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SID 5 Research Project Final Report

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code SF0244

2. Project title

Diffuse pollution and freshwater fish populations

3. Contractororganisation(s)

CefasPakefield RoadLowestoftSuffolkNR33 0HT

54. Total Defra project costs £ (agreed fixed price)

5. Project: start date................. 01 April 2004

SID 5 (Rev. 3/06) Page 1 of 26

end date.................. 31 April 2009


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent

non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

There is now evidence from studies carried out in Europe and North America that contaminants derived principally from intensive agriculture may have significant effects on salmonids at specific periods during the life cycle, often at concentrations frequently found in the environment. In particular, research carried out at the Cefas, Lowestoft Laboratory has indicated that a range of pesticides may compromise Atlantic salmon (Salmo salar L.) sense of smell, reproduction, embryo development and the parr-smolt transformation and/or entry into saltwater. This research has highlighted that in terms of the life cycle of the Atlantic salmon, the freshwater and marine environments cannot be considered in isolation and that exposure to poor water quality in freshwater may be a key factor influencing survival of the fish once they migrate into the sea. However, the majority of this research was based on laboratory experiments and there is a requirement to determine whether exposure to environmentally relevant contaminants within river systems in England and Wales are indeed affecting populations in the wild.

Therefore, the present research programme had two principal aims. Firstly, to validate the results from the laboratory based studies carried out under a previous Defra funded research programme SF0228 – Impacts of agricultural contaminants on wild salmonids, and determine whether exposure to these contaminants within river systems in England and Wales are affecting populations in the wild. Secondly, recent monitoring of the aquatic environment has highlighted the presence of novel contaminants whose chemical structure and toxic mechanisms are known to target important biological processes in fish and which may significantly compromise and regulate populations. These contaminants include specific pharmaceuticals, antibiotics from intensive agriculture and aquaculture and brominated flame retardants from industry. The present research examined the potential impacts of these contaminants on fish at both the individual and population level in order to support the advice to the Policy Customer on the regulation of contaminants within aquatic ecosystems and the conservation and management of fish populations.

The specific objectives of the research programme were:

1. To determine the impact of novel diffuse contaminants on juvenile salmon with specific


reference to development, olfactory imprinting, run-timing and behaviour within the marine environment.2. To determine the impact of novel diffuse contaminants on adult salmon with specific reference to the homing of adult salmon and egg viability in female salmonids.3. Determine the relationship between specific declining salmon stocks, land management changes and occurrence of target contaminants in the aquatic environment.4. To assess the effects of diffuse contaminants on the biology of salmon within wild populations.5. To provide recommendations to Policy Division for any required remedial action to reduce the impacts on diffuse contaminants on fish populations.

Laboratory and field based experiments have formed the basis of the research to determine the impact of contaminants on juvenile and adult salmon. The contaminants that were selected for study are known to routinely occur in rivers during the period of the parr-smolt transformation and seaward migration of the smolts and during the spawning season. The concentrations of the contaminants studied also reflect the levels that may occur routinely in the rivers and tributaries and so are therefore relevant to many salmonid populations. Extensive literature and data based investigations formed the basis for the studies examining the relationship between the decline in salmonid stocks and the occurrence of specific agriculture derived contaminants within river catchments.

The major findings of the research were that contaminants such as the brominated flame retardants, which are known to mimic thyroid hormones, significantly disrupt the parr-smolt transformation process whereby the juvenile salmon undergo a number of physiological and behavioural changes that adapt them to a life in the ocean. Specifically, hexabromocyclododecane reduces the olfactory abilty of the fish to detect odours that are considered important during the imprinting process during which the emigrating fish remember the “smell” of their home river and subsequently use this to home to their natal tributaries as spawning adults. Exposure of salmon smolts to hexabromocyclododecane was also shown to reduce the survival of the fish during the transition from freshwater and into the sea. Exposure of salmon smolts in freshwater to environmental levels of the pesticide atrazine (0.5, 1.0, 2.0 and 5.0 μg l-1) also reduced their ability to detect specific odours during the imprinting period. The results clearly demonstrate that exposure of salmon smolts to environmental levels of a range of diffuse contaminants inhibits olfactory function, which is known to play a pivotal role in the imprinting process and the subsequent homing of adult salmon to their natal river.

Diffuse contaminants were also demonstrated to intefere with female salmonid reproduction and the subsequent survival of the eggs and embryos. Eggs exposed to attrazine during fertilisation had a 66% higher risk of mortality compared to control eggs for every microgram per litre of atrazine in the water. Conet assays also indicated that in those surviving eyed eggs, 30 days after fertilisation, DNA damage was higher in the eggs that had been fertilised in the water containing both 0.5g/l or 2.0g/l atrazine. Exposure to polycyclic aromatic hydrocarbons (PAH) produced modifications to the kidney structure of the female fish, as well as lower levels of intestine Na+K+ ATPase activity. This may indicate that the female fish are under physiological stress as a result of PAH exposure. There was also a significant difference in the subsequent survival of the eggs after 50 days which had been fertilised in PAH water compared to the controls. Once again exposure of eggs to contaminants during fertilisation have a poorer survival rate than those fertilised in “clean” water.

It proved difficult to obtain suitable data with which to investigate relationships between pesticide concentrations in the catchments and variations in salmon stocks. However, in the River Avon rod catches of salmon were lower in years when the atrazine levels were high, and similar correlations were shown between the level of another triazine herbicide (simazine) rod catch in this river. Such results may be informative, but must be interpreted with great care.

The incorporation of the laboratory and field-based experimental data into the life cycle model of the salmon demonstrated that low levels of environmental contaminants can have a serious impact on both individuals and populations of salmonids. As more data is gathered, both from laboratory and field-based research programmes the models and the predictions will become more robust.


Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details

of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Introduction.

There is now evidence from studies carried out in Europe and North America that contaminants derived principally from intensive agriculture may have significant effects on salmonids at specific periods during the life cycle, often at concentrations frequently found in the environment. In particular, research carried out at the Cefas, Lowestoft Laboratory has indicated that a range of pesticides may compromise Atlantic salmon (Salmo salar L.) and reproduction (Moore & Waring, 1995; Waring & Moore, 1997; Moore & Waring, 1998; Moore & Waring, 2000; Moore & Lower, 2001); embryo development (Lower & Moore, 2003) and the parr-smolt transformation and/or entry into saltwater (Moore et al., 2003; Waring & Moore, 2004). This research has highlighted that in terms of the life cycle of the Atlantic salmon, the freshwater and marine environments cannot be considered in isolation and that exposure to poor water quality in freshwater may be a key factor influencing survival of the fish once they migrate into the sea. However, the majority of this research was based on laboratory experiments and there is a requirement to determine whether exposure to environmentally relevant contaminants within river systems in England and Wales are indeed affecting populations in the wild.

Therefore, the present research programme had two principal aims. Firstly, to validate the results from the laboratory based studies carried out under a previous Defra funded research programme SF0228 – Impacts of agricultural contaminants on wild salmonids, and determine whether exposure to these contaminants within river systems in England and Wales are affecting populations in the wild. Secondly, recent monitoring of the aquatic environment has highlighted the presence of novel contaminants whose chemical structure and toxic mechanisms are known to target important biological processes in fish and which may significantly compromise and regulate populations. These contaminants include specific pharmaceuticals, antibiotics from intensive agriculture and aquaculture and brominated flame retardants from industry. The present research examined the potential impacts of these contaminants on fish at both the individual and population level in order to support the advice to the Policy Customer on the regulation of contaminants within aquatic ecosystems and the conservation and management of fish populations.

The specific objectives of the research programme were:

1. To determine the impact of novel diffuse contaminants on juvenile salmon with specific reference to development, olfactory imprinting, run-timing and behaviour within the marine environment.2. To determine the impact of novel diffuse contaminants on adult salmon with specific reference to homing in adult salmon and egg viability in female salmonids.3. Determine the relationship between specific declining salmon stocks, land management changes and occurrence of target contaminants in the aquatic environment.4. To assess the effects of diffuse contaminants on the biology of salmon within wild populations.


5. To provide recommendations to Policy Division for any required remedial action to reduce the impacts on diffuse contaminants on fish populations.

Laboratory and field based experiments have formed the basis of the research to determine the impact of contaminants on juvenile and adult salmon. The contaminants that were all selected for study are known to routinely occur in rivers during the period of the parr-smolt transformation and seaward migration of the smolts and during the spawning season. The concentrations of the contaminants studied also reflect the levels that may occur routinely in the rivers and tributaries and so are therefore relevant to many salmonid populations. Extensive literature and data based investigations formed the basis for the studies examining the relationship between the decline in salmonid stocks and the occurrence of specific agriculture derived contaminants within river catchments. The results of the research are detailed below under each of the five objectives.

Objective 1. To determine the impact of novel diffuse contaminants on juvenile salmon with specific reference to development, olfactory imprinting, run-timing and behaviour within the marine environment.

During the parr-smolt transformation the juvenile salmonids undergo significant changes in their morphology, physiology and behaviour that enables them to survive their subsequent life in the sea (see McCormick & Saunders, 1987). The physiological changes involved in smoltification include modifications to plasma ion concentrations (e.g. chloride Cl- & sodium Na+) and an increase in the activity of gill Na+K+ATPase (Hoar, 1988; Boeuf, 1994). There is evidence that the ability to live in saltwater develops within the freshwater environment prior to entry into saline conditions (Moore et al., 1995), which together with an increase in thyroid hormones (Iwata, 1995; Hutchison & Iwata, 1998), growth hormone (GH), cortisol, insulin-like growth factor-I (Hoar, 1988; Sakamoto et al., 1995) and environmental cues (Riley et al., 2002), may all trigger seaward emigration. It is also thought to be during the seaward emigration in freshwater that the smolts undergo olfactory imprinting to their natal stream. Imprinting later allows the fish as adults to return to the home river to spawn after being at sea for periods of between 1-4 years. (Nordeng, 1977; Hasler and Scholz, 1983; Nevitt et al., 1994; Dittman & Quinn, 1996: Dittman et al., 1996). Salmon smolts emigrate rapidly from the freshwater environment and into the coastal zone generally using an ebbing tide within the estuary (Moore et al., 1995; Moore et al., 1996; Moore et al., 1998). Their subsequent migration within the coastal environment generally involves active directed swimming at speeds in excess of the prevailing water currents (Moore et al., 1995).

In order to assess the impact of contaminants on smolt emigration and their potential to survive in the sea, a number of the physiological changes during the parr-smolt transformation have been used as biomarkers. These include the increase in the gill Na+K+ATPase activity that is a robust physiological indicator of a smolt’s physiological transformation and its ability to tolerate the marine environment together with changes to the plasma levels of thyroid hormones (thyroxine T4, and triiodothyronine T3), cortisol and ion concentrations (e.g. chloride Cl- & sodium Na+). In addition, 24 or 72-hour seawater challenge tests were carried out during a number of experiments. These experiments indicated whether exposure to the contaminants within freshwater would reduce the ability of the fish to adapt to saltwater and predict the possible impact on marine survival.

In order to determine the impact of contaminants on olfactory imprinting in salmon smolts, an electrophysiological recording technique was used which examined how contaminants may reduce the ability of the fish to detect odours from con-specifics which are considered to be part of the imprinting process to the home river.

Telemetry and tracking techniques have been used to assess the impact of contaminants on the migratory behaviour and subsequent survival of smolts as they enter the marine environment. These were field-based studies carried out in collaboration with the Department of the Marine, Ireland. During this research the impacts of a range of contaminants both individually and in mixtures on the parr-smolt transformation, smolt emigration and marine survival were studied. These contaminants included the pharmaceutical ibuprofen, caffeine, brominated flame retardants (BFRs), the pesticide atrazine, the endocrine disrupting chemical 4-nonylphenol (4-NP) and polycyclic aromatic hydrocarbons (PAHs).

1.1 The impact of diffuse contaminants on the parr-smolt transformation and salt water adaptation - ibrupofen and caffeine.


Initially, laboratory studies were undertaken to determine the effect of ibuprofen and caffeine on the parr smolt-transformation. Caffeine (1,3,7-trimethylxanthine) is a methylxanthine whose primary biological effect is the competitive antagonism of the adenosine receptor. It also acts as a phosphodiesterase inhibitor and increases the translocation of calcium in cells. During the parr-smolt transformation the gill plays a major role in this ionic and osmotic regulation through the action of chloride cells, which are rich in the enzyme Na+K+ATPase (a protein directly involved in the excretion of monovalent ions). This activity peaks prior to seawater entry but remains at the higher level while the salmon is in the marine environment. Caffeine has the potential to affect this pre-adaptation by binding to the adenosine receptor, thereby lowering the ability of the salmon to cope and survive in saltwater. Similarly, exposure of adult fish within the coastal zone to caffeine may also modify its ability to readapt to freshwater and inhibit the spawning migration. Caffeine is a naturally occurring and commercially produced organic compound. It is used in food, soft drinks, tea and coffee as well as pharmaceuticals such as aspirin. It is probably the most widely used drug in the world. As a result of its widespread usage, caffeine is discharged into waters from STW in huge amounts and is detected in wastewater, surface water and groundwater worldwide. Caffeine is present in wastewaters up to 300μg/l and remains detectable while other chemicals break down during water treatment or become bound to sediments.

Ibruofen (2-(4-isobutylphenyl)propanoic acid) is a non-steroidal anti-inflammatory drug (NSAID), which is believed to work through inhibition of cyclooxygenase (COX) and thus inhibiting prostaglandin synthesis, which is known to have a role in salmon repropduction. A monitoring study carried out by CEFAS, Burnham on Crouch Laboratory found measurable levels of 13 pharmaceuticals and metabolites at UK sewage works and their receiving waters. Ibuprofen was found at the highest concentrations at all 13 sites.

Salmon smolts were exposed to environmental levels of either ibuprofen (0.5 - 30μg/l) or caffeine (0.01 - 300μg/l) for 5 days in freshwater. Half of the fish were then sampled for physiological parameters associated with smoltification, and the remaining fish were transferred to saltwater for 24hours to monitor survival. Increasing concentrations of ibuprofen were found to significantly affect levels of potassium ions in the plasma, but there was no effect on gill Na+K+ATPase activity. Exposure to caffeine also had no effect on gill Na+K+ATPase activity, although regression analysis indicates that exposure to caffeine at low levels has an effect on plasma ions in saltwater. Exposure to environmental levels of ibuprofen and caffeine individually, did not affect survival of salmon during the 24 hours after transfer to saltwater. However, caffeine’s impact on plasma ions and the subsequent affect on osmotic balance in the smolts, may compromise their ability to tolerate saline conditions for extended periods.

1.2 The impact of diffuse contaminants on the parr-smolt transformation and salt water adaptation – brominated flame retardants and caffeine.

Laboratory-based experiments were carried out which further investigated the impacts of mixtures of contaminants on smoltification and seawater survival. Previous studies have shown that exposure to low levels of caffeine affected the osmotic balance of smolts, which may compromise their ability to tolerate saline conditions for extended periods. Laboratory studies were carried out to assess the impact of caffeine in mixture with a brominated flame retardant (BFR), hexabromocyclododecane (HBCD). BFRs are substances used in the manufacture of a wide range of materials such as plastics and textiles and are very prevalent in freshwater, estuarine and marine environments. The majority of flame retardants contain brominated organic compounds, making them persistent and lipophilic with the ability to bioaccumulate. A European Risk Assessment has concluded that HBCD has a high bioaccumulation potential and is found in increasing levels in the environment and biota. Brominated flame retardants are similar in chemical structure to the thyroid hormones and have been shown to disrupt this endocrine system in many animals. In salmon, thyroid hormones play a vital role in smoltification and migratory behaviour and any modification of thyroid hormone concentrations (thyroxine T4 and tri-iodothyronine T3) are likely to significantly alter physiology and behaviour and may reduce the survival of smolts in the sea.

In a laboratory study, Atlantic salmon smolts were exposed to environmental levels of caffeine (10ng/l); HBCD (5ng/l); or a mixture of the two contaminants, for five days in freshwater. Half of the fish were then sampled for some of physiological parameters associated with smoltification, and the remaining fish were transferred to saltwater for 72hours before being sampled for the same parameters. All fish survived the 72hour seawater challenge test, indicating that the smolts were successfully adapted to saline conditions. Transfer to saltwater also resulted in an increase in gill Na+ K+ ATPase activity in the


blank control, caffeine and mixture of HBCD and caffeine groups as would be expected from a successfully adapted smolt. No such increase was observed in the HBCD group after seawater transfer, although exposure to both contaminants together was found to have no significant effect on the gill Na+ K+ ATPase activity.

In an additional study, the impact of HBCD flame retardant on smoltification was investigated in greater detail. Salmon pre-smolts were exposed to low levels of HBCD (5ng/l) for 30days in freshwater before being switched to clean seawater for a further 20days. The 30 day period was chosen in order to investigate the potential impact of a low level of a contaminant over an extended period in freshwater. A subsample of fish (n=5-8) was taken every 7 days in order to provide data for the first time on the impact of a flame retardant over the whole period of this sensitive life-cycle stage of smoltification. Measurement of physiological parameters in seawater would also indicate whether there is any recovery once exposure to the contaminant ceases. As expected, exposure to HBCD did not deleteriously affect gill Na+

K+ ATPase activity. Both control and HBCD-exposed fish showed an increase in gill Na+ K+ ATPase activity over time and after transfer to seawater, indicating that the fish were fully adapted to the increase in water salinity.

However, HBCD did affect plasma levels of thyroid hormones. On transfer to seawater, levels of both thyroxine (T4) and triiodothyronine (T3) were significantly lower in those fish that had been exposed to HBCD in freshwater, compared to the control group. Plasma levels of thyroxine (T4) are also known to be elevated in migrating smolts and a rise in thyroid concentration has been suggested to be associated with the initiation and control of the seaward emigration of salmonids. Although, there was no evidence from these studies to suggest that survival in the sea may be compromised, it is suggested that exposure to HBCD may inhibit or modify migratory behaviour as a result of a reduction to the thyroid profiles of the smolts. This affect of HBCD on smolt behaviour will be discussed further in the section on contaminants and smolt migration in coastal waters.

1.3 The impact of diffuse contaminants on the olfaction during the parr-smolt transformation – the pesticide atrazine.

Olfaction or the sense of smell plays a major role throughout the life history of salmon being intimately involved in pheromone-mediated reproduction, kin recognition, prey/predator detection and homing migration. A range of contaminants have been shown to compromise the sense of smell and inhibit the ability of male adult salmon to detect the pheromones released by the female which synchronise spawning physiology and behaviour (Moore & Waring, 1995; Waring & Moore, 1997; Moore & Waring, 1998; Moore & Waring, 2000; Moore & Lower, 2001). These same contaminants if present during the early spring when juvenile salmon are undergoing smoltification could also reduce the ability of the fish to imprint to the odours, which subsequently control the final homing migration of the spawning adult fish.

Laboratory studies were carried out to assess the effect of the pesticide atrazine on olfactory function in smolts during the spring smolt migration. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is a water-soluble pre- and post-emergence herbicide for the control of annual and perennial grass and annual broad-leaved weeds. Atrazine is known to have high mobility through soil and is a known contaminant of aquatic ecosystems in England and Wales. In 1992 and 1993, atrazine was one of the 5 pesticides most frequently present in both ground and surface water at levels in excess of the Maximum Admissible Concentration (MAC) of 0.1 g l-1 imposed by the Water Act 1991. In addition, analyses of UK surface waters demonstrated levels exceeding the proposed Environmental Quality Standard (EQS) of 2.0 g l-1 based on the annual combined average of atrazine and simazine. In 1993, the use of atrazine was banned on non-cropped land and as a result there has been a decline in its detection in UK surface waters. In 2004 a total ban on the use of atrazine by implemented by the EU. However, atrazine was included in the present study as it is a good example of a triazine pesticide and to better understand how such chemicals with similar toxic mechanisms may impact salmon populations.

During April, groups of Atlantic salmon smolts were exposed to concentrations of atrazine at 0.5, 1.0, 2.0, and 5.0 g l-1 and a control (no atrazine) for a 5-day period. A 5-day period of exposure was chosen as previous modelling studies have indicated that this is the peak period of a pesticide in a water course after run-off from farmland following a rainfall event. April is considered to be the peak migratory period for smolts and when imprinting is thought to occur as the fish emigrate downstream. At the end of the 5 days the olfactory responses of each fish to two odorants were measured using an electrophysiological


technique (electro-olfactogram: EOG) (Moore & Waring, 1995; Waring & Moore, 1997; Moore & Waring, 1998; Moore & Waring, 2000). EOG recording measures trans-epithelial voltage gradients from the surface of the olfactory epithelium (olfactory receptors) and is considered to reflect multi-unit cell activity. The two odorants tested were salmon smolt urine (104 dilution) and the amino acid L-serine (10-5 M. concentration). Urine was selected as it has previously been shown to elicit a strong response from the olfactory epithelium of salmon and is further suggested to be the source of specific compounds or pheromones that are involved in imprinting and homing by salmon (Moore et al. 1994).

After exposure to atrazine at nominal concentration of 0.5, 1.0, 2.0 and 5.0 μg l-1 electrophysiological responses recorded from the olfactory epithelium of salmon smolts to both L-serine and smolt urine were significantly reduced (P < 0.001). Although at the highest nominal concentration tested (5.0 μg l-1) responses were still recorded from the epithelium, the amplitudes were only 43 ± 8.8% (urine) and 33 ± 9.6% (L-serine) compared to the control. Atrazine itself did not elicit a recordable response from the olfactory epithelium at any of the concentrations used in the experiment. The results clearly demonstrates that exposure of salmon smolts to environmental levels of atrazine inhibits olfactory function which is known to play a pivotal role in the imprinting process and the subsequent homing of adult salmon to their home river.

1.4 The impact of diffuse contaminants on the olfaction during the parr-smolt transformation – the brominated flame retardant HBCD.

Salmon pre-smolts were exposed to low levels of hexabromocyclododecane (HBCD)(5ng/l) for 30days in freshwater during the period prior to the smolt migration of wild populations. The olfactory response of the HBCD-exposed fish to conspecific smolt urine was again measured using an electro-olfactogram (EOG). The olfactory response (n=5) was measured weekly throughout the freshwater dosing period. Smolts exposed to HBCD showed an incremental reduction in the olfactory response to conspecific urine over the 30 day dosing period, with a total reduction of 37% from the start to the end of the dosing period. In comparison, the control group increased incrementally over the same period, with a total


increase of 20% in olfactory function (Figure 1).

Figure 1. The olfactory responses to smolt urine recorded from the olfactory epithelium of salmon smolts after exposure to 5ng/l HBCD for 30 days. * p < 0.01.

During this period other fish exposed to HBCD were transferred to seawater, and the levels of both thyroxine (T4) and triiodothyronine (T3) in these fish were significantly lower compared to the control group. The toxic mechanism of HBCD has not yet been defined and therefore it is difficult to establish its mode of action on olfactory function as well as the thyroid endocrine system in the salmon. However, the imprinting process in salmonids has been linked with thyroid hormones and in a number of studies has shown that the olfactory response and imprinting to artificial odours is enhanced by increased thyroidal status (Scholz 1980; Hasler & Scholz 1983). Therefore, it is suggested that HBCD does not act directly on the olfactory receptors in the salmon, but by reducing the thyroid levels in the fish, there is not the subsequent increase in olfactory sensitivity in the fish during smoltification, which may allow the fish to successfully imprint to the home river. In conclusion, the disruption of thyroid hormone homeostasis and the reduction in olfactory function may have implications for the migratory behaviour of salmon, as well as their ability to return to their natal rivers to spawn.

1.5 The impact of diffuse contaminants on the migratory behaviour of smolts during the transition from the freshwater to marine environment – 4 nonylphenol.

The first study investigated whether exposure to the contaminant 4-nonylphenol (NP), an endocrine disrupting chemical, in freshwater influenced the migratory behaviour of Atlantic salmon smolts as they emigrated from freshwater and into the coastal zone. During April 2004 a group of Burrishoole origin Atlantic salmon smolts were exposed for 6 days to 4-nonylphenol at a nominal concentration of 18 g l-1. On the 20th April twenty of these fish were tagged intraperitoneally with miniature coded acoustic transmitters (Vemco: Model V8). A further twenty fish taken from the same group prior to exposure to NP were also tagged with the miniature transmitters. These fish acted as the control group. On 29th April

SID 5 (Rev. 3/06) Page 10 of 26

both groups of fish were released into Furnace Lough adjacent to the Marine Institute, Newport. The subsequent seaward migration of the smolts was monitored using acoustic receivers deployed between the freshwater release point and coastal waters. Thirty four smolts (17 exposed and 17 control) were recorded on the most seaward receiver and were considered to have successfully migrated out into coastal waters. The remaining six fish, (3 from each group) were considered to have died, although the cause of mortality is not known. There was no significant difference between the times taken by the two groups to migrate past each of the receivers located in freshwater and the coastal zone. The mean time that fish resided within freshwater was ~63 hours. The smolts then took on average a further 6-7 hours to migrate past the most seaward receiver located in the coastal zone. There was also no significant difference between the diurnal movements of the two groups. Movement was generally random with respect to the time of day, fish being detected migrating into coastal waters during both day and night. Further, there was no significant difference between the control and the exposed groups in terms of the tidal movements of the fish. All smolts migrated during the ebbing tide (3-4 hours after High Water).

Exposure of the salmon smolts to NP did not have a significant effect on their subsequent migratory behaviour or the run-timing into coastal waters. The temporal and tidal patterns of migration were similar between the two groups. The results of this study are consistent with the conclusions of a previous laboratory based study which indicated that exposure to environmental levels of NP does not significantly effect the ability of Atlantic salmon smolts to physiologically adapt to saline conditions.

1.6 The impact of diffuse contaminants on the migratory behaviour of smolts during the transition from the freshwater to marine environment – the pesticide atrazine.

The impact of the common pesticide atrazine on the migratory behaviour of salmon smolts within the freshwater and on entering the marine environment was studied in collaboration with the Department of the Marine, Republic of Ireland. During April 2005 a group of Burrishoole origin Atlantic salmon smolts were exposed for 3 days to atrazine at a nominal concentration of 0.1 g l-1. Subsequently, eighteen of these fish were tagged intraperitoneally with miniature coded acoustic transmitters (Vemco: Model V8) together with twenty fish taken from the same group prior to exposure to atrazine, which acted as the control group. On 28th April both groups of fish were released into Furnace Lough adjacent to the Marine Institute, Newport. The subsequent seaward migration of the smolts was again monitored using a number of VEMCO VR2 acoustic receivers deployed between Furnace Lough and Clare Island at the most western point of Clew Bay. Exposure of the salmon smolts to atrazine did not appear to have a significant effect on their subsequent migratory behaviour. The residency time within Lough Furnace of the exposed fish was similar to the controls and individuals from each group migrated through the estuary in close proximity. Further, the temporal and tidal patterns of migration were similar between the two groups. The laboratory-based experiments once again examined the impact of the pesticide on physiological parameters associated with the parr-smolt transformation (plasma T3 and T4, gill Na+K+ATPase activity) and saltwater survival. Exposure to a 0.1 gl-1 concentration of atrazine over a 72-hour period significantly reduced gill Na+K+ATPase activity but not plasma T3 and T4 levels when compared to the control group. However, on transfer to 33‰ saltwater there was 100% mortality in the pesticide exposed smolt group and 0% mortality in the controls.

Previous studies by Waring & Moore (2004) have shown that exposure to environmental levels of atrazine does reduce the ability of salmon smolts to survive in seawater. Therefore, the possible reasons for the differences in survival of the exposed smolts released as part of the present behaviour study and previous studies are not clear. However, in the present study the group of smolts exposed to atrazine and then tagged with acoustic transmitters were placed back into freshwater tanks in the hatchery for a period of 6 days before being released. This was to enable the fish to be released with other hatchery smolts reared as part of the ranching experiment. In previous studies the smolts have been transferred directly into saline conditions at the end of pesticide exposure (Waring & Moore 2004). It is therefore possible that in the present study the smolts were able to recover either partially or fully from the effects of pesticide exposure whilst in freshwater and were therefore not immediately osmotically compromised when they subsequently migrated into the marine environment. However, it is possible that if the smolts had only partially recovered from pesticide exposure, mortality in the marine environment may have occurred after a more extended period when the fish had migrated outside the study area and were not detected by the receivers. Therefore, it is suggested that further studies on the recovery of salmon smolts exposed to atrazine and other contaminants in freshwater are required.

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1.7 The impact of diffuse contaminants on the migratory behaviour of smolts during the transition from the freshwater to marine environment – the brominated flame retardant HBCD.

The impact of the common brominated flame retardant hexabromocyclododecane (HBCD) on the physiology and migratory behaviour of salmon smolts within the freshwater zone and on entering the marine environment was studied in collaboration with the Department of the Marine, Republic of Ireland. During April 2007 a group of Burrishoole origin Atlantic salmon smolts were exposed for 4 days to HBCD at a nominal concentration of 10.0 ng l-1. Subsequently, 10 of these fish were tagged intraperitoneally with miniature coded acoustic transmitters (Vemco: Model V8) together with 10 fish taken from the same group prior to exposure to HBCD, which acted as the control group. A further 10 fish from each group were sacrificed and gill tissue and blood plasma samples taken. Finally, 10 fish from each group underwent a 72-hour saltwater challenge test (32‰) and surviving fish were again sampled for gill and blood. On 27th April the tagged and control fish were released into Lough Furnace adjacent to the Marine Institute, Newport. The subsequent seaward migration of the smolts was monitored using a number of VEMCO VR2 acoustic receivers deployed between Lough Furnace and Clare Island at the most western point of Clew Bay.

Exposure of the salmon smolts to HBCD did not have a significant effect on either gill Na+K+ ATPase activity or the levels of the thyroid hormone T3 in the plasma when the fish were sampled in freshwater and after they had been placed in full strength seawater. However, the plasma levels of T4 in the exposed fish were significantly lower in the exposed group compared to the controls. In addition, 30% of the salmon smolts exposed to HBCD died within 18 hours after being placed in seawater. There were no mortalities in the control group after 72 hours in seawater.

There were no significant differences in the diurnal movements of the exposed or control smolts as they migrated through Lough Furnace and entered the estuary. Movement of the control smolts through the estuary and into Clew bay was predominantly on an ebbing tide whilst the exposed smolts moved randomly with respect to the tidal cycle. Six of the control fish successfully migrated seawards past the lower most receiver in Clew Bay and these fish are considered to have migrated successfully into the marine environment. There was evidence that two of the remaining fish had been predated as they moved through the estuary. However, eight of the smolts exposed to HBCD died as they moved through the estuary and into the marine environment. The results of the study suggest that exposure of smolts to HBCD in freshwater may affect their ability to survive when they migrate into the marine environment. In conclusion, the study provides further evidence that the conditions experienced by juvenile salmon in freshwater does have a significant impact on their subsequent survival in the sea.

Objective 1 of the study has been completed as detailed in the Scientific Objectives of the Contract. However, as a result of the baseline funding cuts (February 2006) it was agreed with the Defra Project Manager that there would be amendments to the contract with the removal of the following study:

Conduct field based studies in an experimental stream on PIT tagged fish exposed to diffuse contaminants (BFRs, caffeine and identified pharmaceuticals) to assess the effects on run-timing and freshwater emigration.

Objective 2. To determine the impact of novel diffuse contaminants on adult salmon with specific reference to freshwater entry, homing, and egg viability in female salmonids.

The majority of the previous research carried out at the Cefas, Lowestoft Laboratory on contaminants and salmon reproduction has focused primarily on the male salmon and pheromonal mediated reproduction (Moore & Waring, 1995; Waring & Moore, 1997; Moore & Waring, 1998; Moore & Waring, 2001; Moore & Lower, 2001). In these studies exposure to a range of pesticides reduced the ability of the male salmon to detect the pheromones released by the spawning female and which played a key role in synchronising the reproductive physiology of the male necessary for successful reproduction. However, adult female salmon are also exposed to the same environmental contaminants as the male and so a series of experiments were undertaken to determine how specific contaminants may affect the female reproductive physiology and possibly explain an additional mechanism controlling salmon populations.

2.1 The impact of a freshwater contaminant on female brown trout egg viability – the pesticide atrazine.

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In the initial experiments, the pesticide atrazine, which although banned by the EU in 2004, was again the chosen contaminant as it is still regularly detected in UK freshwaters that support Atlantic salmon populations. Laboratory-based studies were carried out to determine whether environmental levels of atrazine similarly affected reproductive function in female salmonids. Two related studies were carried out, the first replicating conditions that would be experienced by the early-returning salmon component of the population; and the second investigating the effect of a brief exposure during the actual spawning period. Female brown trout (Salmo trutta L.) were used instead of Atlantic salmon because of the difficulties of conducting laboratory experiments on mature adult salmon; however, the two species have been previously been shown to share the same reproductive physiology and pheromone system.

In the first study, groups of mature female brown trout were exposed to either 0.5g/l or 2.0g/l atrazine (or a blank or carrier control) for a 30day continuous dosing period. In the second study, female brown trout were exposed to the same concentrations of atrazine but for 5 days only, to coincide with ovulation of the fish. After this 5-day period, fish from exposed and control groups were stripped of the eggs and a subsample was mixed with milt to determine fertilisation rates. In addition, groups of eggs were also fertilised in the presence of water spiked with either 0.5g/l or 2.0g/l atrazine (or a blank or carrier control) to mimic the conditions during spawning in a contaminated spawning tributary. In order to assess whether exposure of the adult fish to atrazine and exposure of the eggs during fertilisation significantly affected survival and development of the embryos, the eggs from each group were left to develop until they had reached the eyed-stage and mortality during this period was monitored daily.

The results of the first study suggested that there were no significant differences in a range of reproductive and embryo parameters between the exposed and control groups of fish. After 30 days continual exposure to atrazine there were no differences in ovary weight, GSI, female fecundity or the plasma levels of the female reproductive hormones E2 and 17,20β-P. In addition there was no effect on the egg size and weight after exposure to atrazine at both concentrations. However, there were insufficient eggs to monitor subsequent survival over the 30 day period post-ovulation.

The results of the second study again suggested that there were no significant differences in a range of reproductive and embryo parameters between the exposed and control groups of fish after an exposure period of 5 days to the two atrazine concentrations. However, there was significant impact on the survival following fertilisation of the eggs that had been expose to atrazine. Applying the Kaplan-Meier estimation for survival it was calculated that those eggs exposed to atrazine during fertilisation had a 66% higher risk of mortality compared to control eggs for every microgram per litre of atrazine in the water. A number of the eyed eggs exposed to atrazine were analysed for DNA damage using the comet assay (Singh et al., 1988). The assay indicated that in those surviving eyed eggs, 30 days after fertilisation, DNA damage was higher in the eggs that had been fertilised in the water containing both 0.5g/l or 2.0g/l atrazine. However, it is also known that atrazine can impair sperm quality in fish. Therefore, it is not clear whether the poor survival in the eggs was due to the direct effect of the pesticide on the eggs or as a result of the poor quality of the sperm.

2.2 The impact of a freshwater contaminant on female brown trout egg viability – polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAH) are formed by the incomplete combustion of carbon-containing fuels such as wood, coal, diesel and oil. They are found in both freshwater and marine environments and at levels of between 32 – 1000ng/l in the salmonid-supporting River Test. PAHs have been shown to induce developmental malformations in teleost embryos , as well as reducing circulating levels of plasma sex steroids in a number of fish species. A laboratory study was carried out to assess the effect of environmental levels of PAHs on the egg viability of female brown trout, which shares a similar reproductive hormone-pheromone system with Atlantic salmon. Adult ripe female trout were exposed to a mixture of four commonly-found PAHs, or the carrier control, for six days in freshwater. The 4 PAHs and concentrations used in the study were: Naphthalene, 0.07 μg l–1; Fluorene, 0.011 μg l–1; Pyrene, 0.5 μg l–1; Phenanthrene, 1 μg l–1. After the 6 day exposure the eggs were expressed, weighed and measured. 600 eggs from each treatment group were then fertilised with milt from unexposed males, and left to develop in clean water to the eyed stage. A further sample of 600 eggs from each treatment group was fertilised in PAH-spiked water to determine any effect on water quality during fertilisation. Mortality was monitored daily in both groups for fifty days in order to gain a measure of fertilisation rate.

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Additional parameters measured included the Na+K+ ATPase activity in the gills, pseudobranch, and intestine of the female trout as well as kidney structure.

The results of the study indicated that exposure to all concentrations of PAH produced modifications to the kidney structure of the female fish, as well as lower levels of intestine Na+K+ ATPase activity. This may indicate that the female fish were under physiological stress as a result of PAH exposure. There were no differences in the absolute and relative fecundity in the female trout exposed to the PAH and the controls. However, there was a significant difference in the subsequent survival of the eggs after 50 days, which had been fertilised in PAH water compared to the controls. Once again exposure of eggs to contaminants during fertilisation have a poorer survival rate than those fertilised in “clean” water.

2.3 The impact of a freshwater contaminant on female brown trout egg viability – NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used drugs with analgesic, antipyretic and anti-inflammatory effect. Several groups of drugs fall in this category with the most common being ibuprofen and aspirin. An issue of concern is that these drugs are used in humans and animals and then excreted into wastewater or washed away. In addition, unused drugs are sometimes disposed of in the drain and all end up in water sewage treatment where most of them are not completely eliminated. The unmodified drugs or their metabolites that leave the sewage treatment plants are discharged into the environment where most are not biodegradable but where they even may be phototransformed into products that are more toxic than the parent compound. As a result, discharged pharmaceuticals can be found in surface waters, ground waters and sediments while the terrestrial environment receives only a secondary input as their polar and non-volatile nature prevents their escape from the aquatic environment.

In fish, NSAIDS have been shown to affect the production of prostaglandins, which in adult Atlantic salmon and sea trout have been shown to be the principal female reproductive pheromone that synchronises spawning between the males and females. Therefore, the potential impact of NSAIDs on prostaglandin levels in female trout was investigated. During the spawning season in 2007, gravid female brown trout were exposed for 5 days in experimental tanks to either aspirin (0.5µg l-1), ibuprofen (4.0µg l-1) or a mixture of the two compounds (0.5µg l-1 + 4.0µg l-1). Each treatment had a replicate together with appropriate controls. At the end of the study the fish were sacrificed and a sample of blood taken from each fish. The samples were stored at -20ºC in a laboratory freezer prior to are undergoing analysis to measure the levels of prostaglandins in each of the fish. However, prior to analysis an undetected electrical fault in the storage system over a weekend resulted in the loss of all samples. Unfortunately, there has been insufficient time and resources to repeat this experiments. Warning alarms have now been fitted to all cold storage facilities.

2.4 The impact of a freshwater contaminant on male salmon reproduction – Clofibric acid

Additional studies not originally specified as part of the contract were also undertaken to examine the impact of novel pharmaceuticals on male salmon reproduction. One such novel contaminant is the human use drug, clofibrate, or its metabolite clofibric acid, which are designed to lower total body lipids, particularly cholesterol, in the human body. Clofibric acid is increasingly found in aquatic ecosystems where it enters the surface water via domestic sewage, land application of sludges, and landfill sites. A laboratory-based study was designed to determine whether this contaminant posed any threat to wild populations of salmon, by first looking at the effects on male reproduction. Male parr were exposed to either 20 or 100ng/l for a period of 10 days or to the same concentrations for 21days, replicating likely exposure times in the wild. Parr exposed to 20ng/l clofibric had significantly larger livers compared to the control group, which is an indication that the fish was under stress as the liver is the major site of detoxification of contaminants. This also has implications for the production of hormones such as the thyroid hormone triiodothyronine, and for the production of cholesterol itself, which is the major precursor to steroids. Levels of the sex steroids testosterone and 11ketotesterone were significantly affected by exposure to clofibric acid, as was the size of the fish, indicating that exposure to clofibric acid has the ability to significantly affect reproduction in salmon. Reproduction is an energetically demanding process and it is important that the fish has enough energy to put into sex steroid production, gonadal growth, as well as somatic growth. Clofibric acid had no effect on the quantity of milt produced by the parr, nor testes size, although it is suggested that further research is required to examine milt quality.

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Data and outstanding samples will continue to be analysed in order to provide a more thorough picture of the effects of clofibric acid on salmon reproduction. Further studies are required to determine the levels of clofibric acid in UK waters and whether salmon at other life stages, for example, smolts, are likely to be at risk from exposure to this contaminant.

2.5 The impact of a pyrethroid pesticide on endocrine response to female odours and reproductive behaviour in male brown trout.

Further studies not originally specified as part of the contract as were also undertaken in collaboration with colleagues in Sweden and Norway to examine the impact of a pyrethroid sheep dip insecticide on male salmon reproductive behaviour. Cypermethrin [(R,S)--cyano-3-phenoxybenzly (1 R,S)-cis, trans-3-(2,2-dichlorovinyl)- 2,2-dimethylcyclopropane carboxylate] is a synthetic pyrethroid (SP) insecticide, which is increasingly being used as the active ingredient in sheep dips to replace the organophosphates (OPs). Although less toxic to humans than OPs, SPs are significantly more toxic to aquatic invertebrates and fish. Cypermethrin has a proposed Average Annual Environmental Quality Standard (EQS) of 0.0001µg l-1 and a Maximum Admissible Concentration (MAC) of 0.001 g l-1in the aquatic environment. However, levels of cypermethrin in excess of 0.85 g l-1 have been measured during routine monitoring of surface waters. Environmental levels of cypermethrin have been shown to disrupt the male salmon’s sense of smell and inhibit pheromonal mediated reproduction (Moore & Waring 2000).

The reproductive behaviour of brown trout (Salmo trutta L.) from an anadromous stock was studied in a large stream water aquarium. Four adult males and two ovulated females were placed in the aquarium together with eight mature male parr. Four of the parr had been exposed during the previous four days to two concentrations (0.1 or 1.0 µg l-1) of the pyrethroid pesticide cypermethrin (a disrupter of olfactory receptor function) and four of the parr to the solvent ethanol. The behaviour of all fish was followed for 24 h and then blood and milt was collected. Exposure to the higher concentration of cypermethrin disturbed the reproductive behaviour of the parr. They displayed fewer courting events, spent less time near the nesting females and had lower volumes of strippable milt. They also had significantly lower amounts of 11- ketotestosterone (11-KT) in the blood plasma than the control group. The higher cypermethrin group also had significantly lower levels of all these variables than the lower cypermethrin group, apart from strippable milt that showed no significant differences between two groups. No significant differences in non-reproductive behaviours were observed between any of the groups. In the control fish, there were significant positive correlations between a) the number of courting events and the amount of time spent near the female, b) blood plasma levels of 17,20β-P and time spent near the female and c) plasma levels of 17,20β-P and the number of courting events. Further, in control fish, higher plasma levels of 17,20β-P were observed in parr interacting with a female compared to those with no close female contacts. A priming experiment confirmed the results of the previous study by Moore & Waring (2000) that cypermethrin damages olfactory reception. Parr exposed to cypermethrin had significantly lower blood plasma levels of 17,20β-P and 11-KT than control males after exposure to ovarian fluid and urine (known to contain reproductive priming pheromones).

This study together with the previous work on cypermethrin carried out at the Cefas Laboratory clearly indicated that at levels routinely measured in the environment this particular pesticide could disrupt the spawning of Atlantic salmon. A presentation of the results at the Rivers Trust Annual Meeting in 2005 brought the research to a wider audience of stakeholders and as a result Cefas worked very closely with the Salmon and Trout Association in raising the awareness of the toxicity of cypermethrin to freshwater ecosystems. As a result on February 22nd 2007 the Veterinary Medicines Directorate (VMD) banned the sale of cypermethrin sheep dips throughout the UK. It acknowledged that the toxicological information showing the serious damage cypermethrin sheep dips have on the environment is too overwhelming to ignore and that it did not have the information it needed from the manufacturers even to attempt to provide guidance on the pollution risk.

It was agreed with the Defra Project Manager that as a result of the baseline funding cuts (February 2006) there would be amendments to the contract with the removal of the following study: Conduct field based studies to determine the impact on the migratory behaviour of adult salmonids after exposure to diffuse contaminants within estuaries and freshwater. The study will involve the use of miniature passive samplers (Portsmouth University – SF0241) attached to the salmon to measure contaminant exposure and acoustic telemetry techniques to monitor migratory behaviour.

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Objective 3. Determine the relationship between specific declining salmon stocks, land management changes and occurrence of target contaminants in the aquatic environment.

In the UK, 49% of our chalk rivers and 36% of all rivers are surrounded by arable land. It therefore should come as no surprise that these regions suffer badly from rainwater runoff, sediment erosion and diffuse chemical inputs. Diffuse sources of pollution are described by the Environment Agency as situations where pesticides are applied for agricultural and non-agricultural purposes on a field scale to land where a microbiologically active soil layer is present and where degradation and dissipation processes can take place. The chemicals may then accumulate in the sediment or remain in solution and leach into the soil layers and enter rivers via artificial drainage systems or as surface or subsurface flow, or precipitation from spray drift. Diffuse sources of pollution have been a known problem for the past few decades, with concerns being raised by the Royal Commission in 1992 (Royal Commission on Environmental Pollution, 1992). Our understanding of what effect these chemicals then have on the fish populations in our rivers is currently very limited, but data is starting to show that agriculture is potentially an important source of these contaminants.

Although river quality as a whole has been improving in the UK since the 1990’s salmonid populations in some rivers across England and Wales are still causing concern. For example, stock numbers in the southern chalk rivers had declined dramatically by the 1980’s and have remained low ever since. Reproduction and juvenile survival has been poor in some rivers that appear at first to have good water quality and habitat structure, which suggests that sub-lethal factors from diffuse sources might be inhibiting the restoration of fish populations in those rivers.

Land-use activities such as forestry and agriculture result in a range of physical effects such as erosion and sedimentation, which impact upon the productivity of salmonid populations, and the effect of these impacts is reasonably well studied. However, many river systems also receive large quantities of toxic chemicals, such as pesticides and herbicides, which could have potentially serious impacts on the reproductive health and survival of salmonid populations (Moore & Waring, 1995; Waring & Moore, 1997; Moore & Waring, 1998; Moore & Waring, 2001; Moore & Lower, 2003; Lower & Moore, 2003; (Moore et al., 2003; Waring & Moore, 2004).

Work on this objective focused on Rivers from the South West (Hampshire Avon, Tamar, Dart and Exe), Wales and North East (Yorkshire Esk) regions and sought to relate changes in the salmonid stocks (salmon and sea trout) over the past twenty years to changes in the use of pesticides. A detailed review was undertaken to determine the availability of data on land/water use and the occurrence of specific contaminants and is written up in a separate report. The report concentrated on pesticides, which have been shown experimentally to impact on the growth, egg mortality and olfactory responses in salmonids, namely atrazine, simazine, diazinon, cypermethrin and carbofuran. Attempts were made to establish links between the occurrence of these pesticides in surface waters and measures of the status of wild salmonid populations.

3.1 Land-use changes in England and Wales.

Agriculture is a major source of diffuse pollution in the UK, and this source has increased dramatically in recent years. This is primarily as a result of two major changes that have occurred in the agricultural industry since World War II: (i) intensification and (ii) an increase in average size of farm holding. Defra statistics show that between 1960 and 1990 the average UK farm holding size doubled, with the area of arable crops and temporary grass increasing by 36% (cereal cultivation showed a 60% increase), cattle numbers increased by 70% and poultry by 104%. Fodder crop land area has increased by 32% between1986 and 2001, whereas the area used for grassland has remained reasonably constant with only a small (5%) reduction in land area. Maize is the major fodder crop grown in the UK (52% of fodder land use area by 2001). It increased from 12,000 ha in 1983 to 109,413 ha in 1997 reflecting its popularity as an alternative to silage. Maize is mainly grown in the southern counties; 45% in the south west, 21% in the Midlands and 19% in the South East.

The Pesticide usage survey of 2002 estimated that 35% of the total area of arable crops in Great Britain was grown in Eastern Region, 15% in Northern Region, 13% in Midlands and Western Region, 10% in South Eastern Region, 10% in South Western Region and 1% in Wales. Percentage cover in each

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county varies to reflect differences in the suitability of land and climate with arable crops predominating in the east and grassland in west and uplands of England (Figure 2).

Figure 2. Agricultural land use in England and Wales. Source: (Environment Agency).

In 2000 the area of cereals in the UK had decreased by 2% from 1998 figures, in part due to the reduction in set-aside obligations. Winter barley and rye also decreased by 23% each. However, by 2002 agriculture still comprised more than 70% of the land use in England and Wales, with 36% of that used for growing crops. During the growing season of autumn 2001 to harvest 2002 four crops accounted for almost three quarters of all growing crops by area; wheat (48%), winter barley (13%), spring barley (13%) and oilseed rape (9%).

Over the past 15 years in England and Wales, agricultural use of pesticides for plant protection has shown a decline in the weight of the active substance applied by 19%, but the formulation treated area has increased by 23% perhaps suggesting that there is now a trend towards more frequent applications of lower dose chemicals. There has also been a shift in the types of chemicals being used. Organophosphate insecticides have declined in popularity and have largely been replaced by synthetic pyrethroids, which are more toxic to aquatic life.

Many of the pesticides monitored by the Environment Agency regularly exceed their Environmental Quality Standards (EQSs), however the worst offenders are sheep dip and textile chemicals such as cypermethrin and diazinon. A report by the Environment Agency in 2000 suggests that since the amenity license for atrazine and simazine was revoked in 1993 their detection rate has decreased, which is in sharp contrast to the rise in the number of detections of isoproturon and cypermethrin which have increased in line with their popularity. Since 1993, the primary use of atrazine has been in relation to the production of maize, particularly in SW England.

3.2 Relationship between land use, pesticide concentration and salmon stocks.

A number of problems were encountered when attempting to obtain suitable data with which to examine relationships between pesticide concentrations in the catchments and variations in salmon stocks. Firstly, the levels of pesticides published by the Environment Agency is not always provided as an actual measurement but rather as ‘less than’ a certain figure, due to the limitations of the laboratory

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procedures. For instance, the diazinon levels in the River Dart varied from actual concentrations to an upper figure of <32µg kg-1. Even when the raw measurements of pesticide concentrations in rivers were obtained, they still contained ‘less than’ figures. As procedures and technology improves, the analysis should become more accurate, but there is a need to detect the chemicals at a concentration of 0.1µg l-1 (the legal level) or less, since subtle effects of many of these chemicals have been demonstrated by the Cefas research. Secondly, some information particularly in relation to the types and amounts of chemicals used for aquaculture was restricted. Thirdly, much of the information on fish stocks in rivers has been based on catches from commercial and rod fisheries, which may not always provide a reliable indicator of stock abundance. These issues are explored in more detail in the full report of the review.

Recently, monthly averages of triazine herbicides (atrazine and simazine) in surface waters in the River Avon (Hampshire) have been obtained from the Environment Agency, which has permitted some preliminary analyses of the relationship between pesticides and salmon stocks in this river. Peak levels of the pesticides are measured between May and July, which represents the main period of agricultural application. In examining the relationship between pesticides and salmon stocks in the river, two hypotheses were considered. Firstly, it is known that atrazine inhibits the sense of smell in salmon at concentrations of between 0.04 and 14.0 g l-1 (Moore & Waring 1998). Therefore, the presence of atrazine in the River Avon may interfere with the sense of smell which the returning adult salmon use to home to their natal streams. As a result it is possible that increasing levels of atrazine in the river would reduce the number of returning adults and therefore the overall rod catch for that particular year. Secondly, it is known that exposure of smolts to atrazine reduces their ability to adapt to salt water (Waring & Moore 2004). Therefore, exposure of a particular smolt year class to atrazine during their emigration may be expected to result in a reduction in the return of grilse in the following year. The relationship between the annual average levels of atrazine in the River Avon between 1998 and 2006 and rod catch of salmon during the same period is shown in Figure 3. Higher atrazine concentrations in the river are significantly correlated with a reduced rod catch of salmon (p=0.003).

Figure 3. The annual rod catch of Atlantic salmon in relation to the mean annual concentrations of atrazine in the River Avon. The rod catch is shown as total number of salmon caught together with salmon caught before and after the 1st June.

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Figure 4. The rod catch of Atlantic salmon after 1st June in relation to the mean annual concentrations of atrazine in the River Avon. The rod catch is for salmon caught the year following (grilse) the measurement of atrazine.

Figure 4 shows the relationship between the annual average levels of atrazine in the River Avon between 1998 and 2006 and rod catch of salmon after 1st June the following year. The salmon returning at this time of year should be predominantly the grilse component of the stock which will have emigrated as smolts during the spring of the previous year. Again, higher concentrations of atrazine during the smolt migration is significantly correlated with a reduced rod catch of grilse the following year (p=0.003). These results could support both the hypotheses about the potential effects of atrazine on the number of returning adults. However, these such results need to be viewed with a great deal of caution. In examining these relationships it is very clear that there is a wide range of other problems within the River Avon catchment (e.g. erosion, sedimentation, flow regimes, habitat alterations and erosion of spawning sites), all of which play a role in regulating salmon populations. The present results certainly do not prove that atrazine is one of the principal factors regulating salmon populations, but they do suggest that if more detailed data on land use practices and diffuse contaminants can be obtained, further investigation of their relationship with stock parameters would be useful.

Objective 4. To assess the effects of diffuse contaminants on the biology of salmon within wild populations.

A recent Defra funded study SF0241 – Impact of intensive in-river aquaculture on wild salmonids, examined the potential impacts of fish farm effluents on certain aspects of the Atlantic salmon physiology and behaviour. Much of the study was based on comparative studies of mature male salmon parr and salmon smolts caged a short distance upstream and downstream of the discharge points from fish farms (rainbow or brown trout). In addition, assessments were made of the concentrations of steroids and other contaminants in the river water both upstream and downstream of the farms. Although the results of the study provided only a preliminary indication of the potential impact of such farms on wild salmon, two principal findings of the research were that there were changes to the physiology and ability to adapt to saline conditions in salmon smolts caged downstream of fish farms compared to fish that were caged upstream and aspects of the reproductive systems of mature male parr caged downstream of fish farms

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were significantly different to fish that were caged upstream. In order to assess the effects of diffuse contaminants on the biology of salmon within the wild a further study was carried out to examine the impact of effluents from fish farms on female salmonid biology.

A field study was carried out to determine the impact of the effluents originating from trout farm effluent on the River Test on the fecundity of female brown trout. Mature female brown (n=15 per channel) were placed in three cages and situated in either the control channel (no fish-farm effluent) or in the river channel downstream of the fish farm. Fish were left in the cages for six days before removing and sampling for egg number and size, and osmoregulatory organs. Water temperature was recorded throughout the exposure period in the river, and water samples taken to determine the range of contaminants present in each river channel. From the females that had ovulated, eggs were expressed and groups of 600 fertilised with milt from unexposed male trout. These fertilised eggs were placed in egg boxes and buried in artificial redds created in each of the river channels. The egg boxes were left for 50 days before removing. None of the eggs survived in either channel and each egg box contained a large amount of sediment, suggesting that siltation and depleted oxygen may have been the cause of such poor survival in the wild. Analysis of the plasma ion levels and osmoregulatory function in the fish caged downstream of the fish farms indicated some disruption compared to the control groups. In addition there were impacts on kidney structure and function exposed fish as determined by histopathology. The trout farm effluent-exposed group showed significantly lower plasma chloride concentration (129.9 ± 1.4 mmol/l) compared to the control group (134.5 ± 1.4 mmol/l; P = 0.025). However, there were no significant effects of the exposure to the effluents in kidney and gill Na+,K+-ATPase activities and plasma sodium and potassium concentrations. Histological analyses using image analysis indicated that the exposure to trout farm effluent significantly increased the numbers of nephrons (P = 0.008) and basophilic cell clusters (P = 0.0019) and nephron diameter (P < 0.0001). On the other hand, there were no effects of the exposure to the effluent in other histological parameters of the kidney (numbers of C-type, S-type and developing nephrons) and gill (filament and lamellar chloride cell numbers and lamellar width and length). Moreover, histological observation found no heavy histopathological damage in the kidney (haemorrhage and hyalinous deposition in nephrons) and gill (hyperplasia of the gill epithelium and lamellar fusion). The results of the study supports the previous work on salmon and suggests that short-term exposure to trout farm effluent water can have affects on osmoregulatory function in adult brown trout.

4.1 Modelling the impacts of diffuse contaminants on wild populations.

The modelling of the impacts of diffuse contaminants on wild salmonid populations has continued using two general approaches. Firstly, the further development of the simple model produced for a previous Defra funded research programme (SF0228), which incorporates the results from the laboratory and field-based studies to provide a means to assess and compare the effects at the population level. Secondly, utilising models to determine how contaminants from a point source may distribute throughout the catchment and affect fish population at various distances downstream.

The first model divides the salmon life cycle into five periods and makes the following assumptions about the survival of individuals:

1. Spawner to eggs: density independent (eggs produced per adult)2. Eggs to fry: density independent (intra-gravel survival data)3. Fry to Parr (~1 month): density dependent (Ricker) - Parr = * Fry * e-ß*Fry)4. Parr-smolt: density dependent (Beverton & Holt) - Smolt = * Parr / (1+ ß * Parr)5. Smolt to adult: density independent (smolt return data)

Thus the model assumes that there are two periods during the freshwater phase of the life-cycle in which density-dependant, compensatory mechanisms apply, egg to fry (dome-shaped) and parr to smolt asymptotic. The model was populated using a range of parameter values based on available data (obtained from the Environment Agency) and the scientific literature. The results from the laboratory and field-based research were then incorporated into the model to determine the overall effects on potential production and yield.

Figure 5 shows the effect on the stock-recruitment relationship when a hypothetical salmon population is exposed to a 10ng l-1 concentration of the brominated flame retardant DE71 during the parr to smolt

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stage, resulting in an 80% mortality. In this scenario, maximum recruitment would be reduced by about 29% and maximum yield by about 37%.

Stock Recruitment Relationship Yield Curve

0 200 400 600 800 1,0001,2000

500

1000

1500

2000

2500

Spawning stock (No. of individuals)

Rec

ruitm

ent

0 500 1,0000

500

1000

1500

2000

2500

Spawning stock(No. of individuals)

Yie

ld

--- Unexploited stock--- Exploited stock

Figure 5. Impact of a 10ng l-1 concentration of the brominated flame retardant DE71 during the parr stage.

Although the exposure of smolts to a mixture of atrazine (1μg l-1) and the oestrogenic chemical 4-nonylphenol (5 μg l-1) causes a considerably lower mortality (43%) at this stage than that caused to the parr by the brominated flame retardant, the effects on both maximum recruitment (43%) and yield (47%) are greater. This simple scenario demonstrates how the model provides a means to compare the results from different contaminant studies in a standardised format and thereby provide more effective management advice. Clearly, such models are only as robust as our understanding of the population dynamics of the stock and the data derived from the contaminant investigation. However, this and similar models, which have been proposed for further development, can also be used to highlight what further studies should be undertaken on a contaminant to improve the quantitative assessments.

Models are currently used to predict the distribution of pollutants and help identify critical regions within a river catchment receiving contaminants. Using concentrations of contaminants derived from monitoring programmes, it is possible to generate exposure estimates for the whole catchment. One such model, the Exposure Assessment Modelling System (EXAMS – U.S. Environment protection Agency), can assess the fate, exposure, and persistence of synthetic organic chemicals in surface waters of aquatic ecosystems. This model is being used to provide a preliminary assessments of the distribution of pesticides and contaminants in UK salmon-bearing rivers. It has also been successfully used to predict the fate and exposure levels of natural and synthetic estrogens in UK rivers. The present study has begun to use a similar model to examine the fate of chemicals derived from a particular source (e.g. aquaculture or agricultural facilities) and how they distribute themselves downstream within the catchment. Information from the laboratory studies which has indicated a particular toxic effect at a particular concentration is used to assess how much of the water course may be contaminanted at a level to cause an impact at either the individual or population level. It is proposed that this work is further developed to incorporate the information derived from a previous Defra study (SF0241) on the fate and toxicity of fish farm effluents within the catchment downstream from a number of in-river fish farms.

In addition, studies have also been carried out using the Kaplan-Meier method of survival analysis to estimate the relationship between increasing contaminant concentrations and the probability of survival at the different stages of the life cycle. These are based on analysis of the survival function, which is the probability of survival at a given time. This survivor function is more conveniently expressed for the present study as a hazard function, which is the chance of death within any time interval. This provides the basis for the Cox proportional hazard model in which the baseline hazard describes the common shape of the survival time distribution for all individuals. The relative risk function gives the level of each individual risk. That is the relative risk associated with an increase of the covariate value by 1 unit. In other words it will provide an indication of the mortality of an individual at a defined increase in contaminant concentration. In the study on the impact of atrazine on salmon fertilisation and egg survival

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the analysis showed a highly significant effect of atrazine in the water during fertilisation on the survival of the eggs to the eyed stage (hazard ratio = 1.66; P<0.001). This indicates that the eggs exposed to atrazine during fertilisation had a 66% higher risk of mortality compared to control eggs for every microgram per litre of atrazine. This approach has also been used to assess the impact of contaminants at the parr-smolt stage in order to refine the life-cycle model described above.

In conclusion, the incorporation of the laboratory based experimental data into the life cycle models demonstrates that low levels of environmental contaminants can have a serious impact on both individuals and populations of salmonids. As more data is gathered, both from laboratory and field-based research programmes the models and the predictions will become more robust.

Objective 5. Recommendations for reducing impacts of diffuse contaminants on fish populations.

The results of the research have clearly highlighted that diffuse contaminants can operate on specific, sensitive life history stages of salmonids, in particular reproduction, intragravel development of embryos and smoltification. Each of these stages generally occurs during specific periods of the year. For instance reproduction is generally October-January, the intragravel stage is between October and April and the period of smoltification is March until May in most UK populations. In addition, the research has highlighted that different groups of contaminants may have their main toxic effects at different stages in the life cycle. Thus, the pyrethroid and organophosphate insecticides used as sheep dips interfere with reproduction and therefore affect the survival and development of embryos, whilst the brominated flame retardants modify the endocrine system responsible for the parr-smolt transformation and thus reduce the subsequent survival of smolts in sea water. Therefore, in order to reduce the impact of contaminants on salmonid populations, it is recommended that wherever possible the contaminants are used outside the period of the life cycle where they have their maximum toxicological impact on the fish. Because compensatory mechanisms are operating during the freshwater phase of the life cycle, contaminants causing similar levels of mortality at a later stage will be expected to have a greater effect on the population (e.g. production and yield). Therefore, it is also recommended that contaminants that are likely to interfere with the parr-smolt transformation should be considered for more rigorous assessments.

The ban on the use of cypermethrin in 2007 by the Veterinary Medicines Directorate has reinforced the continued need to disseminate the results of the research to a wider audience of stakeholders. Although all of the work on sheep-dip insecticides had been published in peer-reviewed literature and as Defra reports this was not always accessed by other organisations and NGOs. Regular presentations to stakeholders such as the Rivers Trust and close liaison with other interested parties funding research on species other than fish in the freshwater environment is particularly important in highlighting the impacts of diffuse contaminants and reducing their potential impacts through awareness by the principal users of the chemicals.

The modes of toxicity of a number of diffuse contaminants has now been established, which demonstrate how these compounds may directly or indirectly affect physiological and behavioural pathways which control much of the salmon biology. Increasingly, new and more novel chemicals are being developed and licensed for use, many of which end-up in the freshwater environment through discharge or accident. It is not possible to study every chemical and determine its potential effect on salmonid populations. Therefore, based on their chemical structure, known mode of toxicity and the information derived from Cefas and other research, a salmonid life history “risk assessment” has been developed which will highlight the potential risk of chemicals to salmonids and other fish. For instance, organophosphates, pyrethroids, triazine herbicides and any other chemicals that directly affect the nervous system are also likely to impair the sense of smell of salmon, and this in turn may affect a range of behaviours such as migration and reproduction. Triazine herbicides, brominated flame retardants and other chemicals that modify or inhibit the hormonal system controlling the parr-smolt transformation may modify migratory behaviour and reduce the ability of juvenile salmon to survive in the marine environment. It is recommended that this “risk assessment” be used to understand how new contaminants may affect fish populations thus reducing the need for detailed experimental studies and taking a proactive approach to identifying “high risk” chemicals and thus reducing the impact of diffuse contaminants on the freshwater ecosystem.

General Discussion.

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The principal findings of the research is that a wide range of diffuse contaminants derived from intensive agriculture and industry have deleterious effects on salmonids at specific and sensitive stages in the life cycle. Reproduction in both male and female salmon and the processes whereby the juvenile salmon in freshwater pre-adapt to a life in the marine environment are affected by environmental levels of contaminants to the extent that this may significantly contribute to modifications to the population. Following on from previous studies at Cefas, the present research once again highlights that in terms of the complex life cycle of the salmon, the freshwater and marine life history stages are intimately linked. Although it is believed that conditions in the sea may regulate salmon populations for example through their effects on food availability, exposure to poor water quality in freshwater can have a significant impact on the behaviour and survival of the juvenile fish once they migrate to the marine environment. The present study has focused on water quality in the freshwater environment and reduced marine survival. However, there is now a requirement to understand how other environmental factors during the early life history of the salmon may also affect their ability to survive in the sea and successfully return to spawn. In particular, feeding opportunities in freshwater, subsequent smolt condition, modifications to the flow and temperature regimes within freshwater may all contribute to diminish the ability of the fish to successfully survive the marine phase of the life cycle.

The climate change scenarios recently produced for the United Kingdom predict that there will be a general warming of many rivers particularly in the south with a rise in temperature of 2-3 ºC. In addition, predicted rainfall is expected to decrease in the south of England reducing river flow and discharge. The modifications to the river temperature and flow will be further exacerbated by the increased requirement for water for domestic, industrial, aquaculture agricultural with pressure on local government and the Environment Agency for increases to abstraction licences. Under these changes to the freshwater environment there is a serious concern that diffuse pollution will have a further more significant impact on fish biology. Higher water temperature may increase the toxicological effects of contaminants on fish whilst reduced flows may further concentrate the levels of contaminants as well as increasing the residency time of fish in areas with reduced water quality. Therefore, there is a requirement to fully understand the potential impact of the interactions between temperatures and diffuse contaminants on fish within the context of providing advice to Government, the Agency and other bodies on the potential effects of increased water use (e.g abstraction) and other changes which may impinge upon the freshwater environment.

These studies also need to be extended to other species. For example, recruitment of European eel has fallen below 5% of the peak levels in the late 1970s and catches of both yellow and silver eels in Europe have declined from 40,000t in the 1960s to less than 20,000t today. The stock is judged to be outside safe biological limits and in response to the decline, the EU “Eel Recovery” regulation, published in 2007 was implemented which requires Member States to assess their stocks against target levels based on historic production and implement recovery measures as appropriate. However, the major factors regulating eel populations are still unknown and until there is an understanding of the factors causing the low recruitment of eels, the success of any management plans and conservation measures may be limited. By the very nature of its life cycle the European eel may be exposed during its freshwater residency to a wider range of contaminants and for longer periods than the salmon. Therefore, there is concern that diffuse contaminants may affect the European eel in much the same way as demonstrated for salmon and that freshwater pollution may be one of the factors that is responsible for the declines in the European eel stocks. Further research is required to understand how diffuse pollution may impact both the juvenile and adult stages of the eel.

References:

Boeuf, G. (1994). Salmonid smolting: a pre-adaptation to oceanic environment. In: Rankin, G.C. Jenson, G.B. (Eds.), Fish Ecophysiology. Chapman and Hall, London pp. 105-135.

Dittman, A. H. & Quinn, T. P. (1996). Homing in Pacific salmon: mechanisms and ecological basis. Journal of Experimental Biology 199, 83 -91.

Dittman, A. H., Quinn, T. P. & Nevitt, G. A. (1996). Timing of imprinting to natural and artificial odors by coho salmon, Oncorhynchus kisutch. Canadian Journal of Fisheries and Aquatic Science 53,434 -442.

Hasler, A. D. & Scholz, A. T. (1983). Olfactory Imprinting and Homing in Salmon. New York: Springer-Verlag.

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Hoar, W.S. (1988). The physiology of smolting salmonids. In: Hoar, W.S., Randall, D.J. (Eds.) Fish Physiology XIB Academic Press, San Diego, pp 275-343.

Hutchison, M.J & Iwata, M. (1998). Effect of thyroxine on the decrease of aggressive behaviour of four salmonids during the parr-smolt transformation. Aquaculture 168, 169-175.

Iwata, M. (1995). Downstream migratory behaviour of salmonids and its relationship with cortisol and thyroid hormones: A review. Aquaculture 135, 1-3, 131-139.

McCormick, S. D. & Saunders, R. L. (1987). Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth and metabolism. American Fisheries Society Symposium 1, 211 -229.

Moore, A. & Waring, C.P. (1995). Sub-lethal effects of the pesticide Diazinon on olfactory function in mature male Atlantic salmon (Salmo salar L.) parr. Journal of Fish Biology 48, 758-775.

Moore, A., Potter, E.C.E., Milner, N.J. & Bamber, S. (1995). The migratory behaviour of wild Atlantic salmon smolts in the estuary of the River Conwy, North Wales. Canadian Journal of Fisheries and Aquatic Sciences. 52 (9), 1923-1935.

Moore, A., Stonehewer, R., Kell, L.T., Challiss, M.J., Ives, M. Russell, I.C. Riley, W.D. & Mee, D.M. (1996). The movements of emigrating salmonid smolts in relation to the Tawe barrage, Swansea. In: Barrages: Engineering Design & Environmental Impacts. (N. Burt & J. Watts eds.) HR Wallingford Ltd. John Wiley & Sons Ltd. pp. 409-417.

Waring, C.P. & Moore, A. (1997) Sublethal effects of a carbamate pesticide on pheromonal mediated endocrine function in Atlantic salmon. Fish Physiology and Biochemistry 17, 203-211.

Moore, A. & Waring, C.P. (1998) Mechanistic effects of a triazine pesticide on reproductive endocrine function in mature male Atlantic salmon parr. Pesticide Biochemistry and Physiology 62, 41-50.

Moore, A. & Waring, C.P. (2000) The effects of a synthetic pyrethroid pesticide on some aspects of reproduction in Atlantic salmon. Aquatic Toxicology. 52, 1-12.

Moore, A. & Lower, N. (2001). The impact of two pesticides on olfactory mediated endocrine function in mature male Atlantic salmon parr. Comparative Biochemistry and Physiology Part B 129, 269-276.

Moore, A., Scott, A.P., Lower, N., Katsiadaki, I. & Greenwood, L. (2003). The effects of 4-nonylphenol and atrazine on Atlantic salmon (Salmo salar L.) smolts. Aquaculture. 222, 253-263.

Nevitt, G. A., Dittman, A. H., Quinn, T. P. and Moody, W. J., Jr (1994). Evidence for a peripheral olfactory memory in imprinted salmon. Proceedings of the National Academy of Science USA 91, 4288 -4292.

Nordeng, H. (1977). A pheromone hyposthesis for home ward migration in anadromous salmonids. Oikos 28, 155-159.

Riley, W.D., Eagle, M.O. & Ives, S.J. (2002). The onset of downstream movement of juvenile Atlantic salmon, Salmo salar L. in a chalk stream. Fisheries Management and Ecology 9, 87-94.

Sakamoto, T., Hirano, T., Madsen, S.S., Nishioka, R.S., Bern, H.A. (1995). Insulin-like growth factor I gene expression during the parr-smolt transformation of coho salmon. Zoological Science 12, 249-252.

Waring, C.P. & Moore, A. (2004). The effect of atrazine on Atlantic salmon smolts in freshwater and after saltwater transfer. Aquatic Toxicology 66, 93-104.

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Publications, reports and conference presentations generated by the project.

Publications in scientific journals

1. Moore, A., Lower, N., Mayer, I. & Greenwood, L. (2007). The impact of a pesticide on migratory activity and olfactory function in Atlantic salmon (Salmo salar L.) smolts. Aquaculture 273, 350-359.

2. Jaensson, A., Scott, A.P., Moore, A., Kylin, H. & Olsen K.H. (2007). Effects of a pyrethroid pesticide on endocrine responses to female odours and reproductive behaviour in male parr of brown trout (Salmo trutta L.) Aquatic Toxicology 81, 1-9.

3. Lower, N. and Moore, A. (2007). The effect of a brominated flame retardant on smoltification and olfactory function in Atlantic salmon (Salmo salar L.) smolts. Marine and Freshwater Behaviour and Physiology 40 (4), 267-284.

4. Moore, A., Cotter, D., Quayle, V., Rogan, G., Poole, W.R., Lower, N. and Privitera, L. (2008) The impact of a pesticide on the physiology and behaviour of hatchery-reared Atlantic salmon (Salmo salar L.) smolts during the transition from freshwater to the marine environment. Fisheries Management and Ecology 15, 385-392.

5. Mizuno, S., Lower, N., Privitera, L., Witthames, P., Evans, L., Waring, C.P., Moore, A. (2009). Physiological impacts of short-term exposure to river water contaminated by trout farm effluents on osmoregulatory organs in adult brown trout Salmo trutta L. Submitted.

Meetings and presentations:

1. Moore, A. Diffuse pollution and Atlantic salmon populations. Association of Rivers Trusts Spring Meeting, 5th May 2005.

2. Moore, A. Impact of diffuse poluution on salmonid populations. 15th International Salmonid Conference – Salmonids in the 21st Century. Newcastle, 17-20 October 2006.

3. Lower, N. and Moore, A. The impact of a brominated flame retardant on olfactory function in Atlantic salmon smolts (Salmo salar L.) smolts. Chemical Ecology in Aquatic Systems, Florence, Italy, 16-18 October 2006.

4. Lower, N. & Moore, A. The effect of environmental levels of pesticides on salmonid fecundity and embryo development. VII International Congress on the Biology of Fish, St John’s, Newfoundland, Canada, 18-22 July 2006.

5. Moore et al. The impact of a pesticide on the physiology and behaviour of hatchery-reared Atlantic salmon (L.) smolts during the transition from freshwater to the marine environment. 7th Telemetry Conference held in Europe, Silkeborg, Denmark 17-21 June 2007.

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Documents

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