Scientific insights

Bringing you articles full of in-depth scientific insight on subjects ranging from pharmaceutical development through to crop protection registrations.
UK sales@ukorg.huntingdon.com USA sales@priceton.huntingdon.com Japan +81 (0) 3 3238 6381-7

Pharmaceuticals Biologics Crop protection Chemical

The need for (Higher)2 Tier tests to assess pesticide impact on Ecosystem Services

You can read more of our articles, posters and case studies on our website

www.huntingdon.com

Ecotoxicology

The need for (Higher)2 Tier tests to assess pesticide impact on Ecosystem Services
Removing the doubts from risk assessment by testing parental emergence, reproduction and F1 survival of insects in field microcosms

he benefits that humans receive from ecosystems have been categorised in the Millennium Ecosystem Assessment (MEA, 2005) into provisioning, regulating, supporting and cultural services and this concept has been recognised as a useful framework for identifying specific protection goals (SPGs) for plant protection products and their residues (EFSA 2010, Table 1). Freshwater ecosystems are important components of landscapes and the organisms that make up their food webs are diverse and important both ecologically and economically. The macro-invertebrate communities that inhabit aquatic systems are made up of populations of organisms that have both entirely aquatic life histories and the aquatic larval stages of insects that emerge, reproduce, and return to aquatic systems to lay their eggs. These communities are recognised as providing a wide range of valuable Ecosystem Services (ES) that include improving water quality for drinking, detritus processing, sustaining fisheries, providing leisure and recreational activities (Simaika and Samways, 2008), pest control, cultural and recreational services and food sources for terrestrial organisms such as birds, spiders and lizards (Nakano and Murakami, 2001, Sabo and Power 2002, Baxter et al. 2005). In risk assessment processes, specific protection goals (SPGs) are generally aimed at the population level or higher using predicted no-effect concentrations that are experimentally derived from freshwater outdoor microcosm studies. The community composition and realism of freshwater microcosms is particularly important and the range of field methodologies available should be capable of supplying data to assess pesticide impacts on key ESs. An article in the previous issue of this journal (First Flight, Jenkins, 2011) described a novel total enclosure method that was used to quantitatively assess the effects of pesticides on insect emergence from field microcosms, in particular those with longer, more complex life cycles. This higher, higher tier (Higher2 Tier) method in itself goes well beyond the scope of current regulatory test guidelines for field microcosms (HARAP and CLASSIC, 1999 and 2002 respectively, and OECD 2006), and begins to provide some of the answers that are needed to adequately assess impacts of pesticides on Ecosystem Services and on biodiversity. In this article, the total enclosure methodology is extended 2

even further to allow assessment of effects not only on adult insect emergence, but also of reproduction and adult fecundity by monitoring the numbers of first generation larvae (F1) produced in outdoor freshwater microcosms. The key objectives in this study were to assess the impact of a relatively persistent insecticide whose mode of action was likely to be of concern to aquatic insects (particularly Damselflies and Dragonflies) by assessing its effects on: freshwater macro invertebrate communities adult insect emergence, reproduction and survival of F1 generation larvae in field microcosms

Methods

The study was conducted in 20 outdoor freshwater microcosms (water depth: 30 cm) which were designed to represent the worst case of a shallow static or slowflowing ditch. Microcosms were established in early spring by the addition of clay and lake sediments, detritus (fallen leaves of predominantly Alder) aquatic macrophytes and invertebrates in order to allow time for the establishment of similar and stable invertebrate communities in each before application of the pesticide. During the establishment phase, invertebrates with entirely aquatic life histories and insect larvae were collected from a variety of field sites, populations established in microcosms, and their diversity and abundance regularly assessed. Macro-invertebrates with entirely aquatic life histories e.g. freshwater shrimp, (Crangonyx), the hoglouse (Asellus sp.), beetles, mites, flat worms, annelid worms, leeches and molluscs, were sampled every three weeks throughout the study using substrate samplers placed into each microcosm. In addition to well-established samplers (Hester-Dendy, stone substrate), Artificial Substrate Samplers modified to include detritus (predominantly leaves of the common Alder Anus glutinosa), and floating samplers containing Elodea were designed especially for the study in order to provide refugia for newly hatched insect larvae. In addition to these samplers, underwater viewers were used to count the numbers of the larger caddis larvae in microcosms.

Ecotoxicology
Table 1: Overview of Ecosystem Services in agricultural landscapes potentially affected by pesticides (EFSA, 2010) Ecosystem service category Provisioning In crop area Off crop area Microcosms completely enclosed with insect proof netting after application of the test material

Food Fibre and fuel

Food Genetic resource Freshwater Pollination Pest and disease regulation Water regulation Erosion regulation Water purification

Regulation

Pollination Pest and disease regulation

Cultural

Education and inspiration Recreation and ecotourism Cultural heritage

Education and inspiration Recreation and ecotourism Cultural heritage Aesthetic value Primary production Photosynthesis Habitat provision Soil formation and retention Nutrient cycling Water cycling

The larger insects such as damselflies and dragonflies (Odonata) were allowed to live, and (where possible) reproduce, within the enclosures. The numbers of insects on the walls of the enclosure and in an ultraviolet light trap containing a dilute sucrose solution were determined in twice-weekly live counts. Insects that died during the course of the study were collected and preserved in 70% alcohol for identification. The smaller emerging insects (Diptera) which included the midges, mosquitoes and black flies, were more difficult to assess by the live count method so they were collected using a vacuum sampler on one occasion each week and preserved in 70% alcohol for identification.

Supporting

Primary production Photosynthesis

Results

A single application of a commercial formulation of an insecticide was applied at five treatment levels by addition of aqueous solutions below the water surface. The water in each microcosm was then thoroughly mixed to achieve an even distribution of the active ingredient throughout the water column at each analytically verified nominal concentration. Applications were made in May and effects observed over the subsequent 22-week period during which both invertebrate productivity and insect emergence were at their highest. Once the test material had been applied, each of the control and treated microcosms were totally enclosed in insect-proof netting which essentially formed an aerial microcosm (24m3) that prevented the loss of emerging insects. Each enclosure contained hanging baskets of flowers and patio plants at ground level to provide sources of nectar, resting sites and refugia for newly emerged insects. Fresh fruit and dilute solutions (5%) of sucrose were also provided as sources of nutrients.

The results obtained for invertebrates and insects in the field microcosm study are placed in the context of their roles as Ecosystem Service Providers. Macro-invertebrates with entirely aquatic life histories (detritus shredders, fish food organisms) (Figures 1a and 1b) The numbers of the freshwater hoglouse (Asellus aquaticus) and the freshwater shrimp (Crangonyx pseudogracilis), both of which are omnivorous detritus shredders, showed at least a ten-fold increase in number during the course of the study. These results confirmed that conditions within the field microcosms (e.g. water quality, levels of detritus) were sufficiently realistic to support the growth of populations of key ES providers and from this it was considered that for the purpose of this study, the objective of achieving environmental realism had been achieved. No adverse effects of treatment were observed on these populations.

Ecotoxicology
Insect emergence and reproduction
Small larvae ( ) that were likely to have originated from reproduction during the initial peak of emergence, were found in invertebrate samplers in high numbers in late June after which numbers in samplers remained at a relatively constant level. Adult emergence at the highest treatment level was generally lower than that found at other levels, although there were no obvious adverse effects on the emergence process itself. No small larvae were ) suggesting that found in invertebrate samplers ( although adults had emerged from the aquatic system, egg-laying and larval survival may have been impaired at the highest insecticide treatment level. Dragonflies (Figures 5a and 5b) Dragonfly larvae are generally only present in microcosms in very low numbers and of the two families commonly found (Libellulidae and Aeschnidae), the four spotted chaser, Libellula quadrimaculata, was most abundant. Reproduction within enclosures, which was thought to be unlikely, was not observed in the study because of the low numbers of emerging adults and their typical migration away from sites of emergence for some time before mating takes place.

During the course of the study it became apparent that a number of insect species were able to emerge from the aquatic systems in sufficient numbers to allow reproduction to take place within the enclosures. Trichoptera (Caddis) Limnephilus sp. (nutrient recycling, detritus shredder, fish food organisms) (Figures 2a and 2b) The numbers of caddis larvae found in invertebrate samples and counted using underwater viewers naturally fell over the period April to August as the larvae pupated and the adult insects emerged. Small larvae, (which are identified with a ( ) in all the plots in this article) reappeared in invertebrate samplers from September onwards confirming that emerged adults had successfully reproduced in the aerial microcosms formed by the enclosures and deposited their eggs into back into the aquatic microcosm. Two adult emergence peaks were observed and these were due to the presence of two species (L.marmoratus and L. flavicornis). No treatment-related effects were observed on the larval survival of the parental generation, emergence and reproduction of the adults or the number of the numbers of F1 generation larvae found in invertebrate samplers. Odonata Damselflies and Dragonflies Predators feeding on Diptera (pest control), nutrient recycling, cultural services (aesthetic and recreational value). Damselflies Two families of Damselfly (Lestidae and Coenagrionidae) were found in microcosms during the study. In the period before the application of the insecticide, their larval stages were only found in low numbers in invertebrate colonisers possibly because of their broader distribution throughout the system and their predatory feeding habit. Emerald damselfly (Lestes sponsa) (Figures 3a and 3b) Following emergence of the adult emerald damselfly over the period June to September, small larvae ( ) were found in invertebrate samplers in increasing numbers (possibly because they offer a degree of protection from larger predators) again confirming that adults had reproduced within the enclosures. Blue-tailed damselfly (Ischnura elegans) (Figures 4a and 4b) Emergence of the common blue-tailed damselfly was established almost immediately after the application of the insecticide in late May and continued until early September.

Conclusions and implications for higher tier testing

Current methodologies for assessing insect emergence in field microcosm studies are not fit for the purpose of providing quantitative, reliable impact data. There is a clear need to establish total enclosure methodology (a Higher2 Tier test) to allow quantitative assessments of insect emergence to be made under field conditions. Completely enclosing field microcosms in order to collect all of the insects that emerge from a system can generate sufficient data on a wide range of species to allow statistical analysis of insect emergence at both the community and population levels. Total enclosure methodology is easily adapted to allow assessments of the impact of pesticides on the reproduction and survival of F1 larvae of some members of the Odonata, which are recognised as important Ecosystem providers. There is a clear need to provide criteria for assessing the realism of field microcosms in terms of the performance of their Ecosystem Service Providers (e.g. population increase of detritus shredders) to allow the quality and realism of the system to be assessed. The broad ranging appeal of the Ecosystem Services concept brings into sharp focus our limitations in methodology in field studies. It is clear from this study, that the survival of insect larvae and successful adult

Ecotoxicology
Figure 1: Productivity of the macroinvertebrate detritus shredders a) Asellus aquaticus and b) Crangonyx pseudogracilis in control and treated microcosms a) Asellus aquaticus Figure 2: Counts of a) Limnephilus larvae in microcosms and b) live counts of adults found in enclosures a) Aquatic larvae in microcosms

b) Crangonyx pseudogracilis

b) Adult emergence

Figure 3: Counts of a) emerald damselfly (Lestes sponsa) larvae in microcosms and b) live counts of adults found in enclosures. a) Aquatic larvae in microcosms

Figure 4: Counts of a) blue-tailed damselfly (Ischnura elegans) larvae in microcosms and b) live counts of adults found in enclosures. a) Aquatic larvae in microcosms

b) Adult emergence

b) Adult emergence

Ecotoxicology
emergence, may not guarantee the larval survival of the F1 generation, which impacts on survival and dispersal in the wild. Further work on this is therefore considered to be essential to remove any doubts that may exist in risk assessment. The results of this study satisfied the objective of defining a population No Observed Effect Concentration for Damselflies at a much higher testing tier than is currently employed in internationally accepted methods. The wide range of aquatic invertebrate species and emerging adult insects evaluated in this type of study may obviate the need for application factors in determining the predicted no-effect concentration for a population in the assessment of risk in the freshwater aquatic environment.

Key Points

insect Current methodologies for assessingmay not emergence in field microcosm studies

need total enclosure There is a clearHigher to establishallow quantitative methodology (a Tier test) to
2

provide quantitative, reliable data on the effects of pesticides on insect emerging from the aquatic environment.

There is ofcurrently no means of assessing the realism field microcosm studies. The derivation
of some criteria for this (e.g. population increase of detritus shredders) would allow systems to be assessed to ensure that their composition and realism are capable of supporting healthy populations of organisms that are key Ecosystem Service Providers.

assessments of the effects of pesticides on insect emergence and reproduction to be made under field conditions.

Figure 5: Counts of a) dragonfly (Libelluidae) larvae in microcosms and b) live counts of adults found in enclosures. a) Aquatic larvae in microcosms

of this investigation suggest that insect The results alone may not be a reliable end point on emergence which safe environmental levels can be based. For some sensitive species, effects may be manifest by a failure to produce viable F1 generation larvae.

b) Adult emergence
Simaika, J.P. and Samways, M.J. Valuing Dragonflies as service providers. In Dragonflies and Damselflies: Model organisms for Ecological and Evolutionary Research, Chap 9, Ed Alex CordobaAguilar, Oxford University Press, ISBN 978---19-923069-3. (2008) Nakano, S., Murakami, M. reciprocal subsidies: Dynamic interdependence between terrestrial and aquatic food webs. PNAS 98 (1), 167 170. (2001) Sabo, J.L., Power, M.E. River-watereshed exchange : Effects of riverine subsidies on riparian lizards and their terrestrial prey. Ecology, 83, 1860 1869. (2002) Baxter, C.V., Fausch, K.D., Saunders, W.C. Tanglled webs: reciprocal flows of invertebrate prey link streams and riparian zones. Freshwater Biology 50, 201 220. (2005) Jenkins, W.R. First flight A escriptrion of a new method to assess insect emergence in aquatic field microcosm studies. Developments in Life Sciences, 11, 3, number 2, 2011, page 4 to 7). (2011)

References
Millenium ecosystem Assessment, MEA (2005). Ecosystems and human Well-being: Synthesis, Island Press, Washington, DC, 160 pp. EFSA (European Food Safety Authority,) 2010. Scientific Opinion on the development of specific protection goal options for environmental risk assessment of pesticides, in particular in relation to the revision of the Guidance Document on Aquatic and Terrestrial Ecotoxicology (SANCO/3268/2001 and SANCO/10329/2002). EFSA Panel on Plant Protection Products and their residues (PPR), EFSA Journal, 8, 10, 1821

SETAC. Guidance document on Higher Tier Aquatic Risk Assessment for Pesticides (HARAP), Editors P.J.Campbell et al, ISBN 90-5607011-8. (1999) CLASSIC. Proceedings of the CLASSIC workshop (Community Level Aquatic System Studies Interpretation criteria), held at the Fraunhofer Institute, Schmallenberg, Germany. 30 May 2 June 1999. (2002) OECD. Guidance document on simulated freshwater lentic field tests (outdoor microcosms and mesocosms). OECD Series on testing and assessment No 53, ENV/JM/MONO (2006) 17, 2 May 2006. (2006)