## 4.3.7. Selection of test organisms - Birds

Author: Annegaaike Leopold

Reviewers: Nico van den Brink, Kees van Gestel, Peter Edwards

Learning objectives:

You should be able to

- Understand and argue why birds are an important model in ecotoxicology;
- understand and argue the objective of avian toxicity testing performed for regulatory purposes;
- list the most commonly used avian species;
- list the endpoints used in avian toxicity tests;
- name examples of how uncertainty in assessing the risk of chemicals to birds can be reduced.

Keywords: birds, risk assessment, habitats, acute, reproduction.

Introduction

Birds are seen as important models in ecotoxicology for a number of reasons:

- they are a diverse, abundant and widespread order inhabiting many human altered habitats like agriculture;
- they have physiological features that make them different from other vertebrate classes that may affect their sensitivity to chemical exposure;
- they play a specific role ecologically and fulfill essential roles in ecosystems (e.g. in seed dispersal, as biological control agents through eating insects, and removal of carcasses e.g. by vultures);
- protection goals are frequently focused on iconic species that appeal to the public.

A few specific physiological features will be discussed here. Birds are oviparous, laying eggs with hard shells. This leads to concentrated exposure (as opposed to exposure via the bloodstream as in most other vertrebrate species) to maternally transferred material, and where relevant, its metabolites. It also means that offspring receive a single supply of nutrients (and not a continuous supply through the blood stream). This makes birds sensitive to contaminants in a different way than non-oviparous vertebrates, since the embryos develop without physiological maternal interference. The bird embryo starts to regulate its own hormone homeostasis early on in its development in contrast to mammalian embryos. As a result contaminants deposited in the egg by the female bird may cause disturbance of the regulation of these embryonic processes (Murk et al., 1996). Birds have a higher body temperature (40.6 ºC) and a relatively high metabolic rate, which can impact their response to chemicals. As chicks, birds generally have a rapid growth rate, compared to many vertebrate species. Chicks of precocial (or nidifugous) species leave the nest upon hatching and, while they may follow the parents around, they are fully feathered and feed independently. They typically need a few months to grow to full size. Altricial species are naked, blind and helpless at hatch and require parental care until they fledge the nest. They often grow faster – passerines (such as swallows) can reach full size and fledge 14 days after hatching. Many bird species migrate seasonally over long distances and adaptation to this, changes their physiology and biochemical processes. Internal concentrations of organic contaminants, for example, may increase significantly due to the use of lipids stores during migration, while changes in biochemistry may increase the sensitivity of birds to the chemical.

Birds function as good biological indicators of environmental quality largely because of their position in the foodchain and habitat dependence. Protection goals are frequently focused on iconic species, for example the Atlantic puffin, the European turtle dove and the common barn owl (Birdlife International, 2018).

It was recognized early on that exposure of birds to pesticides can take place through many routes of dietary exposure. Given their association with a wide range of habitats, exposure can take place by feeding on the crop itself, on weeds, or (treated) weed seeds, on ground dwelling or foliar dwelling invertebrates, by feeding on invertebrates in the soil, such as earthworms, by drinking water from contaminated streams or by feeding on fish living in contaminated streams (Figure 1, Brooks et al., 2017). Following the introduction of persistent and highly toxic synthetic pesticides in the 1950s and prior to safety regulations, use of many synthetic organic pesticides led to wildlife losses – of birds, fish, and other wildlife (Kendall and Lacher, 1994). As a result, national and international guidelines for assessing first acute and subacute effects of pesticides on birds were developed in the 1970s. In the early 1980s tests were developed to study long-term or reproductive effects of pesticides. Current bird testing guidelines focused primarily on active ingredients used in plant protection products, veterinary medicines and biocides. In Europe the industrial chemicals regulation REACH only requires information on long-term or reproductive toxicity for substances manufactured or imported in quantities of at least 1000 tonnes per annum. These data may be needed to assess the risks of secondary poisoning by a substance that is likely to bioaccumulate and does not degrade rapidly. Secondary poisoning may occur, for example when raptors consume contaminated fish. In the United States no bird tests are required under the industrial chemicals legislation.

Figure 1. Potential routes of dietary exposure for birds feeding in agricultural fields sprayed with a crop protection product (pesticide). Most of the pesticide will land up in the treated crop area, but some of it may land in neighbouring surface water. Exposure to birds can therefore take place through many routes: by feeding on the crop itself (1), on weeds (2), or weed seeds (3), on ground‐dwelling (4) or foliar‐dwelling (5) invertebrates. Birds may also feed on earthworms living in the treated soil (6). Exposure may also occur by drinking from contaminated puddles within the treated crop area (7) or birds may feed on fish living in neighbouring contaminated surface waters (8). Based on Brooks et al. (2017).

The objective of performing avian toxicity tests is to inform an avian effects assessment (Hart et al., 2001) in order to:

- provide scientifically sound information on the type, size, frequency and pattern over time of effects expected from defined exposures of birds to chemicals.
- reduce uncertainty about potential effects of chemicals on birds.
- provide information in a form suitable for use in risk assessment.
- provide this information in a way that makes efficient use of resources and avoids unnecessary use and suffering of animals.

Bird species used in toxicity testing

Selection of bird species for toxicity testing occurs primarily on the basis of their ecological relevance, their availability and ability to adjust to laboratory conditions for breeding and testing. This means most test species have been domesticated over many years. They should have been shown to be relatively sensitive to chemicals through previous experience or published literature and ideally have available historical control data.

The bird species most commonly used in toxicity testing have all been domesticated:

- the waterfowl species mallard duck (Anas platyrynchos) is in the mid range of sensitivity to chemicals, an omnivorous feeder, abundant in many parts of the world, a precocial species; raised commercially and test birds show wild type plumage;
- the ground dwelling game species bobwhite quail (Colinus virginianus) is common in the USA and is of similar in sensitivity to mallards; feeds primarily on seeds and invertebrates; a precocial species; raised commercially and test birds show wild type plumage;
- the ground dwelling species Japanese quail (Coturnix coturnix japonica) occurs naturally in East Asia; feeds on plants material and terrestrial invertebrates. Domesticated to a far great extent than mallard or bobwhite quail and birds raised commercially (for eggs or for meat) are further removed genetically from the wild type. This species is unique in that the young of the year mature and breed (themselves) within 12 months;
- the passerine, altricial species zebra finch (Taeniopygia guttata) occurs naturally in Australia and Indonesia; they eat seeds; are kept and sold as pets; are not far removed from wild type;
- the budgerigar (Melopsittacus undulates) is also altricial); occurs naturally in Australia; eats seeds eating; is bred in captivity and kept and sold as pets.

Other species of birds are sometimes used for specific, often tailor-designed studies. These species include:

- the canary (Serinus canaria domestica).
- the rock pigeon (Columba livia)
- the house sparrow (Passer domesticus),
- red-winged blackbird (Agelaius phoeniceus) - US only
- the ring-necked pheasant, (Phasianus colchicus),
- the grey partridge (Perdix perdix)

Most common avian toxicity tests:

Table 1 provides an overview of all the avian toxicity tests that have been developed over the past approximately 40 years, the most commonly used guidelines, the recommended species, the endpoints recorded in each of these tests, the typical age of birds at the start of the test, the test duration and the length of exposure.

Table 1: Most common avian toxicity tests with their recommended species and key characteristics.

Avian toxicity test | Guideline | Recommended species | Endpoints | Age at start of test | Length of study | Length of exposure
Acute oral gavage– sequential testing – average 26 birds | OECD 223 | bobwhite quail, Japanese quail, zebra finch, budgerigar | mortality, clinical signs, body weight, food consumption, gross necropsy | Young birds not yet mated, at least 16 weeks old at start of test. | At least 14 days | Single oral dose at beginning of test
Acute oral gavage – 60 bird design | USEPA OCSPP 850.2100 | Bobwhite quail single passerine species recommended. | See above | Young birds not yet mated, at least 16 weeks old at start of test. | 14 days | Single oral dose at beginning of test
Sub-acute dietary toxicity * | OCSPP 850.2200 | Bobwhite quail, mallard | See above | Mallard: 5 days old Bobwhite quail: 10-14 days old | 8 days | 5 days
One-generation reproduction | OECD 206 OCSPP 850.2200 | Bobwhite quail, mallard, Japanese quail** | Adult body weight,food consumption, egg production, fertility, embryo survivial, hatchrate, chick survival. | Approaching first breeding season: Mallard (6 to 12 months old) Bobwhite quail 20 -24 weeks | 20 – 22 weeks | 10 weeks
Avoidance testing (pen trials) | OECD Report | As closely related to species at risk as possible; eg: sparrow rock dove, pheasant, grey partridge | Food intake, mortality, Sublethal effects | Young adults if possible (depends on study design) | One to several days, depending on the study design. | One to several days, depending on the study design.
Two-generation endocrine disruptor test | OCSPP 890.2100 | Japanese quail | In addition to endpoints listed for one-generation study: male sexual behaviour, biochemical, histological, and morphological endpoints | 4 weeks post hatch | 38 weeks | 8 weeks – adult (F0) generation +14 weeks F1 generation.
Field studies to refine food residues in higher tier Bird risk assessments. | Appendix N of the EFSA Bird and Mammal Guidance. | Depends on the species at risk in the are of pesticide use. | Depends on the study design developed. | Uncontrollable in a field study | Depends on the study design developed. | Depends on the study design developed.

* This study is hardly every asked for anymore.

** Only in OECD Guideline

Acute toxicity testing

To assess the short-term risk to birds, acute toxicity tests must be performed for all pesticides (the active ingredient thereof) to which birds are likely to be exposed, resulting in an LD50 (mg/kg body/day) (see section on Concentration-response relationships). The acute oral toxicity test involves gavage or capsule dosing at the start of the study (Figure 2). Care must be taken when dosing birds by oral gavage. Some species can readily regurgitate leading to uncertainty in the the dose given. These include mallard duck, pigeons and some passerine species. Table 1 gives the birds species recommended in the OECD and USEPA guidelines, respectively. Gamebirds and passerines are a good combination to take account of phylogeny and a good starting point to better understand the distribution of species sensitivity.

The OECD guideline 223 uses on average 26 birds and is a sequential design (Edwards et al., 2017). Responses of birds to each stage of the test are combined to estimate and improve the estimate of the LD50 and slope. The testing can be stopped at any stage once the accuracy of the LD50 estimate meets the requirements for the risk assessment, hence using far fewer birds for compliance with the 3Rs (reduction, refinement and replacement). If toxicity is expected to be low, 5 birds are dosed at the limit dose of 2000 mg/kg (which is the highest acceptable dose to be given by oral gavage, from a humane point of view). If there is no mortality in the limit test after 14 days the study is complete and the LD50 >2000 mg/kg body weight. If there is mortality a single individual is treated at each of 4 different doses in Stage 1. With these results a working estimate of the LD50 is determined to select 10 further dose levels for a better estimate of the LD50 in Stage 2. If a slope is required a further Stage 3 is required using 10 more birds in a combination of doses selected on the basis of a provisional estimate of the slope.

The USEPA guideline is a single stage design preceeded by a range finding test (used only to set the concentrations for the main test). The LD50 test uses 60 birds (10 at each of five test concentrations and 10 birds in the control group). Despite the high numbers of birds used, the ability to estimate a slope is poor compared to OECD223 (the ability to calculate the LD50 is similar to the OECD 223 guideline).

Figure 2. Gavage dosing of a zebrafinch – Eurofins Agroscience Services, Easton MD, USA.

Dietary toxicity testing

For the medium-term risk assessment an avian dietary toxicity test was regularly performed in the past exposing juvenile (chicks) of bobwhite quail, Japanese quail or mallard to a treated diet. This test determines the median lethal concentration (LC50) of a chemical in response to a 5-day dietary exposure. Given the scientific limitations and animal welfare concerns related to this test (EFSA, 2009) current European regulations recommend to only perform this test when it is expected that the LD50 value measured by the medium-term study will be lower than the acute LD50 i.e. if the chemical is cumulative in its effect.

Reproduction testing

One-generation reproduction tests in bobwhite quail and/or mallard are requested for the registration of all pesticides to which birds are likely to be exposed during the breeding season. Table 1 presents the two standard studies: OECD Test 206 and the US EPA OCSPP 850.2100 study. The substance to be tested is mixed into the diet from the start of the test. The birds are fed ad libitum for a recommended period of 10 weeks before they begin laying eggs in response to a change in photoperiod. The egg-laying period should last at least ten weeks. Endpoints include adult body weight, food consumption, macroscopic findings at necropsy and reproductive endpoints, with the number of 14-day old surviving chicks/ducklings as an overall endpoint.

The OECD guideline states that the Japanese quail (Coturnix coturnix japonica), is also acceptable.

Avoidance (or repellancy) testing

Avoidance behaviour by birds in the field could be seen as reducing the risk of exposure to a pesticide and therefore could be considered in the risk assessment. However, the occurrence of avoidance in the laboratory has a confounding effect on estimates of toxicity in dietary studies (LD50). Avoidance tests thus far have greatest relevance in the risk assessment of seed treatments. A number of factors need to be taken into account including the feeding rate and dietary concentration which may determine whether avoidance or mortality is the outcome. The following comprehensive OECD report provides an overview of guideline development and research activities that have taken place to date under the OECD flag. Sometimes these studies are done as semi-field (or pen) studies.

Endocrine disruptor testing

Endocrine-disrupting substances can be defined as materials that cause effects on reproduction through the disruption of endocrine-mediated processes. If there is reason to suspect that a substance might have an endocrine effect in birds, a two-generation avian test design aimed specifically at the evaluation of endocrine effects could be performed. This test has been developed by the USEPA (OCSPP 890.210). The test has not, however, been accepted as an OECD test to date. It uses the Japanese quail as the preferred species. The main reasons that Japanese quail were selected for this test were: 1) Japanese quail is a precocial species as mentioned earlier. This means that at hatch Japanese quail chicks are much further in their sexual differentiation and development than chicks of altricial species would be. Hormonal processes occurring in Japanese quail in these early stages of development can be disturbed by chemicals maternally deposited in the egg (Ottinger and Dean, 2011). Conversely altricial species undergo these same sexual development stages post-hatch and can be exposed to chemicals in food that might impact these same hormonal processes. 2) as mentioned above, the young of the year mature and breed (themselves) within 12 months which makes the test more efficient that if one used bobwhite quail or mallard.

It is argued among avian toxicologists, that it is necessary to develop a zebra finch endocrine assay system, alongside the Japanese quail system, as this will allow a more systematic determination of differences between responses to EDC’s in altricial and precocial species, there by allowing a better evaluation and subsequent risk assessment of potential endocrine effects in birds. Differences in parental care, nesting behaviour and territoriality are examples of aspects that could be incorporated in such an approach (Jones et al., 2013).

Field studies:

Field studies can be used to test for adverse effects on a range of species simultaneously, under conditions of actual exposure in the environment (Hart et al, 2001). The numbers of sites and control fields and methods (corpse searches, censusing and radiotracking) need careful consideration for optimal use of field studies in avian toxicology. The field site will define the species studied and it is important to consider the relevance of that species in other locations. For further reading about techniques and methods to be used in avian field research Sutherland et al and Bibby et al. (2000) are recommended.

References

Bibby, C., Jones, M., Marsden, S. (2000). Expedition Field Techniques Bird Surveys. Birdlife International.

Birdlife International (2018). The Status of the World’s Birds. https://www.birdlife.org/sites/default/files/attachments/BL_ReportENG_V11_spreads.pdf

Brooks, A.C., Fryer, M., Lawrence, A., Pascual, J., Sharp, R. (2017). Reflections on bird and mammal risk assessment for plant protection products in the European Union: Past, present and future. Environmental Toxicology and Chemistry 36, 565-575.

Edwards, P.J., Leopold, A., Beavers, J.B., Springer, T.A., Chapman, P., Maynard, S.K., Hubbard, P. (2017). More or less: Analysis of the performance of avian acute oral guideline OECD 223 from empirical data. Integrated Environmental Assessment and Management 13, 906-914.

Hart, A., Balluff, D., Barfknecht, R., Chapman, P.F., Hawkes, T., Joermann, G., Leopold, A., Luttik, R. (Eds.) (2001). Avian Effects Assessment: A Framework for Contaminants Studies. A report of a SETAC workshop on ‘Harmonised Approaches to Avian Effects Assessment’, held with the support of the OECD, in Woudschoten, The Netherlands, September 1999. A SETAC Book.

Jones, P.D., Hecker, M., Wiseman, S., Giesy, J.P. (2013). Birds. Chapter 10 In: Matthiessen, P. (Ed.) Endocrine Disrupters - Hazard Testing and Assessment Methods. Wiley & Sons.

Kendall, R.J., Lacher Jr, T.E. (Eds.) (1994). Wildlife Toxicology and Population Modelling – Integrated Studies of Agrochecosystems. Special Publication of SETAC.

Murk, A.J., Boudewijn, T.J., Meininger, P.L., Bosveld, A.T.C., Rossaert, G., Ysebaert, T., Meire, P., Dirksen, S. (1996). Effects of polyhalogenated aromatic hydrocarbons and related contaminants on common tern reproduction: Integration of biological, biochemical, and chemical data. Archives of Environmental Contamination and Toxicology 31, 128–140.

Ottinger, M.A., Dean, K. (2011). Neuroendocrine Impacts of Endocrine-Disrupting Chemicals in Birds: Life Stage and Species Sensitivities. Journal of Toxicology and Environmental Health, Part B: Critical Reviews. 26 July 2011.

Sutherland, W.J., Newton, I., Green, R.E. (Eds.) (2004). Biological Ecology and Conservation. A Handbook of Techniques. Oxford University Press