Copepod

A Combined Approach of Experiments and Modelling for the Implementation of Freshwater Copepods in Ecological Risk Assessment

Devdutt Kulkarni, PhD project, RWTH Aachen, Germany
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Background

Standardized test guidelines used in ecological risk assessment (ERA) consider a relatively small set of test species For instance in most standard risk assessments, Daphnia magna is the only required species representing freshwater invertebrates. This is done under the assumption that tests with such standard species in combination with relatively large assessment factors are protective for other species in the field. Standard test species are usually selected based on intrinsic sensitivity as well as practicability i.e. the ease of culturing them and conducting experiments in the laboratory. However, species in the field may employ variable life-history strategies which may have consequences concerning the ecological vulnerability of these species to toxicants. The variability in the intrinsic sensitivity of different species can be assessed by testing additional species and constructing species sensitivity distributions while ecological vulnerability can be addressed using community-level studies e.g. mesocosms and ecological modelling.

Copepods are important animals in the aquatic food chain. They are predators of other plankton and act as prey for fish, consequently enabling the transfer of energy and substances though the food chain. Copepods, owing to their complex life-history strategies, are potentially vulnerable organisms and therefore, useful as ecological indicators of risk. For marine risk assessment, copepods are now being considered and an OECD test guideline for bottom-dwelling harpacticoid copepods is under way. However, in freshwater ecotoxicology, copepods are largely ignored except in mesocosm studies. To facilitate the consideration of freshwater copepods in higher-tier ERA there is a need for the development of robust test methods and models to facilitate extrapolation between environmental conditions, exposure patterns and species.

Objectives

The present thesis was part of the work package Aquatic Invertebrates, with a particular focus on the employment of freshwater copepods for ERA. This thesis delivers the basics of a combined approach of laboratory experiments and modelling for better consideration of freshwater copepods in ecotoxicology. The objectives of the study are as follows-

  • To identify a representative species of freshwater copepods suitable for ERA through an exhaustive literature review. This species should not only be a good laboratory species (easy to culture and work with in the laboratory) but also be a relevant and potentially ecologically vulnerable species.
  • To establish a stable culture of the selected test species, as well as the food for this species, in the laboratory.
  • To conduct toxicity tests with this species to test for sensitivity to a model toxicant.
  • To conduct ecological experiments to study the life-cycle processes of this species namely- feeding, development, reproduction, survival, using different food sources and feeding regimes.
  • To develop an individual-based population model (IBM), parameterised with data from ecological and toxicological experiments conducted in the laboratory, for the selected test species including responses to the selected toxicant to facilitate extrapolation of individual-level effects to the population level.
  • Finally, to highlight the applicability of the model through a case study comparing individual and population-level sensitivities to the selected toxicant for the selected test species as well as two other planktonic organisms- D. magna and Chaoborus crystallinus.

Selecting a representative species

With a particular focus on plant protection products, an exhaustive literature review was carried out to identify a representative species of freshwater copepods, which could be a good compromise between a potentially vulnerable relevant species, owing to its complex life-history strategies, as well as a good laboratory species based on the ease of rearing it in the laboratory. We used a top-down approach  to select a relevant species for toxicity tests in order to assess the risk posed by pesticides to freshwater copepods. We chose to focus on cyclopoid copepods which are becoming increasingly important in food webs post eutrophication, are important food items for freshwater aquaculture and are of great economic value, important for control of mosquito larvae and abundant in edge-of-field water bodies. Being littoral and pelagic in occurrence, they are susceptible to pesticide exposure via spray drift and run-off and have the potential for being good indicators of the health of the ecosystem. Due to the concentration of many substances at the surfaces of water bodies, surface dwelling organisms will be more exposed than sub-surface and sediment dwelling forms.

M. leuckarti is one of the most well studied freshwater cyclopoid copepod summer species in Germany, in particular and Europe, in general. A fair amount of ecological research has been done on this species, which has established that M. leuckarti is a common planktonic species in European freshwater habitats, shows herbivory at naupliar and early copepodite stages, switches to carnivory at the copepodite IV stage and grows best with a mixed diet of algae and rotifers. It is a small sized species. Although life cycles in the field may be long, copepods were shown to have relatively shorter life cycles under laboratory conditions. However, these development times depend on the quality and quantity of food provided to the copepods and the temperature. This species also shows an increased egg production when fed a mixed diet of algal and animal food. Owing to its relatively shorter life cycle, planktonic distribution, its summer occurrence and ease of culturing in the laboratory, M. leuckarti qualifies as a suitable laboratory species to conduct ecotoxicity tests.

Laboratory cultures

We established three simultaneously running batch cultures of algae (Cryptomonas obovoidea), rotifers (Brachionus calyciflorus) and copepods (M. leuckarti) using a high quality culture medium, COMBO, which is suitable for both phytoplankton and zooplankton cultures. Cultures were acclimated for more than six weeks to laboratory conditions i.e. 20±1°C and a light: dark rhythm of 16:8 h.

Ecological experiments

Feeding, development, reproduction and survival experiments were conducted for the nauplii, copepodite (early copepodite stages I to III and late copepodite stages IV and V) and adult stages. Two different feeding regimes were tested, a pure algal diet and a mixed diet of algae and rotifers. The mixed diet was only tested with late copepodite stages and adults because pre-tests showed that lower copepodite stages and nauplii did not feed on rotifers. Cannibalism experiments were carried out for the late copepodites and adults with 5 replicates of 10 nauplii each and one set of controls. Cannibalism experiments were not carried out with early copepodites and nauplii because pre-tests showed no cannibalistic behaviour in these stages.

Toxicity experiments

Four different acute toxicity tests were conducted to compare the sensitivities of different life stages of M. leuckarti to a model toxicant triphenyltin. 40 individual nauplii (24 h old), copepodites (CII-CIV), and adults (males and females) respectively, were used in each treatment. Six different TPT concentrations 6.25, 12.5, 25, 50, 100 and 200 μg l-1 were selected. The naupliar dataset was not tested with 100 and 200 µg l-1 because pre-tests showed 100% naupliar mortality at 50 µg l-1.  The different life stages of M. leuckarti were exposed to TPT in 96-well plates with a volume of 250 μl per test vessel for each organism. The endpoint immobility (correspondingly survival) of the copepods was checked at nine different time points during the tests (1, 17, 24, 41, 48, 65, 72, 89 and 96 h).

GUTS application

Recently, the General Unified Threshold model of Survival (GUTS) was proposed. This model integrates “all previously published toxicokinetic and toxicodynamics models”. The model comprises toxicokinetic dose metrics (external concentration, internal concentration and scaled internal concentration) and toxicodynamic assumptions (stochastic death (SD), individual tolerance (IT)). We used this model to mechanistically describe the toxic effects of TPT to the different life stages of M. leuckarti over time.

Individual-based modelling

An individual-based model (IBM) was developed for M. leuckarti to simulate population dynamics under toxicant exposure based on data from laboratory experiments. We used parameters from ecological experiments to parameterise the IBM. Parameters for modelling eco-physiological processes like feeding, development, reproduction and survival were determined under different feeding regimes for the three different life stages of M. leuckarti. Data from toxicity experiments with triphenyltin were used to calibrate the toxicity submodel.

Important findings

Laboratory toxicity experiments confirmed M. leuckarti to be sensitive to TPT with the naupliar stages showing a higher sensitivity compared to older stages. Population dynamics of copepods emerged from individual properties. Long-term simulations showed annual patterns fluctuating with the temperature dependence incorporated into the model. The first large peak observed around April when spring temperatures favour population growth. The second large peak was observed around September. This is followed by diapause through winter. Model simulations identified mixed feeding and cannibalism as the two most important aspects of the biology of M. leuckarti that influenced population dynamics under TPT exposure.

Furthermore, a case study wherein the ecological sensitivity of M. leuckarti was compared to D. magna and Chaoborus crystallinus by means of population modelling was carried out. It was observed that population-level sensitivities of the three species used in the case study were higher than those on the individual level. Also, the sensitivity ranking of the three species on the population level was the converse of that on the individual level i.e. the species that was least sensitive at the individual level (C. crystallinus) was found to be most sensitive on the population level. Furthermore, M. leuckarti was less sensitive than D. magna at the individual level and more sensitive than D. magna at the population level. This thesis confirmed the relevance and practicability of copepods for ERA as well as the significance of population modelling in predicting population-level responses from individual-level data. This approach of combining laboratory experiments and population modelling of a representative vulnerable species to allow mechanistic extrapolation to the population level and to other exposure patterns can also be applied to other taxa in order to build up a set of test species and models useful for refined and more realistic ERA.

Outlook

Although the parameterisation of the toxicity submodel within the model presented here is specific to a single species and toxicant combination, it is not rigid. Different submodels for different toxicants can be integrated into the model. The General Unified Threshold model for Survival is able to describe toxic effects based on relatively basic survival data from laboratory tests.  Furthermore, the model can be re-parameterised to describe the life cycles of different copepod species to enable extrapolation across other species and exposure scenarios. The model is veritably intended to support and improve the interpretation of results of toxicity tests. This approach which includes the employment of more relevant species for short and long-term laboratory tests combined with mechanistic modelling should be useful tool for the ERA of chemicals. There are no data on population experiments dealing with toxic effects on M. leuckarti. Therefore, future tasks would include conducting population experiments as well as validating the model with independent datasets from population experiments to make the model more reliable.  The experimental work and the modelling within this study fit well within the tiered risk assessment scheme proposed by the European commission. Data from laboratory experiments with copepods constitute Tier 2 and be can easily implemented into a model which constitutes Tier 3. The population models should be validated on data from population experiments and mesocosm studies. After successful testing, the model could be used as a tool to predict the population-level effects on copepods when exposed to toxicants. To facilitate the consideration of freshwater copepods in ERA there is a need for the development of robust methods and guidelines for studies on important freshwater representative species understandable for risk assessors. This study is one such example which confirms that a combined approach of laboratory experiments and population modelling can prove to be powerful resource for the ERA of plant protection products in freshwater ecosystems.

Publications

Supervisor: Thomas G. Preuß (RWTH Aachen)
Co-Supervisor: Udo Hommen (IME)
Associated partners: gaiac; Bayer; BASF; UBA