (4) were scored in treatment 40 µg/kg and 320 µg/kg. The densities in other treatments varied between 9 and 16 (see figure 37). Tanypodinae (figure 38) varied between 2 (80 µg/kg) and 11 specimens (20 µg/kg) while the control and highest treatment (320 µg/kg) had the same mean value. Chironomini spec. had been found with the highest numbers in treatments 320 µg/kg and 40 µg/kg although error bars indicate a strong variation (figure 39).
Chironomus sp.
30 20 10
Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 37. Abundance of Chironomus in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
Tanypodinae
30 20 10
Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 38. Abundance of Tanypodinae in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
31
Chironomini
15 10 5
Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 39. Abundance of Chironomini in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
Sialis sp. is on average quite evenly distributed over the different treatments but variation between the replicates was relatively large (figure 40). Caenis sp. (figure 41) was found in low numbers in all treatments. Juveniles of Erpobdella were found in low numbers in the controls and 160 µg/kg treatment. They were more abundant in the 320 µg/kg and 80 µg/kg treatments (figure 42).
Sialis sp.
15 10 5
Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level
figure 40. Abundance in Sialis spec in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
Caenis sp.
20 15 10 5 Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 41. Abundance of Caenis spec in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch.
32
Erpobdella juvenile
20 15 10 5 Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 42. Abundance of Erpobdella juveniles in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
The mean numbers of taxa (figure 43) found in the different treatments were almost the same. For the main taxonomic groups, Diptera (midges) did not show distinct differences between treatments although their number were highest in the 20 µg/kg treatment (see figure 44). Ephemeroptera (mayflies) showed more variability in total numbers between the treatments and the 320 µg/kg and 40 µg/kg treatments had the lowest abundance of Ephemeroptera (see figure 45).
Taxa
20 15 10 5 Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level
figure 43. Abundance of taxa in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
Diptera
60 40 20
Abundance 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 44. Abundance of Diptera (flies and midges) in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
33
Ephemeroptera
30 20 10 0 Abundance Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 320 µg/kg µg/kg Treatment level figure 45. Abundance of Ephemeroptera (mayflies) in the containers with lufenuron-spiked sediment that were incubated in an outdoor experimental ditch
Biomass In figure 46 the biomass without snails and leaches is presented which gives a no distinct treatment related trend. When snails and leaches were included, particular treatments (control, 160, 320) showed an increase in biomass while the ones without any snails or leaches remained low (see figure 47). Still no treatment related trend is observed (see annex C-3).
Biomass excl. snails/leaches
8
6
4
Biomass(mg) 2
0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 46. Dry weight exclusive snails and leaches
Biomass incl. snails/leaches 70
60
50 40 30
20 Biomass(mg) 10 0 Controls 20 µg/kg 40 µg/kg 80 µg/kg 160 µg/kg 320 µg/kg Treatment level figure 47. Dry weight inclusive snails and leaches
34
4 Discussion and conclusion
4.1 Discussion
Water quality variables In all test vessels oxygen level never dropped below 5 mg/L which is above the minimal required concentration found by (Williams 1962). The oxygen showed some variation due to the aeration that had been turned off and on as a trial to reduce turbidity. The increased electrical conductivity values during the toxicity experiment might be related to the observed turbidity. Water quality parameters measured during the behaviour experiments showed only minor variation.
Toxicity experiment De results show that after 5 days of exposure to lufenuron no lethal effects had occurred to A. aquaticus that can be related to the lufenuron application. At day 21 pronounced effects were observed with 100% death at the highest concentration and 50 % at the second highest. Exact concentrations in the sediment were unknown at time of writing which give uncertainty to the LC50 calculated. Note, however, that due to the turbidity it could not be seen whether for example lufenuron exposure resulted in immobility during the first 5 days of exposure. It could also be that the intake in the first 5 days was enough to cause death overtime for instance when A. aquaticus moulds. After the water and specimens were poured over a sieve to get them out, it was not easy to distinguish between immobile and mobile individuals. Living and dead individuals, however, could be distinguished very well. Specimens that were not found back were assumed to be dead because A. aquaticus is known for cannibalism and scavenging and only one dead specimen was found back in a test vessel (Bloor 2010 and personal observation). On day 21 dead individuals were observed in the test vessels that received spiked sediment with the highest lufenuron concentration but they fell apart when the water was poured over the sieve. Due to the turbidity, the remark was made that it could very well be that A. aquaticus not only got exposed via the sediment, but also via the water phase and suspended solids (Lawrence and Mason 2001; Eggleton and Thomas 2004). Measurements were not conducted by the end of this report and data concerning concentrations in the water phase and suspended solids are consequently unknown. Exposure via the sediment compartment can be expected due to the hydrophobic properties of lufenuron. This suggests that lufenuron was adsorbed to the organic matter of the sediment in particular. Most likely A. aquaticus foraged on the sediment organic matter during the toxicity experiment which is also assumable because otherwise starvation and consequently more dead specimens would be expected in the controls. The latency of effect that has been observed during the toxicity experiment and the ditch mesocosm experiment of Brock and co-workers (2009) can be related to the mode of action of lufenuron which inhibits the synthesis of chitin. Due to the turbid test vessels, however, we could not investigate the onset of toxic lethal effects in the different treatments. Since at the highest treatment in particular the dead animals fell apart during sampling makes it reasonable that they were dead for several days. It cannot be excluded that at lower treatment levels the time required to show effect is longer than 21 days.
35
Avoidance experiment Avoidance of lufenuron-contaminated sediment was not observed in the avoidance behaviour experiment with A. aquaticus. However, some variation in presence scores was observed in 6 out of 16 aquaria. This variation, however, could not be attributed to the presence of lufenuron-contaminated sediment. The responses observed do not suggest that there is an external factor (outside the aquaria) that is responsible for the variation in presence-scores observed. A possible explanation is that there might be more peat particles lying at the surface on those sections of the aquaria that were preferred by A. aquaticus. It was tried to make an equal distribution of the particles but, possibly, a perfect distribution of peat particles in the left and right sections was not present in all aquaria. The exact exposure concentration is uncertain but in any case the species had the opportunity to choose between contaminated and uncontaminated sediment. If the route of exposure predominantly is via ingesting of food, it is more important to know where individual A. aquaticus feed than where they walked. A small food choice experiment with A. aquaticus using contaminated and non-contaminated Populus leaves may provide useful insight whether contaminated food is avoided. To test the general concept and methodology for the chosen test design, Anisoptera, Zygoptera and Agrypnia sp. were used in the avoidance behaviour experiment as well. Anisoptera and Zygoptera do not feed on organic matter in the sediment since they predominantly prey on organisms that live in the water column. Avoidance of contaminated food is from that point of view not expected in our experimental set-up. Anisoptera showed no significant differences between left and right sections in any of the four aquaria. Zygoptera however showed a preference for one section in the aquaria but this was not correlated with the presence of contaminated sediment. During the manual scoring of the positions of Zygoptera it already draw attention that their positions did hardly change. Zygoptera sits most of the time on a single position waiting for a prey (see annex B-5 for its ecology). This ambush hunting behaviour caused the difference amongst sides and is clearly presented in the distribution graphs presented for Zygoptera (see figure 33-36). Agrypnia sp. is bound to vegetation instead to sediment which makes this species in essence inappropriate for sediment avoidance. Agrypnia sp. feed amongst others on detritus/organic matter which makes them potentially more susceptible to exposure via food than Anisoptera or Zygoptera. However, no avoidance of lufenuron-contaminated sediment was observed for Agrypnia sp.
Colonization experiment This experiment was closely related to Brock and co-workers (2009;2010) since it includes colonization and recovery in the same type of outdoor experimental ditch. Note, however, that in the colonization experiment presented here the containers with contaminated sediment were “small islands” in a “sea” of unpolluted sediment. In the experiment of Brock et al (2009; 2010) a large part (33%; 67% or 100%) of the surface area of the ditches was contaminated with lufenuron. In the incubated containers with lufenuron-spiked sediment a clear concentration response relationship could not be observed for the endpoints abundance and biomass of macroinvertebrates that colonized the sediment present in the containers. This also suggests that there is no avoidance by a larger array of macro invertebrates (other than A. aquaticus). If the highest concentration is lethal for several macro invertebrates, which is observed for A. aquaticus during the laboratory toxicity experiment and for a wide array of
36 benthic macroinvertebrates in the ditch experiment performed by Brock and co-workers (2009;2010) the question at stake is: why could a treatment-related response not be observed in our colonization experiment? An obvious explanation is that the macroinvertebrates found in the containers did not actively avoid lufenuron-contaminated sediment, a large surface of non-contaminated sediment was present in the ditch that acted as source of colonization (resulting in a more or less constant inflow), hereby masking toxicity. Another explanation might be that colonized benthic macroinvertebrates predominantly fed on fresh organic matter settling down above the contaminated sediment, which in fact was a non-contaminated food source. Additional experiments are needed to test this hypothesis.
OECD sediment This sediment is used as a standard for Chironomus riparius toxicity experiments and is spiked via the water phase. In natural sediments the organic matter content of the upper layer usually is higher and spiking via the water phase will contaminate this upper layer in particular. OECD sediment however has organic matter that is completely mixed, so that the upper layer on average will be different in organic matter content than natural undisturbed sediment. Consequently, the type of sediment used in a toxicity experiment will affect the exposure profile of the toxicant in the sediment, and consequently the exposure-response relationship (Lacey, Watzin et al. 1999; Goedkoop and Peterson 2003). For this reason we decided to spike the total sediment in our experiments to get a homogeneous exposure to lufenuron in the sediment compartment. This procedure, however, was laborious. In future experiments it may be very interesting to compare the concentration-response relationship for lufenuron and Asellus aquaticus in sediment toxicity tests using artificial and natural sediment and spiking via water or sediment.
4.2 Conclusion The lufenuron concentrations in the spiked sediments tested do not cause mortality of A. aquaticus within 5 days. After 21 days of sediment exposure, however, 100% mortality was observed in the 320 µg/kg treatment, and partial mortality in the 80 and 160 µg/kg treatments. Avoidance of lufenuron-contaminated sediment was not observed for Asellus aquaticus in the laboratory avoidance behaviour experiment. In addition small containers with lufenuron-spiked sediment that were incubated in an experimental ditch were readily colonised by macroinvertebrates, irrespective of the concentration of lufenuron in the spiked sediment. Coming back to the research question it can be concluded that avoidance, although food avoidance is not fully addressed yet, was not the driving factor for the absence of sediment dwelling organisms in the treated sections. Toxicity and slow migration are the remaining explanations. Taking a closer look to the organisms of concern in the ditch experiment of Brock and co-workers. (2009;2010), it appears that the vulnerable taxa of Tanypodinae, Chironomus and Orthocladiinae are midges. Their migration is indeed minor under water, but this is only one way to migrate (EDGAR 1968; Williams 1989). A probably more important way of colonization occurs via adult midges that deposit eggs in the ditches. Taking this into account migration is probably not the driving factor that causes the absence of midge larvae in the treated ditches. The only remaining explanation is toxicity, most probably via the ingestion of lufenuron-contaminated detritus. Additional research is recommended for testing possible food avoidance and the inflow of macroinvertebrates to contaminated sediment.
37
5 References
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"Sublethal and sex-specific cypermethrin effects in toxicity tests with the midge Chironomus riparius Meigen." Ecotoxicology 19(7): 1201-1208. Higler, L. W. G. (2008). "Verspreidingsatlas Nederlandse kokerjuffers (Trichoptera)." 280. Hooper, H. L., R. M. Sibly, et al. (2005). "Joint effects of density and a growth inhibitor on the life history and population growth rate of the midge Chironomus riparius." Environmental Toxicology and Chemistry 24(5): 1140-1145. Johnson, D. M. (1991). "Behavioral ecology of larval dragonflies and damselflies." Trends in Ecology & Evolution 6(1): 8-13. Lacey, R., M. C. Watzin, et al. (1999). "Sediment organic matter content as a confounding factor in toxicity tests with Chironomus tentans." Environmental Toxicology and Chemistry 18(2): 231-236. Lagauzere, S., R. Terrail, et al. (2009). "Ecotoxicity of uranium to Tubifex tubifex worms (Annelida, Clitellata, Tubificidae) exposed to contaminated sediment." Ecotoxicology and Environmental Safety 72(2): 527- 537. Lawrence, A. L. and R. P. Mason (2001). "Factors controlling the bioaccumulation of mercury and methylmercury by the estuarine amphipod Leptocheirus plumulosus." Environmental Pollution 111(2): 217-231. Li, Z. H., V. Zlabek, et al. (2010). "Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout, Oncorhynchus mykiss." Chemico-Biological Interactions 185(3): 241-246. Maltby, L. (1991). "Pollution as a Probe of Life-History Adaptation in Asellus aquaticus (Isopoda)." Oikos 61(1): 11-18. Marklund, O., I. Blindow, et al. (2001). "Distribution and diel migration of macroinvertebrates within dense submerged vegetation." Freshwater Biology 46(7): 913-924. McLaughlin, A. and P. Mineau (1995). "The impact of agricultural practices on biodiversity." Agriculture Ecosystems & Environment 55(3): 201-212. Menchaca, I., M. J. Belzunce, et al. (2010). "Sensitivity Comparison of Laboratory-Cultured and Field-Collected Amphipod Corophium multisetosum in Toxicity Tests." Bulletin of Environmental Contamination and Toxicology 84(4): 390-394. OECD (2004). "Test No. 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment ". Perez, J. R., S. Loureiro, et al. (2010). "Assessment of water quality in the Alqueva Reservoir (Portugal) using bioassays." Environmental Science and Pollution Research 17(3): 688-702.
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Rask, M. and C. Hiisivuori (1985). "The predation on Asellus aquaticus (L.) by perch, Perca fluviatilis (L.), in a small forest lake." Hydrobiologia 121(1): 27-33. Sardo, A. M. and A. M. V. M. Soares (2010). "Assessment of the effects of the pesticide imidacloprid on the behaviour of the aquatic oligochaete lumbriculus variegatus." Archives of Environmental Contamination and Toxicology 58(3): 648-656. Singh, B. B., R. Chandra, et al. (2008). "Effect of Pyridine and Formaldehyde on a macrophyte (Lemna Minor L.) and a sludge worm (Tubifex tubifex muller) in fresh water microcosms." Applied Ecology and Environmental Research 6(2): 21-35. Suiter, K. and F. Gould (1994). "Physiological resistance and behavioral avoidance responses to residues of four pesticides by six spider mite populations." Entomologia Experimentalis et Applicata 71(1): 1-14. van Tol, J. (1988). "The protection of dragonflies (Odonata) and their biotopes." Vogt, C., M. Hess, et al. (2010). "Effects of cadmium on life-cycle parameters in a multi-generation study with Chironomus riparius following a pre-exposure of populations to two different tributyltin concentrations for several generations." Ecotoxicology 19(7): 1174-1182. Whitehurst, I. T. (1991). "TheGammarus: Asellus ratio as an index of organic pollution." Water Research 25(3): 333-339. Wiggins, G. B. (1998). "The Caddisfly family Phrychaneidae (Trichoptera)." Williams, C. (1989). "Downstream drift of the larvae of Chironomidae (Diptera) in the River Chew, S.W. England." Hydrobiologia 183(1): 59-72. Williams, W. D. (1962). "Notes on the ecological similarities of Asellus aquaticus (L.) and A. meridianus Rac. (Crust., Isopoda)." Hydrobiologia 20(1): 1-30.
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6 Appendix
A. Toxicity experiment A-1 Asellus aquaticus characterization A-2 Water quality variables A-3 Results toxicity experiment
B. Avoidance behaviour experiment B-1 Asellus aquaticus characterization B-2 Water quality variables A. aquaticus B-3 Results behaviour experiment A. aquaticus B-4 Characterization Agrypnia sp., Anisoptera (Odonata) and Zygoptera (Odonata) B-5 Ecology Agrypnia sp., Anisoptera and Zygoptera B-6 Water quality variables Agrypnia sp., Anisoptera and Zygoptera B-7 Results behaviour experiment Agrypnia sp., Anisoptera and Zygoptera
C. Colonization experiment C-1 Abundance taxa C-2 Biomass
40
A Toxicity experiment
A-1 Weight and length characterization of Asellus aquaticus used in the toxicity experiment (n=20)
Asellus aquaticus characterization toxicity experiment
Weight Weight with wet Weight with Wet weight Dry weight Length Wet Dry weight Dry weight/length ID empty Asellus dry Asellus Asellus Asellus Asellus weight/length [weight%] [g/mm] [g] [g] [g] [g] [g] [mm] [g/mm]
1 0.0457 0.0565 0.0485 0.0108 0.0028 25.9259 5.3185 0.00203 0.00053 2 0.0463 0.0680 0.0504 0.0217 0.0041 18.8940 6.7801 0.00320 0.00060 3 0.0453 0.0758 0.0504 0.0305 0.0051 16.7213 6.3741 0.00478 0.00080 4 0.0453 0.1013 0.0568 0.0560 0.0115 20.5357 8.1957 0.00683 0.00140 5 0.0459 0.0726 0.0516 0.0267 0.0057 21.3483 7.6232 0.00350 0.00075 6 0.0453 0.1030 0.0542 0.0577 0.0089 15.4246 9.0746 0.00636 0.00098 7 0.0461 0.1161 0.0546 0.0700 0.0085 12.1429 8.3208 0.00841 0.00102 8 0.0450 0.1040 0.0540 0.0590 0.0090 15.2542 8.3880 0.00703 0.00107 9 0.0458 0.0838 0.0507 0.0380 0.0049 12.8947 6.8132 0.00558 0.00072 10 0.0455 0.0759 0.0491 0.0304 0.0036 11.8421 5.8678 0.00518 0.00061 11 0.0460 0.0744 0.0512 0.0284 0.0052 18.3099 5.9462 0.00478 0.00087 12 0.0456 0.0885 0.0519 0.0429 0.0063 14.6853 6.9355 0.00619 0.00091 13 0.0453 0.1182 0.0562 0.0729 0.0109 14.9520 8.1168 0.00898 0.00134 14 0.0461 0.1084 0.0539 0.0623 0.0078 12.5201 7.5166 0.00829 0.00104 15 0.0456 0.0663 0.0490 0.0207 0.0034 16.4251 5.6997 0.00363 0.00060 16 0.0456 0.0979 0.0511 0.0523 0.0055 10.5163 7.8730 0.00664 0.00070 17 0.0457 0.0754 0.0498 0.0297 0.0041 13.8047 6.8507 0.00434 0.00060 18 0.0458 0.1190 0.0551 0.0732 0.0093 12.7049 8.6245 0.00849 0.00108 19 0.0456 0.1220 0.0563 0.0764 0.0107 14.0052 8.5410 0.00895 0.00125 20 0.0457 0.0644 0.0483 0.0187 0.0026 13.9037 5.2452 0.00357 0.00050
A-2 Water quality variables toxicity experiment
Water quality variables measured per vessel during the toxicity experiment: Temperature Temperature ID 14-mrt 15-mrt 16-mrt 18-mrt 24-mrt 31-mrt 4-apr Average Ca 20.8 21.6 20.9 20.8 21.0 Cb 20.4 21 20.9 20.8 20.8 Cc 20.4 20.9 20.9 20.8 20.8 Cd 20.3 21 20.8 20.8 20.8 20.8 20.9 20.8 Ce 20.2 20.8 20.8 20.8 20.8 20.8 20.9 20.7
20a 20.7 20.9 20.8 20.8 20.8 20b 20.5 20.9 20.8 20.8 20.8 20c 20.5 20.8 20.8 20.9 20.8 20d 20.5 20.8 20.8 20.9 20.8 20.8 20.9 20.8 20e 20.5 20.8 20.8 20.8 20.8 20.8 20.9 20.8
40a 20.5 20.8 20.8 20.8 20.7 40b 20.7 20.9 20.8 20.8 20.8 40c 20.6 20.9 20.9 20.9 20.8 40d 20.6 20.9 20.9 20.9 20.8 20.8 20.9 20.8 40e 20.6 20.9 20.8 20.9 20.8 20.8 20.9 20.8
80a 20.5 20.8 20.8 20.9 20.8 80b 20.6 20.8 20.8 20.9 20.8 80c 20.7 20.8 20.8 20.8 20.8 80d 20.7 20.9 20.8 20.8 20.8 20.8 20.9 20.8 80e 20.7 20.9 20.9 20.8 20.8 20.8 20.9 20.8
160a 20.7 20.8 20.8 20.8 20.8 160b 20.7 20.9 20.8 20.8 20.8 160c 20.8 20.9 20.8 20.8 20.8 160d 20.8 20.9 20.9 20.9 20.8 20.8 20.8 20.8 160e 20.8 20.9 20.9 20.9 20.8 20.8 20.8 20.8
320a 20.7 20.8 20.8 20.9 20.8 320b 20.8 20.8 20.8 20.9 20.8 320c 20.8 20.7 20.9 20.9 20.8 320d 20.8 20.7 20.9 20.9 20.8 20.8 20.8 20.8 320e 20.8 20.7 20.9 20.9 20.9 20.8 20.8 20.8
Average 20.6 20.9 20.8 20.8 20.8 20.8 20.9 20.8 SD 0.2 0.2 0.0 0.1 0.0 0.0 0.0 0.1 SD % 0.8 0.8 0.2 0.2 0.1 0.0 0.2 0.3
A-2 Water quality variables toxicity experiment
Water quality variables measured per vessel during the toxicity experiment: Oxygen Oxygen ID 14-mrt 15-mrt 16-mrt 18-mrt 24-mrt 31-mrt 4-apr Average Ca 9.6 8 7.5 9.7 8.7 Cb 9.9 8.7 6.5 9.6 8.7 Cc 9.5 8.6 6.4 10.2 8.7 Cd 9.6 9.3 8.4 10 9.4 11.7 9.6 9.7 Ce 9.6 8.6 7.4 10.3 9.5 11.9 9.5 9.5
20a 10.4 10 6.1 9.9 9.1 20b 10.2 9 5.6 10.1 8.7 20c 10.1 8.9 5.8 10.3 8.8 20d 10.1 9 6.4 10.2 9.6 11.9 9.7 9.6 20e 9.9 9.3 7 10.1 9.4 12.1 9.6 9.6
40a 10.4 8.5 5.4 10.4 8.7 40b 10.7 9.4 6.5 10.7 9.3 40c 9.7 8.8 5.7 10.7 8.7 40d 9.9 9.3 6.7 10.7 9.7 12.1 9.6 9.7 40e 9.4 9.5 7.7 10.6 9.4 11.9 9.3 9.7
80a 9.9 9.4 5.6 10.7 8.9 80b 9.5 9.3 5.7 10.8 8.8 80c 9.4 9.7 5.1 10.8 8.8 80d 9.5 9.3 6.4 10.9 10.5 11.9 9.4 9.7 80e 9.3 9.4 5.7 8.3 10.6 8.9 9.2 8.8
160a 9.3 9 5.6 10.7 8.7 160b 9.6 9.1 6.6 10.4 8.9 160c 9.6 9.4 7.3 10.1 9.1 160d 10 9.8 7.1 10.1 10.4 12.1 9.5 9.9 160e 9.6 9.7 6.5 9.5 10.3 11.6 9 9.5
320a 9.9 9.1 7.1 10.2 9.1 320b 10.1 9.7 5.7 10 8.9 320c 10.1 9.9 5.9 10.1 9.0 320d 9.4 9.3 5.9 10 10.5 11.1 9.6 9.4 320e 8.8 9.5 5.4 9.6 10.3 11.3 9.2 9.2
Average 9.8 9.2 6.4 10.2 10.0 11.5 9.4 9.1 SD 0.4 0.5 0.8 0.5 0.5 0.9 0.2 0.4 SD % 4.1 4.9 12.6 5.2 5.0 7.7 2.3 4.4
A-2 Water quality variables toxicity experiment
Water quality variables measured per vessel during the toxicity experiment: pH pH ID 14-mrt 15-mrt 16-mrt 18-mrt 24-mrt 31-mrt 4-apr Average Ca 7.92 8.16 7.95 8.37 8.10 Cb 7.94 8.28 8.01 8.38 8.15 Cc 7.94 8.23 8.07 8.32 8.14 Cd 7.95 8.25 8.05 8.35 7.98 8.3 7.92 8.11 Ce 7.89 8.24 8.1 8.37 7.97 8.31 7.95 8.12
20a 7.94 8.26 8.11 8.27 8.15 20b 7.93 8.25 8.07 8.25 8.13 20c 7.98 8.25 8.08 8.24 8.14 20d 7.99 8.25 8.08 8.25 8.01 8.31 7.96 8.12 20e 7.98 8.25 8.12 8.23 8.02 8.28 7.99 8.12
40a 8.03 8.22 8.14 8.23 8.16 40b 8.03 8.26 8.15 8.25 8.17 40c 8.02 8.25 8.15 8.25 8.17 40d 8.07 8.22 8.17 8.23 8.01 8.3 7.95 8.14 40e 8 8.25 8.16 8.24 8.03 8.31 7.94 8.13
80a 8.01 8.23 8.14 8.39 8.19 80b 7.99 8.25 8.06 8.35 8.16 80c 7.97 8.23 8.04 8.33 8.14 80d 8.01 8.23 8.06 8.33 7.97 8.23 7.96 8.11 80e 8.05 8.25 8.1 8.29 7.99 8.14 8.04 8.12
160a 7.94 8.23 8.05 8.29 8.13 160b 7.91 8.23 8.06 8.27 8.12 160c 7.97 8.22 8.08 8.3 8.14 160d 7.96 8.2 8.12 8.29 8.01 8.26 8.06 8.13 160e 7.99 8.21 8.13 8.27 8 8.21 8.06 8.12
320a 7.96 8.22 8.16 8.19 8.13 320b 7.85 8.23 8.17 8.16 8.10 320c 7.92 8.22 8.16 8.18 8.12 320d 7.91 8.21 8.15 8.19 8.02 8.28 8.09 8.12 320e 7.89 8.23 8.17 8.19 8.09 8.29 8.04 8.13
Average 8.0 8.2 8.1 8.3 8.0 8.3 8.0 8.1 SD 0.1 0.0 0.1 0.1 0.0 0.1 0.1 0.0 SD % 0.6 0.3 0.7 0.8 0.4 0.6 0.7 0.3
A-2 Water quality variables toxicity experiment
Water quality variables measured per vessel during the toxicity experiment: Electrical conductivity Electrical conductivity ID 14-mrt 15-mrt 16-mrt 18-mrt 24-mrt 31-mrt 4-apr Average Ca 169 180 192 205 186.5 Cb 163 166 178 195 175.5 Cc 169 173 170 201 178.3 Cd 167 170 176 200 220 243 238 202.0 Ce 170 172 174 202 216 258 239 204.4
20a 171 172 179 204 181.5 20b 165 167 175 202 177.3 20c 168 171 175 202 179.0 20d 164 167 169 199 216 251 243 201.3 20e 165 167 168 197 214 245 239 199.3
40a 172 174 184 211 185.3 40b 168 170 174 201 178.3 40c 173 176 179 202 182.5 40d 173 176 178 199 217 259 249 207.3 40e 168 171 177 201 214 252 244 203.9
80a 171 163 179 207 180.0 80b 173 175 176 207 182.8 80c 166 172 184 210 183.0 80d 183 184 173 209 227 268 264 215.4 80e 163 168 171 203 221 274 254 207.7
160a 168 167 178 202 178.8 160b 170 171 177 201 179.8 160c 168 171 175 202 179.0 160d 163 165 169 204 223 258 246 204.0 160e 174 177 179 205 227 263 250 210.7
320a 178 178 185 211 188.0 320b 164 169 175 204 178.0 320c 159 167 171 205 175.5 320d 168 173 177 201 226 267 255 209.6 320e 164 170 175 204 230 263 254 208.6
Average 168.6 171.4 176.4 203.2 220.9 258.4 247.9 190.8 SD 4.9 4.7 5.2 3.8 5.6 9.4 7.9 13.4 SD % 2.9 2.7 2.9 1.9 2.5 3.6 3.2 7.0
A-3 Results toxicity experiment
Toxicity results with in the left column a total overview of the number of Asellus aquaticus still alive at the start, day 5 and the end of the experiment per vessel. The middle and right column presents the number of specimens found alive and dead for day 5 and day 21.
Alive Asellus Day 5 Day 21
ID Alive Dead total ID Alive Dead total ID Day 0 Day 5 Day 21
Ca 10 10 Ca 10 0 10 Cd 8 2 10
Cb 10 10 Cb 10 0 10 Ce 7 3 10
Cc 10 10 Cc 10 0 10 20d 8 2 10
Cd 10 8 20a 10 0 10 20e 8 2 10
Ce 10 7 20b 10 0 10 40d 4 6 10
20c 9 1 10 40e 10 0 10
20a 10 10 40a 10 0 10 80d 10 0 10
20b 10 10 40b 10 0 10 80e 5 5 10
20c 10 9 40c 10 0 10 160d 7 3 10 20d 10 8 80a 9 1 10 160e 3 7 10
20e 10 4 80b 9 1 10 320d 0 10 10
80c 9 1 10 320e 0 10 10
40a 10 10 160a 9 1 10
40b 10 10 160b 10 0 10
40c 10 10 160c 10 0 10
40d 10 4 320a 8 2 10
40e 10 10 320b 10 0 10
80a 10 9
80b 10 9
80c 10 9 80d 10 10
80e 10 5
160a 10 9
160b 10 10
160c 10 10 160d 10 7
160e 10 3
320a 10 8
320b 10 10
320c 10 10 320d 10 0
320e 10 0
B. Avoidance behaviour experiment
B-1 Weight and length characterization of A. aquaticus used in avoidance behaviour experiment
Wet Weight Weight with wet Weight with dry Dry weight Length Wet Dry weight Dry weight ID empty Asellus Asellus Asellus Asellus weight/length weight/length Asellus [weight%] [g] [g] [g] [g] [mm] [g/mm] [g/mm] [g]
1 0.0452 0.0672 0.0507 0.0220 0.0055 25.0000 9.289 0.00237 0.00059 2 0.0459 0.0677 0.0494 0.0218 0.0035 16.0550 9.397 0.00232 0.00037 3 0.0450 0.0625 0.0496 0.0175 0.0046 26.2857 8.216 0.00213 0.00056 4 0.0454 0.0600 0.0493 0.0146 0.0039 26.7123 7.369 0.00198 0.00053 5 0.0457 0.0689 0.0499 0.0232 0.0042 18.1034 8.315 0.00279 0.00051 6 0.0452 0.0620 0.0492 0.0168 0.0040 23.8095 7.794 0.00216 0.00051 7 0.0451 0.0694 0.0493 0.0243 0.0042 17.2840 8.969 0.00271 0.00047 8 0.0455 0.0604 0.0487 0.0149 0.0032 21.4765 7.526 0.00198 0.00043 9 0.0450 0.0859 0.0522 0.0409 0.0072 17.6039 11.707 0.00349 0.00062 10 0.0459 0.0707 0.0510 0.0248 0.0051 20.5645 9.416 0.00263 0.00054 11 0.0456 0.0657 0.0480 0.0201 0.0024 11.9403 7.808 0.00257 0.00031 12 0.0456 0.0982 0.0516 0.0526 0.0060 11.4068 9.912 0.00531 0.00061 13 0.0454 0.0641 0.0478 0.0187 0.0024 12.8342 6.413 0.00292 0.00037 14 0.0458 0.0708 0.0505 0.0250 0.0047 18.8000 9.714 0.00257 0.00048 15 0.0453 0.0674 0.0492 0.0221 0.0039 17.6471 7.986 0.00277 0.00049 16 0.0457 0.0778 0.0494 0.0321 0.0037 11.5265 8.155 0.00394 0.00045 18 0.0044 0.0177 0.0067 0.0133 0.0023 17.2932 5.809 0.00229 0.00040 19 0.0091 0.0348 0.0121 0.0257 0.0030 11.6732 7.760 0.00331 0.00039 20 0.0084 0.0186 0.0124 0.0102 0.0040 39.2157 9.065 0.00113 0.00044
B-2 Water quality variables
Water quality variables measured over time per aquaria during the avoidance behaviour experiment with A. aquaticus Temp. O2 El. conduct. Date ID pH [˚C] (O2) [mg/L] [µS/cm] 1-6-2011 Control 1 19.6 7.9 8.08 325 1-6-2011 Control 2 19.6 8.0 8.08 317 1-6-2011 Control 3 19.7 8.0 8.02 319 1-6-2011 Control 4 19.5 8.2 8.10 314 1-6-2011 Control 5 19.6 7.9 7.87 310 1-6-2011 Control 6 19.6 7.8 8.04 346 1-6-2011 Control 7 19.6 8.3 7.91 319 1-6-2011 Control 8 19.8 8.2 8.07 322 1-6-2011 320 A 19.5 8.1 8.15 285 1-6-2011 320 B 19.5 8.2 8.13 304 1-6-2011 320 C 19.6 8.3 8.12 295 1-6-2011 320 D 19.6 8.0 8.06 296 1-6-2011 320 E 19.6 7.8 8.09 295 1-6-2011 320 F 19.7 7.7 8.12 297 1-6-2011 320 G 19.6 8.5 8.14 283 1-6-2011 320 H 19.6 8.2 8.12 290 4-6-2011 Control 1 19.3 8.3 8.10 338 4-6-2011 Control 2 19.4 8.3 8.17 331 4-6-2011 Control 3 19.3 8.3 8.12 335 4-6-2011 Control 4 19.5 8.4 8.12 329 4-6-2011 Control 5 19.6 8.8 7.95 320 4-6-2011 Control 6 19.4 8.3 8.14 361 4-6-2011 Control 7 19.5 8.7 8.04 332 4-6-2011 Control 8 19.3 8.6 8.12 338 4-6-2011 320 A 19.3 8.1 8.13 296 4-6-2011 320 B 19.4 8.3 8.13 317 4-6-2011 320 C 19.5 8.4 8.17 308 4-6-2011 320 D 19.3 8.5 8.13 309 4-6-2011 320 E 19.3 8.6 8.18 309 4-6-2011 320 F 19.3 8.3 8.13 311 4-6-2011 320 G 19.5 8.1 8.14 294 4-6-2011 320 H 19.5 8.6 8.13 301
Average 19.5 8.3 8.1 313.4 SD 0.1 0.3 0.1 20.2 SD % 0.7 3.2 0.8 6.4
B-3 Manual scoring results avoidance behaviour experiment – Control 1. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 2. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 3. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 4. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % has taken place in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 5. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 6. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 7. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – Control 8. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 A. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 B. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 C. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 D. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 E. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 F. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 G. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-3 Manual scoring results avoidance behaviour experiment – 320 H. The number of A. aquaticus observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
B-4 Length weight characterization of Zygoptera, Agrypnia sp. and Anisoptera used in the avoidance behaviour experiment.
Weight Weight Weight Wet Dry Wet Dry Dry weight Length ID Species empty with wet with dry weight weight weight/length weight/length [g] [mm] [g] [g] [g] [g] [weight%] [g/mm] [g/mm]
1 Zygoptera 0.0502 0.0758 0.0555 0.0256 0.0053 20.7031 13.630 0.00188 0.00039 2 Zygoptera 0.0499 0.0726 0.0536 0.0227 0.0037 16.2996 13.940 0.00163 0.00027 3 Zygoptera 0.0498 0.0817 0.0580 0.0319 0.0082 25.7053 14.820 0.00215 0.00055 4 Zygoptera 0.0489 0.0751 0.0540 0.0262 0.0051 19.4656 14.860 0.00176 0.00034 5 Zygoptera 0.0494 0.0782 0.0527 0.0288 0.0033 11.4583 14.930 0.00193 0.00022 6 Zygoptera 0.0498 0.0852 0.0590 0.0354 0.0092 25.9887 17.040 0.00208 0.00054 7 Zygoptera 0.0497 0.0859 0.0588 0.0362 0.0091 25.1381 16.090 0.00225 0.00057 Mean 0.0497 0.0792 0.0559 0.0295 0.0063 20.6798 15.0443 0.0020 0.0004 SD 0.0004 0.0052 0.0026 0.0051 0.0025 5.4622 1.1827 0.0002 0.0001 SD % 0.8359 6.5204 4.7134 17.3853 40.1569 26.4132 7.8618 11.2698 34.8511 8 Agrypnia sp. 0.0492 0.1961 0.0831 0.1469 0.0339 23.0769 21.430 0.00685 0.00158 9 Agrypnia sp. 0.0489 0.1895 0.0828 0.1406 0.0339 24.1110 14.730 0.00955 0.00230 10 Agrypnia sp. 0.0496 0.1787 0.0823 0.1291 0.0327 25.3292 18.470 0.00699 0.00177 11 Agrypnia sp. 0.0491 0.1181 0.0703 0.0690 0.0212 30.7246 16.220 0.00425 0.00131 12 Agrypnia sp. 0.0492 0.1273 0.0660 0.0781 0.0168 21.5109 15.960 0.00489 0.00105 13 Agrypnia sp. 0.0494 0.1064 0.0594 0.0570 0.0100 17.5439 21.770 0.00262 0.00046 14 Agrypnia sp. 0.0490 0.1026 0.0617 0.0536 0.0127 23.6940 20.660 0.00259 0.00061 Mean 0.0492 0.1455 0.0722 0.0963 0.0230 23.7129 18.4629 0.0054 0.0013 SD 0.0002 0.0409 0.0104 0.0409 0.0104 3.9843 2.8809 0.0025 0.0007 SD % 0.4838 28.1226 14.4027 42.4718 45.1444 16.8021 15.6038 47.2789 50.1297 15 Anisoptera 0.0499 0.1952 0.0757 0.1453 0.0258 17.7564 13.950 0.01042 0.00185 16 Anisoptera 0.0493 0.1862 0.0777 0.1369 0.0284 20.7451 13.890 0.00986 0.00204 17 Anisoptera 0.0484 0.2372 0.0872 0.1888 0.0388 20.5508 15.430 0.01224 0.00251 18 Anisoptera 0.0494 0.2409 0.0848 0.1915 0.0354 18.4856 15.830 0.01210 0.00224 19 Anisoptera 0.0492 0.2067 0.0838 0.1575 0.0346 21.9683 14.720 0.01070 0.00235 20 Anisoptera 0.0498 0.1803 0.0730 0.1305 0.0232 17.7778 13.390 0.00975 0.00173 Mean 0.0493 0.2078 0.0804 0.1584 0.0310 19.5473 14.5350 0.0108 0.0021 SD 0.0005 0.0259 0.0057 0.0262 0.0061 1.7759 0.9572 0.0011 0.0003 SD % 1.0853 12.4459 7.0672 16.5402 19.7482 9.0850 6.5858 10.0141 14.1619
B-5 Ecology Ecology Zygoptera, Anisoptera and Agrypnia
After the experiments with A. aquaticus it was decided to make use of the existing experimental setup and test three other species for method development purposes. The actual choice for the species was dependent on the availability of suitable species. While searching for species in ditches at the Sinderhoeve, it appeared that the only species with a by camera recordable size and that could be caught in suitable numbers within a reasonable time, were Zygoptera (Odonata) (Damselfly), Anisoptera (Odonata) (Dragonfly) and the Trichoptera Agrypnia sp.
Ecology Odonata Dragonflies (Anisoptera) and Damselfies (Zygoptera) are part of the group Odonata of which approximately 6000 species are known (see figure 1 and 2). The larvae live mostly in aquatic ecosystems with a few exceptions of species that live in tropical rain forests. Their habitat varies between stagnant and running waters. What all species have in common is that they are predacious during their larval stage and feed on several preys varying from water fleas to small fish and is also known for cannibalism(van Tol 1988). Their life histories differ per habitat where slow movement behaviour was found in habitats with fish, their main predator. Specimens showed sit and wait behaviour (ambush hunters) in order to reduce the risk of predation. The fast movement behaviour was found in fish-free habitats, where Odonata was actively looking for prey and consequently grew much faster than specimens with the slow movements (Johnson 1991). Odonata used in the experiments showed the slow movement behaviour. In addition Zygoptera moved less than Anisoptera but clearly showed ‘ambush’ behaviour.
figure 1. Zygoptera (Odonata) (www.nwnature.net) Zygoptera (Odonata) (Adult) (www.richard-seaman.com
figure 2. Anisoptera (Odonata) (www.nwnature.net) Anisoptera (Odonata) (Adult)
Ecology Agrypnia sp. The insect Trichoptera, also called caddisfly, is closely related to Lepidoptera (butterfly). It thanks its name to the house where they are living in during their larval stage, a caddis (see figure 12). The materials for their caddis differ per species varying between small stones, leafs and pieces of wood and reed (Wiggins 1998). Larvae live in all kinds of fresh and brackish waters and are common in many continents over the world. There are about 180 species living in the Netherlands. Of these species, only a few species are able to swim what makes that they live on substrate such as stones and vegetation. The caddis from Agrypnia sp. is spiral shaped and often made of a piece of reed. Agrypnia sp. larvae live entirely on vegetation where it feeds amongst others on periphyton and organic matter. It lives predominantly in still standing water although it has been found in small streams as well (Higler 2008). Vegetation that grows through the water surface is important for the final metamorphosis to an adult (see figure 3).
figure 3. Larvae Agrypnia sp. (www.biologie.swordfish-diving.nl) and adult Agrypnia sp. (www.biopix.com)
B-6 Water quality variables
Water quality variables measured during avoidance behaviour experiment per aquarium with Anisoptera, Zygoptera and Agrypnia sp.
Anisoptera O2 Temp Date Treatment [mg/l] [˚C] EC [µS/cm] pH 14-jun C 5 8.4 20.3 338 7.6 14-jun 320 H 8.6 20.3 318 8.2 14-jun C 7 8.6 20.3 343 8.2 14-jun 320 G 8.7 20.3 300 8.1 17-jun C 5 8.6 19.9 355 8.02 17-jun 320 H 8.8 19.9 333 7.97 17-jun C 7 8.6 19.9 359 7.96 17-jun 320 G 8.2 19.9 314 8.01
Average 8.56 20.10 332.50 8.01 SD 0.18 0.21 20.56 0.19 SD % 2.2 1.1 6.2 2.4 Zygoptera O2 Temp Date Treatment [mg/l] [˚C] EC [µS/cm] pH 14-jun C 6 8.5 20.3 381 8 14-jun 320 E 8.8 20.3 321 8.2 14-jun C 2 8.6 20.3 351 8 14-jun 320 D 8.8 20.3 323 8.2 17-jun C 6 8.4 19.8 398 8 17-jun 320 E 9 19.8 337 7.99 17-jun C 2 8.5 19.8 367 7.98 17-jun 320 D 9.1 19.8 338 7.99
Average 8.71 20.05 352.00 8.05 SD 0.25 0.27 27.79 0.10 SD % 2.9 1.3 7.9 1.2 Agrypnia sp. O2 Temp Date Treatment [mg/l] [˚C] EC [µS/cm] pH 14-jun C 1 8.4 20.3 360 7.9 14-jun 320 B 8.7 20.3 336 8.2 14-jun C 4 8.6 20.3 345 8.1 14-jun 320 C 8.8 20.3 322 8.2 17-jun C 1 8.2 19.8 359 7.98 17-jun 320 B 8.6 19.8 353 7.96 17-jun C 4 8.5 19.8 362 7.97 17-jun 320 C 8.6 19.8 339 7.95
Average 8.55 20.05 347.00 8.03 SD 0.19 0.27 14.08 0.12 SD % 2.2 1.3 4.1 1.5
B-7 Manual scoring results avoidance behaviour experiment – Control 2. The number of Zygoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total % Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R L M R 14-6-2011 16.00 Control_2 Zygoptera 2 3 5 2 0 3 40 0 60 14-6-2011 17.00 Control_2 Zygoptera 3 2 5 3 0 2 60 0 40 14-6-2011 18.00 Control_2 Zygoptera 2 3 5 2 0 3 40 0 60 15-6-2011 9.00 Control_2 Zygoptera 2 1 1 4 2 1 1 50 25 25 15-6-2011 10.00 Control_2 Zygoptera 3 1 4 3 0 1 75 0 25 15-6-2011 11.00 Control_2 Zygoptera 3 1 4 3 0 1 75 0 25 15-6-2011 12.00 Control_2 Zygoptera 2 2 4 2 0 2 50 0 50 15-6-2011 13.00 Control_2 Zygoptera 2 1 1 4 2 0 2 50 0 50 15-6-2011 14.00 Control_2 Zygoptera 2 1 1 4 2 0 2 50 0 50 15-6-2011 15.00 Control_2 Zygoptera 2 1 1 4 2 1 1 50 25 25 15-6-2011 16.00 Control_2 Zygoptera 2 1 1 4 2 1 1 50 25 25 15-6-2011 17.00 Control_2 Zygoptera 2 1 1 4 2 1 1 50 25 25 16-6-2011 9.15 Control_2 Zygoptera 2 2 4 2 0 2 50 0 50 16-6-2011 10.15 Control_2 Zygoptera 2 1 1 4 2 0 2 50 0 50 16-6-2011 11.15 Control_2 Zygoptera 2 1 1 4 2 0 2 50 0 50 16-6-2011 12.15 Control_2 Zygoptera 2 1 1 4 2 0 2 50 0 50 16-6-2011 13.15 Control_2 Zygoptera 2 2 4 2 0 2 50 0 50 16-6-2011 14.15 Control_2 Zygoptera 3 1 4 3 1 0 75 25 0 16-6-2011 15.15 Control_2 Zygoptera 2 1 1 4 3 0 1 75 0 25 16-6-2011 16.15 Control_2 Zygoptera 2 1 1 4 3 1 0 75 25 0 16-6-2011 17.15 Control_2 Zygoptera 1 1 1 1 4 2 0 2 50 0 50 17-6-2011 9.00 Control_2 Zygoptera 3 1 4 3 0 1 75 0 25 17-6-2011 10.00 Control_2 Zygoptera 1 1 1 1 4 1 1 2 25 25 50 17-6-2011 11.00 Control_2 Zygoptera 2 1 1 4 2 0 2 50 0 50
B-7 Manual scoring results avoidance behaviour experiment – Control 6. The number of Zygoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R.
Total % Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R L M R 14-6-2011 16.00 Control_6 Zygoptera 2 3 5 2 0 3 40 0 60 14-6-2011 17.00 Control_6 Zygoptera 2 2 1 5 2 0 3 40 0 60 14-6-2011 18.00 Control_6 Zygoptera 2 1 2 5 2 1 2 40 20 40 15-6-2011 9.00 Control_6 Zygoptera 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 10.00 Control_6 Zygoptera 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 11.00 Control_6 Zygoptera 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 12.00 Control_6 Zygoptera 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 13.00 Control_6 Zygoptera 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 14.00 Control_6 Zygoptera 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 15.00 Control_6 Zygoptera 1 1 2 1 5 1 3 1 20 60 20 15-6-2011 16.00 Control_6 Zygoptera 2 1 2 5 2 0 3 40 0 60 15-6-2011 17.00 Control_6 Zygoptera 1 1 1 2 5 1 1 3 20 20 60 16-6-2011 9.15 Control_6 Zygoptera 1 1 2 1 5 1 1 3 20 20 60 16-6-2011 10.15 Control_6 Zygoptera 1 1 2 1 5 1 1 3 20 20 60 16-6-2011 11.15 Control_6 Zygoptera 1 2 2 5 1 0 4 20 0 80 16-6-2011 12.15 Control_6 Zygoptera 1 2 2 5 1 0 4 20 0 80 16-6-2011 13.15 Control_6 Zygoptera 1 2 2 5 1 0 4 20 0 80 16-6-2011 14.15 Control_6 Zygoptera 1 1 3 5 1 0 4 20 0 80 16-6-2011 15.15 Control_6 Zygoptera 1 1 1 2 5 1 1 3 20 20 60 16-6-2011 16.15 Control_6 Zygoptera 1 2 2 5 1 0 4 20 0 80 16-6-2011 17.15 Control_6 Zygoptera 1 3 1 5 1 0 4 20 0 80 17-6-2011 9.00 Control_6 Zygoptera 2 1 1 4 2 0 2 50 0 50 17-6-2011 10.00 Control_6 Zygoptera 2 1 1 4 2 0 2 50 0 50 17-6-2011 11.00 Control_6 Zygoptera 3 1 1 5 3 0 2 60 0 40
B-7 Manual scoring results avoidance behaviour experiment – 320 D. The number of Zygoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total % Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R L M R 14-6-2011 16.00 320_D Zygoptera 4 1 5 4 1 0 80 20 0 14-6-2011 17.00 320_D Zygoptera 4 1 5 4 0 1 80 0 20 14-6-2011 18.00 320_D Zygoptera 4 1 5 4 0 1 80 0 20 15-6-2011 9.00 320_D Zygoptera 4 1 5 4 0 1 80 0 20 15-6-2011 10.00 320_D Zygoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 11.00 320_D Zygoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 12.00 320_D Zygoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 13.00 320_D Zygoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 14.00 320_D Zygoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 15.00 320_D Zygoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 16.00 320_D Zygoptera 1 2 1 1 5 3 1 1 60 20 20 15-6-2011 17.00 320_D Zygoptera 2 1 1 1 5 3 1 1 60 20 20 16-6-2011 9.15 320_D Zygoptera 2 1 1 1 5 3 1 1 60 20 20 16-6-2011 10.15 320_D Zygoptera 2 1 1 1 5 3 1 1 60 20 20 16-6-2011 11.15 320_D Zygoptera 2 1 1 4 3 1 0 75 25 0 16-6-2011 12.15 320_D Zygoptera 3 1 1 5 4 1 0 80 20 0 16-6-2011 13.15 320_D Zygoptera 2 2 1 5 4 1 0 80 20 0 16-6-2011 14.15 320_D Zygoptera 2 2 1 5 4 1 0 80 20 0 16-6-2011 15.15 320_D Zygoptera 2 2 1 5 4 1 0 80 20 0 16-6-2011 16.15 320_D Zygoptera 2 2 1 5 4 1 0 80 20 0 16-6-2011 17.15 320_D Zygoptera 2 2 1 5 4 1 0 80 20 0 17-6-2011 9.00 320_D Zygoptera 1 1 1 1 1 5 2 1 2 40 20 40 17-6-2011 10.00 320_D Zygoptera 1 1 1 1 1 5 2 1 2 40 20 40 17-6-2011 11.00 320_D Zygoptera 2 1 1 1 5 3 1 1 60 20 20
B-7 Manual scoring results avoidance behaviour experiment – 320 E. The number of Zygoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total % Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R L M R 14-6-2011 16.00 320_E Zygoptera 3 1 1 5 3 0 2 60 0 40 14-6-2011 17.00 320_E Zygoptera 3 1 1 5 3 1 1 60 20 20 14-6-2011 18.00 320_E Zygoptera 3 1 1 5 3 0 2 60 0 40 15-6-2011 9.00 320_E Zygoptera 2 1 2 5 2 1 2 40 20 40 15-6-2011 10.00 320_E Zygoptera 1 1 2 4 2 0 2 50 0 50 15-6-2011 11.00 320_E Zygoptera 2 1 2 5 2 0 3 40 0 60 15-6-2011 12.00 320_E Zygoptera 2 2 4 2 0 2 50 0 50 15-6-2011 13.00 320_E Zygoptera 2 2 4 2 0 2 50 0 50 15-6-2011 14.00 320_E Zygoptera 2 1 1 4 3 0 1 75 0 25 15-6-2011 15.00 320_E Zygoptera 2 1 1 1 5 3 0 2 60 0 40 15-6-2011 16.00 320_E Zygoptera 2 1 1 1 5 3 0 2 60 0 40 15-6-2011 17.00 320_E Zygoptera 3 1 1 5 3 0 2 60 0 40 16-6-2011 9.15 320_E Zygoptera 1 2 2 5 3 0 2 60 0 40 16-6-2011 10.15 320_E Zygoptera 1 1 1 2 5 2 0 3 40 0 60 16-6-2011 11.15 320_E Zygoptera 1 1 2 1 5 2 0 3 40 0 60 16-6-2011 12.15 320_E Zygoptera 1 1 1 2 5 2 1 2 40 20 40 16-6-2011 13.15 320_E Zygoptera 2 3 5 2 0 3 40 0 60 16-6-2011 14.15 320_E Zygoptera 1 1 1 1 1 5 2 1 2 40 20 40 16-6-2011 15.15 320_E Zygoptera 1 1 1 2 5 1 2 2 20 40 40 16-6-2011 16.15 320_E Zygoptera 2 1 2 5 3 0 2 60 0 40 16-6-2011 17.15 320_E Zygoptera 2 1 2 5 3 0 2 60 0 40 17-6-2011 9.00 320_E Zygoptera 2 2 1 5 4 0 1 80 0 20 17-6-2011 10.00 320_E Zygoptera 1 1 2 4 2 0 2 50 0 50 17-6-2011 11.00 320_E Zygoptera 2 1 1 1 5 3 0 2 60 0 40
B-7 Manual scoring results avoidance behaviour experiment – Control 1. The number of Agrypnia sp. observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total Total Total Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R % L % M % R 14-6-2011 16.00 Control_1 Agrypnia sp. 2 2 1 5 2 2 1 40 40 20 14-6-2011 17.00 Control_1 Agrypnia sp. 1 1 3 5 1 0 4 20 0 80 14-6-2011 18.00 Control_1 Agrypnia sp. 1 2 1 1 5 3 0 2 60 0 40 15-6-2011 9.00 Control_1 Agrypnia sp. 1 2 2 5 3 0 2 60 0 40 15-6-2011 10.00 Control_1 Agrypnia sp. 1 1 3 5 1 0 4 20 0 80 15-6-2011 11.00 Control_1 Agrypnia sp. 3 1 1 5 3 0 2 60 0 40 15-6-2011 12.00 Control_1 Agrypnia sp. 1 1 3 5 0 1 4 0 20 80 15-6-2011 13.00 Control_1 Agrypnia sp. 1 2 1 1 5 3 0 2 60 0 40 15-6-2011 14.00 Control_1 Agrypnia sp. 1 1 1 2 5 1 1 3 20 20 60 15-6-2011 15.00 Control_1 Agrypnia sp. 3 2 5 3 0 2 60 0 40 15-6-2011 16.00 Control_1 Agrypnia sp. 5 5 0 0 5 0 0 100 15-6-2011 17.00 Control_1 Agrypnia sp. 2 2 1 5 2 0 3 40 0 60 16-6-2011 9.15 Control_1 Agrypnia sp. 3 1 1 5 3 0 2 60 0 40 16-6-2011 10.15 Control_1 Agrypnia sp. 3 2 5 3 0 2 60 0 40 16-6-2011 11.15 Control_1 Agrypnia sp. 3 2 5 3 0 2 60 0 40 16-6-2011 12.15 Control_1 Agrypnia sp. 3 2 5 3 0 2 60 0 40 16-6-2011 13.15 Control_1 Agrypnia sp. 1 3 1 5 4 0 1 80 0 20 16-6-2011 14.15 Control_1 Agrypnia sp. 1 1 3 5 1 1 3 20 20 60 16-6-2011 15.15 Control_1 Agrypnia sp. 1 2 2 5 3 0 2 60 0 40 16-6-2011 16.15 Control_1 Agrypnia sp. 1 4 5 0 0 5 0 0 100 16-6-2011 17.15 Control_1 Agrypnia sp. 5 5 0 0 5 0 0 100 17-6-2011 9.00 Control_1 Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 17-6-2011 10.00 Control_1 Agrypnia sp. 2 1 1 1 5 2 1 2 40 20 40 17-6-2011 11.00 Control_1 Agrypnia sp. 3 1 1 5 3 1 1 60 20 20
B-7 Manual scoring results avoidance behaviour experiment – Control 4. The number of Agrypnia sp. observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total Total Total Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R % L % M % R 14-6-2011 16.00 Control_4 Agrypnia sp. 2 2 1 5 2 0 3 40 0 60 14-6-2011 17.00 Control_4 Agrypnia sp. 4 1 5 4 0 1 80 0 20 14-6-2011 18.00 Control_4 Agrypnia sp. 2 2 1 5 2 0 3 40 0 60 15-6-2011 9.00 Control_4 Agrypnia sp. 3 1 1 5 4 0 1 80 0 20 15-6-2011 10.00 Control_4 Agrypnia sp. 3 1 1 5 3 1 1 60 20 20 15-6-2011 11.00 Control_4 Agrypnia sp. 3 1 1 5 3 0 2 60 0 40 15-6-2011 12.00 Control_4 Agrypnia sp. 2 3 5 2 0 3 40 0 60 15-6-2011 13.00 Control_4 Agrypnia sp. 2 2 1 5 2 2 1 40 40 20 15-6-2011 14.00 Control_4 Agrypnia sp. 1 1 1 1 1 5 2 1 2 40 20 40 15-6-2011 15.00 Control_4 Agrypnia sp. 2 1 2 5 3 0 2 60 0 40 15-6-2011 16.00 Control_4 Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 15-6-2011 17.00 Control_4 Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 16-6-2011 9.15 Control_4 Agrypnia sp. 1 1 3 5 2 0 3 40 0 60 16-6-2011 10.15 Control_4 Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 16-6-2011 11.15 Control_4 Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 16-6-2011 12.15 Control_4 Agrypnia sp. 2 2 1 5 2 0 3 40 0 60 16-6-2011 13.15 Control_4 Agrypnia sp. 2 2 1 5 2 0 3 40 0 60 16-6-2011 14.15 Control_4 Agrypnia sp. 1 1 1 2 5 1 1 3 20 20 60 16-6-2011 15.15 Control_4 Agrypnia sp. 1 3 1 5 1 0 4 20 0 80 16-6-2011 16.15 Control_4 Agrypnia sp. 1 2 1 1 5 3 1 1 60 20 20 16-6-2011 17.15 Control_4 Agrypnia sp. 1 1 2 1 5 2 0 3 40 0 60 17-6-2011 9.00 Control_4 Agrypnia sp. 3 1 1 5 4 0 1 80 0 20 17-6-2011 10.00 Control_4 Agrypnia sp. 3 1 1 5 4 0 1 80 0 20 17-6-2011 11.00 Control_4 Agrypnia sp. 4 1 5 4 0 1 80 0 20
B-7 Manual scoring results avoidance behaviour experiment – 320 B. The number of Agrypnia sp. observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total Total Total Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R % L % M % R 14-6-2011 16.00 320_B Agrypnia sp. 1 1 2 1 5 1 1 3 20 20 60 14-6-2011 17.00 320_B Agrypnia sp. 2 3 5 5 0 0 100 0 0 14-6-2011 18.00 320_B Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 15-6-2011 9.00 320_B Agrypnia sp. 1 1 3 5 2 0 3 40 0 60 15-6-2011 10.00 320_B Agrypnia sp. 1 2 2 5 3 0 2 60 0 40 15-6-2011 11.00 320_B Agrypnia sp. 1 2 1 1 5 3 0 2 60 0 40 15-6-2011 12.00 320_B Agrypnia sp. 1 1 2 1 5 2 0 3 40 0 60 15-6-2011 13.00 320_B Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 15-6-2011 14.00 320_B Agrypnia sp. 2 1 2 5 3 0 2 60 0 40 15-6-2011 15.00 320_B Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 15-6-2011 16.00 320_B Agrypnia sp. 1 1 1 2 5 2 0 3 40 0 60 15-6-2011 17.00 320_B Agrypnia sp. 1 4 5 1 0 4 20 0 80 16-6-2011 9.15 320_B Agrypnia sp. 1 4 5 1 0 4 20 0 80 16-6-2011 10.15 320_B Agrypnia sp. 2 1 1 1 5 2 1 2 40 20 40 16-6-2011 11.15 320_B Agrypnia sp. 1 4 5 0 0 5 0 0 100 16-6-2011 12.15 320_B Agrypnia sp. 2 1 2 5 3 2 0 60 40 0 16-6-2011 13.15 320_B Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 16-6-2011 14.15 320_B Agrypnia sp. 1 3 1 5 4 0 1 80 0 20 16-6-2011 15.15 320_B Agrypnia sp. 1 1 3 5 1 1 3 20 20 60 16-6-2011 16.15 320_B Agrypnia sp. 1 2 2 5 3 0 2 60 0 40 16-6-2011 17.15 320_B Agrypnia sp. 3 1 1 5 4 0 1 80 0 20 17-6-2011 9.00 320_B Agrypnia sp. 1 1 3 5 1 0 4 20 0 80 17-6-2011 10.00 320_B Agrypnia sp. 2 2 1 5 4 0 1 80 0 20 17-6-2011 11.00 320_B Agrypnia sp. 1 3 1 5 4 0 1 80 0 20
B-7 Manual scoring results avoidance behaviour experiment – 320 C. The number of Agrypnia sp. observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R % L M R 14-6-2011 16.00 320_C Agrypnia sp. 2 2 1 5 2 0 3 40 0 60 14-6-2011 17.00 320_C Agrypnia sp. 3 2 5 3 0 2 60 0 40 14-6-2011 18.00 320_C Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 15-6-2011 9.00 320_C Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 15-6-2011 10.00 320_C Agrypnia sp. 1 3 1 5 0 1 4 0 20 80 15-6-2011 11.00 320_C Agrypnia sp. 2 1 2 5 2 0 3 40 0 60 15-6-2011 12.00 320_C Agrypnia sp. 1 2 2 5 1 2 2 20 40 40 15-6-2011 13.00 320_C Agrypnia sp. 3 2 5 0 0 5 0 0 100 15-6-2011 14.00 320_C Agrypnia sp. 2 1 2 5 3 0 2 60 0 40 15-6-2011 15.00 320_C Agrypnia sp. 1 1 3 5 2 0 3 40 0 60 15-6-2011 16.00 320_C Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 15-6-2011 17.00 320_C Agrypnia sp. 1 4 5 1 0 4 20 0 80 16-6-2011 9.15 320_C Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 16-6-2011 10.15 320_C Agrypnia sp. 1 2 2 5 1 0 4 20 0 80 16-6-2011 11.15 320_C Agrypnia sp. 1 3 1 5 1 0 4 20 0 80 16-6-2011 12.15 320_C Agrypnia sp. 1 2 2 5 0 1 4 0 20 80 16-6-2011 13.15 320_C Agrypnia sp. 3 1 1 5 3 0 2 60 0 40 16-6-2011 14.15 320_C Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 16-6-2011 15.15 320_C Agrypnia sp. 2 1 1 1 5 3 0 2 60 0 40 16-6-2011 16.15 320_C Agrypnia sp. 1 2 2 5 3 0 2 60 0 40 16-6-2011 17.15 320_C Agrypnia sp. 1 3 1 5 1 0 4 20 0 80 17-6-2011 9.00 320_C Agrypnia sp. 2 1 2 5 2 0 3 40 0 60 17-6-2011 10.00 320_C Agrypnia sp. 1 1 2 1 5 2 2 1 40 40 20 17-6-2011 11.00 320_C Agrypnia sp. 1 3 1 5 1 0 4 20 0 80
B-7 Manual scoring results avoidance behaviour experiment – Control 5. The number of Anisoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R % L M R 14-6-2011 16.00 Control_5 Anisoptera 3 1 1 5 3 1 1 60 20 20 14-6-2011 17.00 Control_5 Anisoptera 3 2 5 3 0 2 60 0 40 14-6-2011 18.00 Control_5 Anisoptera 3 1 1 5 4 0 1 80 0 20 15-6-2011 9.00 Control_5 Anisoptera 2 1 1 4 2 1 1 50 25 25 15-6-2011 10.00 Control_5 Anisoptera 1 2 1 4 3 0 1 75 0 25 15-6-2011 11.00 Control_5 Anisoptera 3 1 4 3 0 1 75 0 25 15-6-2011 12.00 Control_5 Anisoptera 1 2 1 4 3 0 1 75 0 25 15-6-2011 13.00 Control_5 Anisoptera 2 1 1 4 2 1 1 50 25 25 15-6-2011 14.00 Control_5 Anisoptera 1 1 1 1 4 1 1 2 25 25 50 15-6-2011 15.00 Control_5 Anisoptera 2 1 2 5 2 1 2 40 20 40 15-6-2011 16.00 Control_5 Anisoptera 2 2 4 4 0 0 100 0 0 15-6-2011 17.00 Control_5 Anisoptera 2 1 1 4 2 0 2 50 0 50 16-6-2011 9.15 Control_5 Anisoptera 1 2 3 1 0 2 33.33 0 66.66 16-6-2011 10.15 Control_5 Anisoptera 2 2 4 2 0 2 50 0 50 16-6-2011 11.15 Control_5 Anisoptera 2 2 4 2 0 2 50 0 50 16-6-2011 12.15 Control_5 Anisoptera 1 2 1 4 1 0 3 25 0 75 16-6-2011 13.15 Control_5 Anisoptera 1 2 1 4 1 0 3 25 0 75 16-6-2011 14.15 Control_5 Anisoptera 1 2 3 1 0 2 33.33 0 66.66 16-6-2011 15.15 Control_5 Anisoptera 1 3 4 1 0 3 25 0 75 16-6-2011 16.15 Control_5 Anisoptera 2 1 1 4 2 0 2 50 0 50 16-6-2011 17.15 Control_5 Anisoptera 1 2 1 4 1 2 1 25 50 25 17-6-2011 9.00 Control_5 Anisoptera 2 2 4 0 0 4 0 0 100 17-6-2011 10.00 Control_5 Anisoptera 1 2 1 4 1 0 3 25 0 75 17-6-2011 11.00 Control_5 Anisoptera 1 3 4 0 0 4 0 0 100
B-7 Manual scoring results avoidance behaviour experiment – Control 7. The number of Anisoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total Total Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R % L % M R 14-6-2011 16.00 Control_7 Anisoptera 3 1 1 5 3 0 2 60 0 40 14-6-2011 17.00 Control_7 Anisoptera 1 2 2 5 1 0 4 20 0 80 14-6-2011 18.00 Control_7 Anisoptera 1 1 1 2 5 1 2 2 20 40 40 15-6-2011 9.00 Control_7 Anisoptera 2 3 5 2 0 3 40 0 60 15-6-2011 10.00 Control_7 Anisoptera 1 1 2 1 5 1 1 3 20 20 60 15-6-2011 11.00 Control_7 Anisoptera 1 4 5 5 0 0 100 0 0 15-6-2011 12.00 Control_7 Anisoptera 1 3 1 5 0 1 4 0 20 80 15-6-2011 13.00 Control_7 Anisoptera 4 1 5 4 0 1 80 0 20 15-6-2011 14.00 Control_7 Anisoptera 2 1 1 1 5 3 1 1 60 20 20 15-6-2011 15.00 Control_7 Anisoptera 1 3 1 5 4 0 1 80 0 20 15-6-2011 16.00 Control_7 Anisoptera 1 1 2 1 5 1 1 3 20 20 60 15-6-2011 17.00 Control_7 Anisoptera 4 1 5 0 0 5 0 0 100 16-6-2011 9.15 Control_7 Anisoptera 3 2 5 5 0 0 100 0 0 16-6-2011 10.15 Control_7 Anisoptera 1 2 1 1 5 1 2 2 20 40 40 16-6-2011 11.15 Control_7 Anisoptera 2 1 2 5 2 0 3 40 0 60 16-6-2011 12.15 Control_7 Anisoptera 2 3 5 2 0 3 40 0 60 16-6-2011 13.15 Control_7 Anisoptera 2 2 1 5 2 0 3 40 0 60 16-6-2011 14.15 Control_7 Anisoptera 2 2 1 5 4 1 0 80 20 0 16-6-2011 15.15 Control_7 Anisoptera 1 3 1 5 4 0 1 80 0 20 16-6-2011 16.15 Control_7 Anisoptera 1 3 1 5 1 0 4 20 0 80 16-6-2011 17.15 Control_7 Anisoptera 2 2 1 5 4 0 1 80 0 20 17-6-2011 9.00 Control_7 Anisoptera 1 1 1 2 5 1 1 3 20 20 60 17-6-2011 10.00 Control_7 Anisoptera 1 1 1 1 1 5 2 1 2 40 20 40 17-6-2011 11.00 Control_7 Anisoptera 1 2 2 5 3 0 2 60 0 40
B-7 Manual scoring results avoidance behaviour experiment – 320 G. The number of Anisoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total % Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R L M R 14-6-2011 16.00 320_G Anisoptera 3 2 5 3 0 2 60 0 40 14-6-2011 17.00 320_G Anisoptera 2 1 2 5 3 0 2 60 0 40 14-6-2011 18.00 320_G Anisoptera 2 1 2 5 3 0 2 60 0 40 15-6-2011 9.00 320_G Anisoptera 3 1 1 5 3 0 2 60 0 40 15-6-2011 10.00 320_G Anisoptera 3 2 5 3 0 2 60 0 40 15-6-2011 11.00 320_G Anisoptera 1 2 2 5 3 0 2 60 0 40 15-6-2011 12.00 320_G Anisoptera 1 2 2 5 3 0 2 60 0 40 15-6-2011 13.00 320_G Anisoptera 3 1 1 5 3 1 1 60 20 20 15-6-2011 14.00 320_G Anisoptera 1 2 1 1 5 3 1 1 60 20 20 15-6-2011 15.00 320_G Anisoptera 2 1 1 4 3 0 1 75 0 25 15-6-2011 16.00 320_G Anisoptera 2 1 1 4 3 0 1 75 0 25 15-6-2011 17.00 320_G Anisoptera 2 1 1 4 3 0 1 75 0 25 16-6-2011 9.15 320_G Anisoptera 3 1 4 3 0 1 75 0 25 16-6-2011 10.15 320_G Anisoptera 3 1 4 4 0 0 100 0 0 16-6-2011 11.15 320_G Anisoptera 3 1 4 3 0 1 75 0 25 16-6-2011 12.15 320_G Anisoptera 2 1 1 4 2 1 1 50 25 25 16-6-2011 13.15 320_G Anisoptera 1 1 1 1 4 2 0 2 50 0 50 16-6-2011 14.15 320_G Anisoptera 1 2 1 4 1 0 3 25 0 75 16-6-2011 15.15 320_G Anisoptera 1 2 1 4 1 0 3 25 0 75 16-6-2011 16.15 320_G Anisoptera 1 1 1 1 2 0 1 200 0 100 16-6-2011 17.15 320_G Anisoptera 1 2 1 4 1 0 3 25 0 75 17-6-2011 9.00 320_G Anisoptera 1 1 1 3 1 0 2 33 0 67 17-6-2011 10.00 320_G Anisoptera 1 2 3 0 0 3 0 0 100 17-6-2011 11.00 320_G Anisoptera 1 2 3 0 0 3 0 0 100
B-7 Manual scoring results avoidance behaviour experiment – 320 H. The number of Anisoptera observed are shown per section L1,L2 – left high and low, M1,M2 – middle high and low, R1,R2 – right high and low and summed per section in Total L,M and R. Distribution in % is shown in Total % L,M and R. Total % Total % Total % Date Time aqua ID Species L1 L2 M1 M2 R1 R2 Total Total_L Total_M Total_R L M R 14-6-2011 16.00 320_H Anisoptera 2 1 2 5 2 0 3 40 0 60 14-6-2011 17.00 320_H Anisoptera 2 1 1 1 5 2 1 2 40 20 40 14-6-2011 18.00 320_H Anisoptera 1 1 3 5 2 0 3 40 0 60 15-6-2011 9.00 320_H Anisoptera 1 1 2 1 5 1 1 3 20 20 60 15-6-2011 10.00 320_H Anisoptera 1 3 1 5 1 0 4 20 0 80 15-6-2011 11.00 320_H Anisoptera 2 1 2 5 2 1 2 40 20 40 15-6-2011 12.00 320_H Anisoptera 1 4 5 5 0 0 100 0 0 15-6-2011 13.00 320_H Anisoptera 1 3 1 5 4 0 1 80 0 20 15-6-2011 14.00 320_H Anisoptera 1 1 1 1 1 5 2 1 2 40 20 40 15-6-2011 15.00 320_H Anisoptera 2 1 2 5 2 1 2 40 20 40 15-6-2011 16.00 320_H Anisoptera 2 2 1 5 2 0 3 40 0 60 15-6-2011 17.00 320_H Anisoptera 2 3 5 2 0 3 40 0 60 16-6-2011 9.15 320_H Anisoptera 1 1 2 4 1 1 2 25 25 50 16-6-2011 10.15 320_H Anisoptera 1 1 3 5 2 0 3 40 0 60 16-6-2011 11.15 320_H Anisoptera 1 1 3 5 2 0 3 40 0 60 16-6-2011 12.15 320_H Anisoptera 2 1 2 5 3 0 2 60 0 40 16-6-2011 13.15 320_H Anisoptera 3 2 5 3 0 2 60 0 40 16-6-2011 14.15 320_H Anisoptera 3 2 5 3 0 2 60 0 40 16-6-2011 15.15 320_H Anisoptera 3 2 5 3 0 2 60 0 40 16-6-2011 16.15 320_H Anisoptera 3 2 5 3 0 2 60 0 40 16-6-2011 17.15 320_H Anisoptera 1 2 2 5 3 0 2 60 0 40 17-6-2011 9.00 320_H Anisoptera 1 1 3 5 2 0 3 40 0 60 17-6-2011 10.00 320_H Anisoptera 1 1 3 5 2 0 3 40 0 60 17-6-2011 11.00 320_H Anisoptera 1 1 2 4 1 1 2 25 25 50
C. Colonization experiment
C-1 Abundance of specimens per taxa per container sampled in the colonization experiment (1) Chironomus Chironomidae Chironomini Chaoborus Sialis Caenis Erpobdella Start date End date code conc. replica spec Tanypodinae pop spec spec spec. spec. Tangtarsini octuculata 15-4-2011 9-6-2011 c1 A 0 1 15 10 1 2 2 15-4-2011 9-6-2011 c1 B 0 2 8 9 2 1 15-4-2011 9-6-2011 c1 C 0 3 12 5 2 1 4 2 15-4-2011 9-6-2011 c2 A 0 1 4 18 2 2 1 15-4-2011 9-6-2011 c2 B 0 2 6 4 18 5 6 15-4-2011 9-6-2011 c2 C 0 3 4 4 3 2 2 15-4-2011 9-6-2011 20 A 20 1 17 12 1 4 6 15-4-2011 9-6-2011 20 B 20 2 26 11 2 2 5 1 15-4-2011 9-6-2011 20 C 20 3 5 11 8 3 15-4-2011 9-6-2011 40 A 40 1 16 1 11 1 1 15-4-2011 9-6-2011 40 B 40 2 6 6 8 1 1 15-4-2011 9-6-2011 40 C 40 3 6 6 1 3 5 15-4-2011 9-6-2011 80 A 80 1 15 3 2 1 1 15-4-2011 9-6-2011 80 B 80 2 16 3 3 1 15-4-2011 9-6-2011 80 C 80 3 14 2 4 3 3 15-4-2011 9-6-2011 160 A 160 1 16 6 5 3 2 1 15-4-2011 9-6-2011 160 B 160 2 21 3 1 5 15-4-2011 9-6-2011 160 C 160 3 5 3 2 3 2 15-4-2011 9-6-2011 320 A 320 1 11 10 5 8 1 15-4-2011 9-6-2011 320 B 320 2 1 4 6 2 1 15-4-2011 9-6-2011 320 C 320 3 9 12 2
Total 196 140 1 119 1 52 48 2 6
C-1 Abundance of specimens per taxa per container sampled in the colonization experiment (2) Erpobdellea Dytiscidae Cloeon Amisopleza Start date End date code conc. replica juv larvae Orthocladinae dipterum Ceratopogonidae larvae Tanytarsini Planorbis 15-4-2011 9-6-2011 c1 A 0 1 1 1 15-4-2011 9-6-2011 c1 B 0 2 15-4-2011 9-6-2011 c1 C 0 3 1 3 15-4-2011 9-6-2011 c2 A 0 1 15-4-2011 9-6-2011 c2 B 0 2 4 1 2 15-4-2011 9-6-2011 c2 C 0 3 4 15-4-2011 9-6-2011 20 A 20 1 3 15-4-2011 9-6-2011 20 B 20 2 4 1 1 15-4-2011 9-6-2011 20 C 20 3 7 1 1 15-4-2011 9-6-2011 40 A 40 1 1 11 1 15-4-2011 9-6-2011 40 B 40 2 1 1 15-4-2011 9-6-2011 40 C 40 3 5 15-4-2011 9-6-2011 80 A 80 1 2 15-4-2011 9-6-2011 80 B 80 2 2 1 15-4-2011 9-6-2011 80 C 80 3 9 1 15-4-2011 9-6-2011 160 A 160 1 15-4-2011 9-6-2011 160 B 160 2 2 15-4-2011 9-6-2011 160 C 160 3 15-4-2011 9-6-2011 320 A 320 1 4 1 15-4-2011 9-6-2011 320 B 320 2 9 15-4-2011 9-6-2011 320 C 320 3 5 5 1 1 1
Total 63 2 21 2 5 1 3 2
C-2 Biomass
Dry weight determination of the macroinvertebrates per container sampled during the colonization experiment. The column boat shows the weight of the aluminium boat. The column with boat and macro fauna represents the total weight of the boat and the macro fauna. The dry macro fauna column is the actual dry weight of macro fauna per container.
ID Boat [g] Boat + dry macro fauna [g] Dry macro fauna [g] C1 A 0.0498 0.0566 0.0068 C1 B 0.0504 0.0534 0.003 C1 C 0.0498 0.0562 0.0064 C2 A 0.0505 0.0555 0.005 C2 B 0.0501 0.0576 0.0075 C2 C 0.0501 0.0513 0.0012 C1 C 0.0497 0.1107 0.061 C2 A 0.0491 0.1005 0.0514 20 A 0.0499 0.0532 0.0033 20 B 0.0501 0.0578 0.0077 20 C 0.0505 0.0539 0.0034 40 A 0.0501 0.0546 0.0045 40 B 0.0502 0.0527 0.0025 40 C 0.0501 0.0528 0.0027 80 A 0.0496 0.0551 0.0055 80 B 0.0501 0.0543 0.0042 80 C 0.0492 0.0536 0.0044 80 A 0.0497 0.0621 0.0124 Leech 160 A 0.0503 0.0531 0.0028 160 B 0.05 0.0562 0.0062 160 C 0.0492 0.0505 0.0013 160 A 0.0497 0.0954 0.0457 Leech 320 A 0.0491 0.0536 0.0045 320 B 0.0499 0.0512 0.0013 320 C 0.049 0.053 0.004 320 A 0.0491 0.0803 0.0312 Snail 320 B 0.0502 0.0764 0.0262 Leech
320 C 0.05 0.0649 0.0149 Snail