DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit DEFRA, Area 301 Cromwell House, Dean Stanley Street, London, SW1P 3JH. An electronic version should be e-mailed to [email protected]

Project title Investigation of the causes of heron deformities in North Nottinghamshire

DEFRA project code WM0101

Contractor organisation Central Science Laboratory and location Sand Hutton York YO41 1LZ

Total DEFRA project costs £ 20,054

Project start date 23/01/03 Project end date 30/04/03

Executive summary (maximum 2 sides A4)

1. Dead and deformed heron (Ardea cinerea) chicks have been reported at a large heronry at Besthorpe Nature Reserve, North Notts, since 1996 (when systematic visits to the heronry started). Many of the birds died for no obvious reason but deformities in others included multiple fractures of the tarsus and tibia and metacarpal bones (angel wings). Work at the Institute of Zoology has shown weaknesses in the long bones of the heron chicks that are consistent with low mineralization of growing bones, a syndrome similar to rickets.

2. Predatory water birds such as herons are susceptible to bioaccumulation of pollutants through ingestion of contaminated food sources and as they are at the top of the food chain they are at risk of contaminant bioaccumulation. Two major classes of environmental contaminants have been reported to result in deformities in growing birds: trace elements, e.g. selenium, and PCBs/dioxins

3. Tissue samples were taken from nestlings collected from the Besthorpe heronry in 2002 (undeformed, dead and deformed) and eggs were collected in 2003 from Besthorpe and from control sites in Cheshire and Hertfordshire. Fat samples from nestlings (and adults found dead nearby) and eggs were analysed for PCBs (non-ortho- and ortho-) and dioxins (PCDDs/PCDFs) and liver samples from the nestlings, adults and eggs were analysed for trace elements.

4. Some trace elements, mercury, lead, copper and selenium were close to or above background levels in the nestling or adult liver samples and this was probably related to the proximity of the coal-fired power plants to the heronry. The levels could not be directly related to the deformities in the herons but they may contribute to an effect.

5. The profile of PCBs in the heron fat and egg samples were typical of those other species of heron and for other fish -eating birds such as cormorants. Thus PCB153 is the dominant PCB congener and PCB77 and PCB126 are the dominant non-ortho congeners.

6. The PCB/dioxin residue data show that the nestlings which were euthanised due to the severity of deformity had higher concentrations of PCBs/dioxins in their fat deposits (610 ng/kg fat, expressed as (TCDD) WHO-TEQ) than birds from the same colony that were not deformed (260 ng/kg fat); the levels were similar to those in adult birds from the same area (655 ng/kg fat). The concentrations of PCB77 (a non-ortho-congener) in the nestling fat (4306-6730 ng/kg fat) were well above

CSG 15 (Rev. 6/02) 1 Project Investigation of the causes of heron deformities in North DEFRA WM0101 title Nottinghamshire project code

those in eggs (819-2099 ng/kg fat), which are similar in profile to the adult fat sample (1109 ng/kg fat) and are deposited in eggs from in adult liver.

7. The levels of PCBs/dioxins in eggs from nests which had contained nestlings with deformities in 2002 were also far higher (945 ng WHO-TEQ/kg fat) than levels in other eggs from other nests in the colony (320-442 ng WHO-TEQ /kg fat) and from the Tring control site (324-338 ng WHO-TEQ/kg fat). It is known that there is little variation in contaminant levels between eggs in the same clutch and there is also no loss of contaminant mass from heron eggs to chicks. This suggests that the female birds in these nests had been exposed to significantly higher levels of non-ortho and ortho-PCBs than other females in the same heronry. There is even greater concern raised by the “control” egg sample from Cheshire which contained a total WHO-TEQ of 2233 ng/kg fat. The eggs from nests which contained deformed nestlings in 2002 (and the “control” eggs from Cheshire) have levels of accumulated PCBs/dioxins which are similar to those reported to have effects in other studies.

8. There are a number of observations in this study that support the hypothesis that the deformities in the heron nestlings are due to reduced calcium deposition in bone and suggest that PCBs/dioxins are associated with these effects.

a) The levels of calcium in plasma were lower in deformed than in apparently normal nestlings in the same colony and there is significant of eggshell thinning in eggs from the Besthorpe colony when compared to eggs from other sites. b) Concentrations of PCBs/dioxins expressed as total WHO-TEQ (TCDD equivalents) were higher in deformed nestlings and in nestlings found dead than those apparently unaffected. c) The WHO-TEQ was also higher in eggs from nests that contained deformed nestlings in 2002 (this assumes birds are nest-loyal from year to year) than from other nests in the Besthorpe colony or the control Tring colony. d) These contaminants may affect vitamin D dependent calcium mobilisation from the shell across the chorioallantoic membrane thus result in insufficient calcium for mineralization; TCDD has also been observed to impact on calcium deposition in bone. A negative correlation has been reported between TCDD levels in eggs with plasma calcium, tibia length, wet, dry and ash weight in the hatched great blue heron chicks. Lead has also been shown to affect vitamin D and calcium metabolism in some cases. Lead levels in nestlings were higher in the deformed than apparently unaffected nestlings.

9. The levels of PCBs/dioxins in heron nestlings and eggs from nests affected in 2002 were sufficiently high to suggest that this is the underlying cause of the deformities. The eggs from Cheshire colonies also raise concerns due to their very high residue levels of PCBs/dioxins suggesting the problem is not localised. Further work is required to identify the source of the pollution, e.g. sampling fish and other food sources (the diet of grey herons is diverse) in the vicinity of the colony and further sampling to determine individual variations in residue levels and correlation with deformities. The diverse diet of herons means that a number of food sources should be considered, e.g. earthworms due to the location of the site, and consideration should be given to other species which may be using similar food sources.

CSG 15 (Rev. 6/02) 2 Project Investigation of the causes of heron deformities in North DEFRA WM0101 title Nottinghamshire project code

Scientific report (maximum 20 sides A4)

Introduction

Dead and deformed heron (Ardea cinerea) chicks have been reported at a large heronry at Besthorpe Nature Reserve, North Notts, since 1996 (when systematic visits to the heronry started) (Blackburn and Kent 1999). Many of the birds died for no obvious reason but deformities in others included multiple fractures of the tarsus and tibia and metacarpal bones (angel wings) (Grantham 2000).

Work at the Institute of Zoology has shown weaknesses in the long bones of the heron chicks that are consistent with low mineralization of growing bones, a syndrome similar to rickets (Rodriguez 2001). These deformities are most obvious at 3-4 weeks of age. Studies on the stomach contents have shown remains of rodents, earthworms, and vegetation but this may be due to the rapid metabolism of fish by herons; stomach contents of birds from other, unaffected, sites showed similar contents. Higher levels of deformities have been reported in years with dry springs but this may be due to higher levels of mortality in wet springs (Grantham 2000).

Predatory water birds such as herons are susceptible to bioaccumulation of pollutants through ingestion of contaminated food sources and as they are at the top of the food chain they are at risk of contaminant biomagnification (De Luca-Abbott et al 2001). Two major classes of environmental contaminants may result in deformities in growing birds.

Trace elements Trace elements, such as selenium, have been widely reported to cause deformities (Fairbrother et al 1999, Ohlendorf 1999, Spahn and Sherry 1999, Spallholz and Hoffman 2002, Hernandez et al 1999), e.g. herons in the Great Lakes, USA where selenium has been reported as a contaminant from sewage sludge disposal, emissions from smelting plants and in agricultural runoff (Ohlendorf 1999). Arsenic, cadmium, mercury and lead are environmental contaminants that find their way into the food chain from historical use of the metals and industrial pollution (Hernandez et al 1999). These elements have no clear nutritional value, but considerable evidence exists indicating their toxicity. Analysis of livers from some of the nestlings in 1997 and 1998 showed levels of selenium in liver similar to those in birds from unaffected sites. However birds are most sensitive to selenium at the egg stage and therefore this study analysed a range of trace elements in the livers of nestling birds and in eggs laid in 2003.

PCBs/Dioxins Polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs)/ polychlorinated dibenzofurans (PCDFs) are two groups of synthetic chlorinated hydrocarbons that have been detected as residues throughout the global ecosystem. Their presence in birds is thoroughly reviewed by Hoffman et al. (1996). Residues of PCBs have resulted from their use primarily as insulating and cooling agents in closed electrical transformers and capacitors but also as heat transfer agents, lubricants, flame retardants, plasticizers and waterproofing materials. PCDDs/PDDFs have been manufactured as impurities of chlorphenols and production of herbicides such as 2,4,5T and are also produced during incineration leading to their presence in flyash.

The environmental persistence of the PCBs and PCDDs/PCDFs is due to their resistance to bacterial and chemical breakdown are they are readily absorbed from water into fat of plankton and thus transferred through food chains. Fish and then piscivorous birds, and predatory birds and mammals, accumulate progressively higher concentrations (bioaccumulation) and residues are deposited in fat. Although residue levels vary between tissues (highest in liver), the congener profiles do not vary between tissues in piscivorous birds (Boumphrey et al 1993). The high levels of the contaminants in the liver of birds results in the residues in eggs being similar in congener profile and content (on a lipid basis) as lipid is mobilised from liver and deposited in eggs.

PCBs and dioxins (a term used here to cover both PCDDs and PCDFs) are closely associated with incidences of bird deformities and mortality. These highly lipophilic compounds are likely to be deposited into eggs during the mobilisation of female fat and are well established as endocrine (hormone) disrupters in birds. Endocrine disruption can result in a wide range of effects including disruption of growth, deformities, disruption of sexual maturation. Oestrogen is associated with the deposition of calcium in bone and antiestrogens such as TCDD have been associated with effects on bone calcium deposition in mammals (Allen and Leamy 2001). Although the bone ash calcium levels were reported as “normal” there are no control values for grey heron nestlings with which to compare (Grantham 2000). The presence of fly-ash lagoons which were used until the 1970s (the site is in an area which formerly had many coal-fired power stations, e.g. Cottam, High Marnham may be a source of dioxins and PCBs leaching into areas where the herons feed.

There are 209 possible isomers of PCBs which are based on chlorination of 10 possible degrees of chlorination (mono to decachlorobiphenyl) with various positional isomers (congeners) within each group (Figure 1). Some of these congeners are more readily metabolised in vivo whereas others bioaccumulate. In addition, these congeners vary in toxicity depending on the position and content of chlorine. Congeners with chlorines in positions 4 and 4’ or 3,4 or 3,4,5 on one or both rings tend to be more toxic and bioaccumulate.

CSG 15 (Rev. 6/02) 3 Project Investigation of the causes of heron deformities in North DEFRA WM0101 title Nottinghamshire project code

PCDDs have 75 positional isomers with 1 to 8 chlorine atoms per molecule (Figure 2) and PCDFs have 135 positional isomers. Those with chlorine substitution in the 2,3,7,8 positions are the most toxic.

Both PCBs and PCDDs/PCDFs may be biologically toxic with a wide range of responses including immunotoxicity, reproductive impairments, porphyria and liver damage. The most toxic PCB congeners are those that have 4 or more chlorine atoms in the para- and meta-positions on the biphenyl ring but no chlorines in the ortho positions, i.e. non-ortho PCBs. These PCBs can assume a relatively coplanar conformation similar to that of 2,3,7,8-TCDD and include 3,3’,4,4’,5,-penta-CB (PCB 126), 3,3’,4,4’- tetra-CB (PCB 77) and 3,3’,4,4’,5,5’-hexa-CB (PCB 169).

Comparison of residue data for PCBs and PCDDs/PCDFs is complicated by the dependence of residue levels on the numbers of congeners for which residues have been quantified. In addition the impact of the PCBs and PCDDs/PCDFs depends not only on the residue level but also the congeners’ toxicity. Therefore, there have been several approaches to quantifying residues based on both the measured residue and the relative toxicity of the congener. The most commonly used in the toxic equivalents approach in which the residue level of the PCB or PCDD/PCDF are multiplied by their toxicity relative to TCDD and these are summed to give a total toxic equivalent (WHO-TEQ) (Kennedy et al 1986, Van Leeuwen and Younes, 2000).

Figure 1 Generalised structure of biphenyl and possible positions for chlorine (ortho (positions 2 or 6), meta (3 or 5), para (4)) on PCB congeners

3 2 2’ 3’

4 1 1’ 4’

5 6 6’ 5’

Figure 2 Generalised structure of dibenzo-p-dioxin and possible positions for chlorine

9 1 O 8 2

7 3 O

6 4

Materials and methods Nestling samples Samples of liver and fat were obtained from the Institute of Zoology for heron nestlings sampled in 2002 (Table 1). Liver samples from each bird were analysed for trace elements and fat samples were pooled according to their cause of death (Table 1) for PCB/dioxin analysis. Thus nestlings in Group 1 were euthanised due to causes other than deformities, group 2 birds were found dead in nest and group 3 birds were euthanised due to the scale of their deformities. Group 4 birds were adults that were found dead in the local area.

Data for blood samples analysed in 2002 were collated and assessed to determine any significant differences between deformed and apparently unaffected birds in the Besthorpe colony and also unaffected control colonies in Cheshire.

Egg samples Egg samples were collected under licence from English Nature in early 2003 (Licence 20030202). The eggs were sampled from Besthorpe and from 2 control sites in Cheshire (Green Wood, Keckwick, Runcorn and Pitts Heath, Keckwick, Runcorn) and in Tring, Hertfordshire. Eggs were taken from individual nests (Table 2). Eggs were grouped randomly from the Besthorpe colony other than

CSG 15 (Rev. 6/02) 4 Project Investigation of the causes of heron deformities in North DEFRA WM0101 title Nottinghamshire project code

the 2 eggs in Group 2 that were from nests which had contained deformed chicks in 2002. These pooled samples were analysed for PCBs/dioxins and for trace elements.

The thickness of the eggshells on the sampled eggs (and 4 addled eggs collected in April 2003) were analysed to determine whether any eggshell thinning was evident compared to the control sites. A micrometer was used to measure the thickness of each eggshell (including membrane) at 4 points around the midpoint after the eggs had been washed and allowed to dry for 24 hours.

Trace element analysis Analysis for metals in liver from individual nestlings and pooled egg samples was undertaken using induction coupled mass spectrometry (ICPMS) with identification of 10 elements (selenium, chromium, mercury, zinc, arsenic, lead, aluminium, nickel, cadmium, copper).

PCB/dioxin analysis 13 The sample was ground, fortified with known amounts of surrogate ( C12-labelled) analogues of the target analytes and exhaustively extracted using mixed organic solvents. The extract was cleaned up using adsorption chromatography. Ortho-PCBs, non-ortho-PCBs and PCDDs/PCDFs were segregated into three separate fractions. Each fraction was concentrated and further cleaned up before the inclusion of additional surrogate standards. Final determination was by high resolution gas chromatography with either low resolution mass spectrometric detection (ortho-PCBs) or high resolution mass spectrometric detection (non-ortho-PCBs and PCDDs/PCDFs) (Ambidge et al 1990, Krokos et al 1997, Fernandes et al 2002). The standard dioxin and dioxin-like PCB analysis suite was determined to provide data on the following compounds: (a) the seventeen laterally (2,3,7,8-) chlorinated dibenzo-p-dioxins and dibenzofurans, (b) non-ortho-substituted PCBs (IUPAC numbers 77, 81, 126 and 169) and (c) ortho-substituted PCBs (IUPAC numbers 18, 28, 31, 47, 49, 51, 52, 99, 101, 105, 114, 118, 123, 128, 138, 153, 156, 157, 167, 180 and 189). Since dioxins and furans are found as complex mixtures of congeners, systems of Toxic Equivalents (WHO-TEQs) have been devised. Therefore the data were also expressed using WHO toxic equivalents (WHO-TEQs) of 2,3,7,8-TCDD, considered to be the most toxic of the dioxins. To express the toxicity of a congener relative to 2,3,7,8-TCDD an internationally agreed weighting factor (known as a Toxic Equivalent Factor (TEF)) is applied to each of the congeners (Van Leeuwen and Younes 2000). This gives a result as a Toxic Equivalent (WHO- TEQ) and summation of the WHO-TEQs gives a 'Total WHO-TEQ' for a mixture.

Table 2 Identification of heron egg samples collected in 2003 CSL no Site Sample id. Comments Date group 8177 Besthorpe Egg A Tree 18 nest 1 16.02.03 1 8178 Besthorpe Egg B Tree 18 nest 5 16.02.03 1 8183 Besthorpe Egg E Tree 18 new nest 01.03.03 1 8182 Besthorpe Egg 2 Tree 23 nest 1 dead chick in 2002 16.02.03 2 8187 Besthorpe Egg 4 Tree 22 nest 1 dead chick in 2002 01.03.03 2 8179 Besthorpe Egg C Tree 11 nest 1 16.02.03 3 8180 Besthorpe Egg D Tree 16 nest 3 16.02.03 3 8181 Besthorpe Egg 1 Tree 33 nest 1 16.02.03 3 8184 Besthorpe Egg F Tree 20 nest 7 01.03.03 4 8185 Besthorpe Egg G Tree 27 nest 1 01.03.03 4 8186 Besthorpe Egg 3 Tree 22 nest 4 01.03.03 5 8188 Besthorpe Egg 5 Tree 4 nest 2 01.03.03 5

8189 Cheshire A Green Wood 09.03.03 6 8190 Cheshire C Pitts Heath 09.03.03 6

8191 Tring Tring 1 23.02.03 7 8192 Tring Tring 2 23.02.03 7 8193 Tring Tring 3 22.02.03 8 8194 Tring Tring 4 12.03.03 8

Table 1 Identification of tissue samples analysed from herons collected in 2002. ZSL Mass of Mass of Comments as written on Group PM No. Ring no. No. heron (g)liver (g) Site Date Tree Nest list or with specimen 1 138/03 1296226 1 1067 44.98 Besthorpe 12.05.02 30 D 1 well grown pullus from Notts nest 30/D. Bird jumped & fell through the branches. Euthanised as it appeared

CSG 15 (Rev. 6/02) 5 Project Investigation of the causes of heron deformities in North DEFRA WM0101 title Nottinghamshire project code

concussed. Besthorpe Dead chick from collapsed 1 160/03 36 719 37.89 Notts 27.03.02 27 nest Besthorpe 2 113/03 1293381 41 1545 49.23 Notts 27.03.02 23A 1 Dead chick Dead in nest >21 days old. Decomposing. With fractures of right wing, Besthorpe right femur and left 2 139/03 1293387 17 1190 24.32 Notts 20.04.02 20 2 tibiotarsal bone Besthorpe 2 145/03 46 686 31.93 Notts 27.03.02 25 2 Deformed Dead in nest Besthorpe 2 157/03 6 889 40.49 Notts 10.04.02 25 2 Deformed Dead in nest Besthorpe 3 114/03 1293383 42 966 46.76 Notts 27.03.02 16 3 Deformed - euthanised

Besthorpe 3 140/03 1293386 34 983 36.53 Notts 10.04.02 Deformed - euthanised Besthorpe 3 141/03 35 1238 43.84 Notts 27.03.02 16 3 Deformed - euthanised Besthorpe 3 143/03 1293399 32 1154 40.8 Notts 10.04.02 22 1 Deformed - euthanised Besthorpe 3 161/03 33 729 32.94 Notts 27.03.02 20 2 Deformed - euthanised Tiln Wood 4 156/03 38 1462 36.8 Retford Adult heron 1 of 2 adult herons picked up presumed shot. (Empty Tiln Wood cartridge nearby + local 4 158/03 31 1150 15.9 Retford 23.04.02 information). 1 of 2 adult herons picked up presumed shot. (Empty Tiln Wood cartridge nearby + local 4 159/03 30 1642 24.76 Retford 23.04.02 information).

CSG 15 (Rev. 6/02) 6 Results

Trace elements The levels of trace elements in the livers from nestling and adult birds and in pooled egg samples are shown in Table 3. There were no apparent differences in trace element levels between the eggs from Besthorpe and the control sites. Assessment of the levels of trace elements in the liver samples is more complicated in the absence of parallel control samples.

Table 3 Trace elements detected in liver and egg samples HERON LIVER Mean (mg/kg we weight)

Group ID Al Cr Ni Cu Zn As Se Cd Hg Pb 1 XT0138/03 0.35 <0.01 0.07 57 58 0.04 3.7 0.022 0.29 0.021 1 XT160/03 0.31 <0.01 (0.01) 30 30 0.21 1.1 (0.005) 0.32 0.105 2 0113/03 0.10 0.04 (0.02) 72 34 0.87 1.3 (0.007) 0.27 0.092 2 XT139/03 1.61 0.03 (0.02) 106 113 0.08 1.3 0.017 0.68 0.143 2 XT145/03 0.15 <0.01 (0.02) 62 79 0.12 1.0 0.019 0.44 0.116 2 XT157/03 0.62 (0.01) 0.03 43 60 0.11 1.1 0.012 0.43 0.081 3 0114/03 0.13 (0.01) 0.07 44 51 0.39 1.0 (0.004) 0.29 0.109 3 XT140/03 0.15 (0.01) (0.02) 95 88 0.22 1.1 0.011 0.43 0.092 3 XT141/03 0.15 (0.01) (0.01) 67 43 0.28 1.2 (0.007) 0.26 0.221 3 XT143/03 0.30 <0.01 (0.01) 132 123 0.27 1.7 0.030 0.65 0.122 3 XT161/03 0.21 <0.01 (0.02) 54 48 0.17 1.1 (0.004) 0.28 0.064 4 XT156/03 1.88 (0.02) 0.10 11 35 0.09 2.7 2.085 0.43 0.590 4 XT158/03 1.09 <0.01 (0.03) 495 43 0.07 37.9 1.524 0.82 0.772 4 XT159/03 1.01 <0.01 0.04 9 71 0.07 16.3 0.141 16.02 0.032

HERON EGGS Mean (mg/kg wet weight)

Group ID Al Cr Ni Cu Zn As Se Cd Hg Pb Besthorpe Egg A,B+E Tree 18 nest 1+5 and new 1 nest (0.03) <0.01 (0.01) 0.80 8.28 (0.03) (0.7) <0.003 0.12 0.015 Besthorpe Egg 2+4. Tree 23+22 nest 1 (dead chicks 2 in 2002) <0.02 <0.01 (0.01) 0.84 8.47 (0.02) 0.8 <0.003 0.07 0.024 Besthorpe Egg C,D+1. 3 Tree 11,16,33 nest 1+3 (0.06) <0.01 (0.01) 0.73 9.16 (0.01) 0.8 <0.003 0.14 0.016 Besthorpe Egg F+G Tree 4 20+27 nest 7+1. 0.13 <0.01 (0.01) 0.72 8.22 (0.03) 0.8 <0.003 0.18 0.011 Besthorpe Egg 3+5 Tree 5 22+4 nest 4+2. <0.02 <0.01 (0.02) 0.81 9.49 (0.03) 0.7 <0.003 0.16 (0.008) Cheshire A Green Wood 6 and C Pitts Heath <0.02 <0.01 (0.01) 0.84 10.34 0.04 0.8 <0.003 0.20 0.037

7 Tring 1, Tring 2 <0.02 <0.01 (0.01) 0.81 8.90 (0.03) 0.8 <0.003 0.30 (0.003)

8 Tring 3, Tring 4 <0.02 <0.01 (0.01) 0.75 8.50 (0.02) 0.9 <0.003 0.10 (0.004)

( ) below the limit of quantitation shaded cells show data where replicates were poor PCBs/dioxins Detailed data for the non-ortho-PCBs, ortho-PCBs and PCDDs/PCDFs summarised in Table 4 show that the nestlings euthanised due to problems other than deformities had the lowest levels of contaminants (expressed as total WHO-TEQ) with the highest levels in the birds euthanised due to the severity of their deformities (the concentrations of contaminants in fat were comparable to that of adult birds found dead in the area).

The control eggs from Cheshire had higher levels of PCBs/dioxins than the eggs from Tring and the highest levels of all samples (Table 4) suggesting that the Cheshire site is contaminated and therefore these data are excluded from any further comparisons. The eggs from the Besthorpe heronry showed 2-3 fold higher levels of PCBs/dioxins in eggs taken from nests that had contained deformed chicks in 2002 (pool 2) than from other nests in the heronry but the other eggs had similar levels of contaminants to the eggs from the CSG 15 (Rev. 6/02) 7 Project DEFRA title project code

Tring control site (there are insufficient control data for a statistical comparison) (Table 4). The profile of contribution of each of the classes of PCB and dioxin to the WHO-TEQ are shown in Figure 3. The major differences between the eggs for Pool 2 and the other eggs from the Besthorpe site were in the levels of ortho- and non-ortho-PCBs.

The non-ortho PCB congener profiles differed between the nestling fat samples, egg samples from Besthorpe and egg samples from Tring (Figure 4). This suggests that either PCB77 metabolism differs between adults (shown by the profile of the congeners in eggs) and nestlings or the nestlings are exposed to a differing congener profile at the Besthorpe site. As exposure of the adult birds determines the PCB/dioxin profile in eggs it is unsurprising that the profile in eggs from the control and Besthorpe sites are similar.

Although the absolute levels of PCBs/dioxins were lower in apparently unaffected nestlings than in dead and deformed nestlings the overall PCB congener profile did not differ (Figure 5). The congener profiles are similar from apparently unaffected nestlings (pool 1) and from deformed birds (pool 3) but the relative amounts of congeners differs in fat samples from adult birds (pool 4) although PCB138 and PCB153 remain the highest.

The PCB congener profiles are similar in the eggs pooled from the Besthorpe colony (pool 1 and pool 2 (deformed nestlings in 2002)) and the profiles only differ slightly from the pooled eggs from the control colony (Figure 5). Again the major congeners present are PCB138 and PCB153.

Table 4 PCB/Dioxin residues in fat and egg samples Besthorpe nestling fat samples Adult Besthorpe egg samples heron fat Sample Details: pool 1 pool 2 pool 3 pool 4 pool 1 pool 2 pool 3 pool 4 pool 5 Dioxins ng/kg whole 88 182 259 252 16 12 9 13 12 ng/kg fat 154 251 391 414 289 192 135 234 199 WHO-TEQ ng/kg fat 55 102 156 196 87 75 56 97 67

Non-ortho PCBs ng/kg whole 3651 6370 7198 3653 227 475 211 247 423 ng/kg fat 6339 8787 10857 5999 4035 7754 3088 4289 6906 WHO-TEQ ng/kg fat 137 217 304 341 219 547 172 215 251

Ortho PCBs ug/kg whole 1433 1764 4620 2067 274 214 214 221 254 ug/kg fat 2488 2433 6969 3395 4869 17580 3152 3842 4151 WHO-TEQ ng/kg fat 69 51 159 118 117 323 91 103 124

Total PCBs ug/kg whole 1437 1770 4627 2071 274 215 214 221 255 ug/kg fat 2495 2441 6980 3401 4873 17588 3155 3847 4158 WHO-TEQ ng/kg fat 205 268 463 459 337 870 263 318 375

Total WHO-TEQ ng/kg whole 150 268 410 399 24 58 22 24 27 Total WHO-TEQ ng/kg fat 260 370 619 655 423 945 320 415 442

Mean Besthorpe Sample Details: Cheshire nestling fat Mean Besthorpe Mean Tring egg pool 6 Tring egg samples (SE) Adult fat egg (SE) Pool 7 Pool 8 Dioxins ng/kg whole 111 8 13 176 (49) 252 13 (1) 11 (2) ng/kg fat 1542 117 152 265 (69) 414 210 (25) 135 (18) WHO-TEQ ng/kg fat 651 45 58 104 (29) 196 76 (7) 51 (6)

CSG 15 (1/00) 8 Project DEFRA title project code

Non-ortho PCBs ng/kg whole 1079 248 270 5740 (1071) 3653 317 (55) 259 (11) ng/kg fat 15015 3469 3154 8661 (1306) 5999 5214 (897) 3311 (157) WHO-TEQ ng/kg fat 992 188 194 219 (48) 341 281 (68) 191 (3)

Ortho PCBs ug/kg whole 458 181 216 2606 (1012) 2067 235 (12) 198 (17) ug/kg fat 19246 2536 2518 3963 (1503) 3395 6719 (2729) 2527 (9) WHO-TEQ ng/kg fat 590 90 86 93 (33) 118 152 (43) 88 (2)

Total PCBs ug/kg whole 459 181 216 2611 (1013) 2071 236 (12) 199 (17) ug/kg fat 19261 2540 2522 3972 (1504) 3401 6724 (2730) 2531 (9) WHO-TEQ ng/kg fat 1582 278 280 312 (77) 459 433 (111) 279 (1)

Total WHO-TEQ ng/kg whole 161 23 29 276 (75) 399 31 (7) 26 (3) Total WHO-TEQ ng/kg fat 2233 324 338 416 (106) 655 509 (111) 331 (7)

Figure 3 Contribution of dioxins, non-ortho and ortho-PCBs to the total WHO-TEQ

70.0

60.0

50.0 n o i t

u 40.0 Dioxin b i r

t non ortho-PCB n

o 30.0 ortho-PCB c

% 20.0

10.0

0.0

2 3 1 4 2 3 5 8

1 4 6 7 l l l l l l l l

l l l l o o o o o o o o o o o o o o o o o o o o o o o o p p p p p p p p

p p p p t t t t

g g g g a a a a g g g g g g g g F F F F g g g g e e e e e e e e n n n n n n n n o o o o n n n n o o o o r r r r o o o o r r r r e e r r e e r r e e e e e e e e H H H H H H H H H H H H

Figure 4 Non-ortho-PCB congener profile for nestling heron fat and eggs from the Besthorpe site and from eggs at a control site.

CSG 15 (1/00) 9 Project DEFRA title project code

8000.00

7000.00

6000.00

5000.00

t Heron fat a f

g 4000.00 Heron egg k / g

n Egg control 3000.00

2000.00

1000.00

0.00 PCB77 PCB81 PCB126 PCB169

Figure 5 PCB congener profile for eggs and fat samples for unaffected nestlings (Fat pool 1), deformed nestlings (Fat pool 3), adult birds (Fat pool 4), Eggs from Besthorpe (Egg pool 1), from nests with deformed nestlings in 2002 (Egg pool 2) and control eggs (Egg pool 7).

H e r o n F a t p o o l 1

3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0

CSG 15 (1/00) 10 Project DEFRA title project code

H e r o n F a t P o o l 3

3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0

H e r o n F a t P o o l 4

3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0

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H e r o n e g g p o o l 1

3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0

H e r o n e g g p o o l 2

3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0

H e r o n e g g p o o l 7

3 5 . 0 0

3 0 . 0 0

2 5 . 0 0

2 0 . 0 0

1 5 . 0 0

1 0 . 0 0

5 . 0 0

0 . 0 0

Discussion

Trace elements In all cases except lead, the levels of trace elements analysed in eggs from the Besthorpe site and from the control sites were similar suggesting that the levels detected were within normal background levels. In the absence of control data for nestling livers the data were compared with published data. There are no relevant published data available for toxic levels of aluminium and nickel in liver.

Arsenic Most living organisms normally contain measurable concentrations of arsenic, but except for marine biota, these are usually less than 1 mg/kg fresh weight. Arsenic is bioconcentrated by organisms, but not biomagnified in the food chain. The early developmental CSG 15 (1/00) 12 Project DEFRA title project code

stages are the most sensitive to arsenic and it is a known teratogen in several classes of vertebrates and abnormalities include skeletal anomalies.

Background levels in San Francisco Bay (Hui et al 2001, Ohlendorf et al 1986) ranged from 0.11-3.9 mg/kg dry weight (liver is approx 75% moisture content, i.e. 0.03-1.0 mg/kg wet wt) in the livers of a range of shorebirds. Shorebirds (seven species) wintering in the Corpus Christi, Texas, area contained an average of only 0.3 mg As/kg wet weight in livers (maximum of 1.5 mg/kg), despite the presence of smelters and the heavy use of arsenical herbicides and defoliants; these values probably reflect normal background concentrations (White et al 1980).

In this study only one nestling liver sample exceeded 0.3 mg/kg fresh weight and this did not exceed what are considered background levels.

Cadmium Cadmium can accumulate over a lifetime (Hui et al 2001) and one of the principal physiological effects of cadmium is to interfere with renal function (Nicholson and Osborn 1993) and are strongly correlated with lowered chick growth rates (Spahn and Sherry 1999). Vertebrates exposed to elevated levels of metals, particularly cadmium, also can experience enhanced zinc uptake (Doganoc 1996) and cadmium concentrates in the viscera of vertebrates, especially the liver and kidneys. In vitro cadmium has been shown to induce bone damage through osteoporosis, inhibition of calcification and inhibition of collagen synthesis. In vivo exposure to cadmium has been associated with eggshell thinning and chronic dietary exposure to cadmium may affect zinc, iron and calcium metabolism (Furness 1996).

A review of 32 studies showed concentration of cadmium (0.002-0.6 mg/kg wet wt, median 0.015 mg/kg) in eggs of raptors, seabirds and other fish eating birds which showed no adverse effects (Burger 2002). Background levels reported in shorebirds (Hui 2001) ranged from 0.87-33.9 mg/kg dry weight in liver (0.21-8.5 mg/kg wet wt) and in a range of avian species (0.1-32 mg/kg wet weight in liver (Furness 1996). Hontelez et al (1992) reported cadmium levels in grey heron liver in the Netherlands of 0.2-4.7 mg/kg dry wt. Cadmium concentrations in livers of breeding puffins were higher in two declining colonies of puffins in St. Kilda and Clo Mor (12.9- 22.3 mg/kg, dry weight, 3.3-5.6 mg/kg wet wt) than in colonies of puffins from other areas, or in livers of other seabirds examined (Parslow et al 1972).

In little blue herons (Egretta caerulea) chicks exposed to cadmium had significantly slower growth rates than non-exposed chicks (bone length used to calculate growth rate in this study). Of 31 eggs sampled only one contained cadmium in albumin or yolk (0.2 μg/g). Although some cadmium may be deposited in the eggs, chick tissue levels were determined primarily, if not exclusively by local food sources (Spahn and Sherry 1999).

In this study higher levels of cadmium (1-2 mg/kg wet weight) were detected in livers from adult birds than in nestlings (0.004-0.03 mg/kg wet weight) and the levels of cadmium were below those which are considered to have adverse effects.

Chromium Under laboratory conditions, chromium is mutagenic, carcinogenic, and teratogenic to a wide variety of organisms, and Cr 6+ has the greatest biological activity. A review of 21 studies of the concentration of chromium in unaffected birds ranged from 0.01-1.0 mg/kg wet wt (median 0.21 mg/kg) in eggs of raptors, seabirds and other fish eating birds (Burger 2002). In this study all residues were well below those reported as background.

Copper Background levels reported in shorebirds (Hui et al 2001) range from 14-129 mg/kg dry weight (3.5-32 mg/kg wet wt) in shorebirds in San Francisco bay. In this study levels in nestling birds were 30-132 mg/kg wet weight liver. Levels in adult birds were up to 495 mg/kg wet weight. Both these levels are above usual background levels which is probably associated with the proximity of coal fired power stations (Llacuna et al 1995).

Lead Lead is deposited in hard tissues that are difficult to digest, such as bone, so it has a low biomagnification potential (Henny et al 1991). Lead has been shown to have adverse effects on vitamin D and calcium metabolism and growth under some circumstances (Hart et al 1991). In little blue herons (Egretta caerulea) exposure to lead correlated with increased nestling mortality (Spahn and Sherry 1999). Of 31 eggs sampled only two contained lead in albumin or yolk (1.2 + 1.6 mg/kg). Burger (2002) reviewed 29 studies of background concentration of lead 0.02-6.7 mg/kg wet wt (median 0.190) in eggs of raptors, seabirds and other fish eating birds. In this study the maximum lead residue in the Besthorpe eggs was 0.024 mg/kg (and in a control egg pool from Cheshire was 0.037 mg/kg) which is within the levels considered as background.

Ohlendorf et al (1986) reported background levels in American coot liver of 0.21-0.31mg/kg dry wt (0.05-0.8 mg/kg wet wt) and Hontelez et al (1992) reported levels of 0.4-2.1 mg/kg dry wt (0.1-0.5 mg/kg wet wt) in grey heron livers in the Netherlands. The Patuxent Wildlife Research Centre (http://www.pwrc.usgs.gov/cgi-bin) and Franson (1996) provides threshold values for effects of elevated lead as >2 mg/kg fresh wt liver, and poisoned > 5 mg/kg fresh wt liver in birds. The residues detected in nestling livers in

CSG 15 (1/00) 13 Project DEFRA title project code

this study were 0.021-0.221 mg/kg wet weight and in adult liver were up to 0.772 mg/kg which are below the identified threshold levels although they may be high enough to affect vitamin D and calcium metabolism (Hart et al 1991, Pain 1996, Franson 1996) .

Mercury Mercury is primarily a neurotoxin and has been shown to impair reproduction when concentrations in livers were 3.0mg/kg dry weight.(Hui et al 2001, Fimreeite 1971, Heinz 1979) and mercury levels of 1mg/kg are known to decrease hatchability and to produce behavioral effects in hatchlings (Eisler 1987, Conners et al 1975). Great white herons that died from acute causes had less mercury in their livers 0.6 - 4.0mg/kg than did those that died from chronic diseases (Erwin and Custer 2000, Spalding et al 1994). Mortality from chronic diseases in great white herons was associated with mercury concentrations in livers that exceeded 6mg/kg (wet weight), (Erwin and Custer 2000). Background levels were reviewed in 68 studies by Burger (2002) and ranged from 0.070-7.92 mg/kg wet wt (median 0.34) in eggs of raptors, seabirds and other fish eating birds and 0.25 mg/kg in eggs has been suggested as a background level in birds (King et al 1991, Allen et al 1998)

The levels of mercury in many of the eggs in this study were below the 0.25 mg/kg identified background levels (max 0.18 mg/kg wet weight from Besthorpe) although one of the control egg pool’s had levels of 0.30 mg/kg.

The levels of mercury in nestling livers range from 0.27- 0.68 mg/kg but one adult bird contained 16 mg/kg wet weight in liver. The highest levels in nestlings were in those found dead in the nest suggesting both nestling and adult birds may be affected. These levels of mercury may be associated with the proximity of coal burning power stations (Thompson 1996, Llacuna et al 1995).

Selenium In wild aquatic birds selenium exposure has been associated with mortality, impaired reproduction with teratogenesis, reduced growth, histopathological lesions and alterations in hepatic glutathione (Hoffman 2002). For several species of aquatic birds, levels of selenium as selenmethionine in the egg above 3mg/kg wet wt result in reduced hatchability and deformed embryos (Spallholz and Hoffman 2002). Background levels reported in shorebirds and aquatic birds ranged from 2.42-20.5 mg/kg dry weight in eggs (Hui et al 2001, Ohlendorf et al 1988, Ohlendorf et al 1986, Fairbrother et al 1994). Concentration of selenium in eggs of raptors, seabirds and other fish eating birds in 13 studies reviewed by Burger (2002) showed 0.3-7.1 mg/kg wet wt (median 1.1 mg/kg). Selenium residues in heron eggs were lower than those reported in mallards fed the same amount of selenium, suggesting that herons may be less sensitive to the effects of selenium than mallards (Smith et al 1988).

In this study levels in eggs were all less than 1mg/kg wet weight suggesting these were background levels.

Control levels in mallards were 5.1 ± 0.3 mg/kg dry wt in liver (1.3 ± 0.1 mg/kg wet wt) (Ohlendorf et al 1986). Background levels reported in shorebirds and aquatic birds ranged from 3.9-31.5 mg/kg dry weight in liver (1.0-8.0 mg/kg wet wt) (Hui et al 2001, Ohlendorf et al 1988, Ohlendorf et al 1986, Fairbrother et al 1994). Reduced growth in ducklings (15mg/kg Se wet wt in liver), diminished immune function in adults (5mg/kg Se in liver) and histopathological lesions (29 mg/kg Se in liver) were observed in mallards (Heinz et al 1989).

In this study the levels in nestling livers ranged from 1-3.7 mg/kg wet weight and in adult livers from 2.7 to 37.9 mg/kg wet weight. The majority of the nestlings had low levels of selenium but one nestling (3.7 mg/kg) had levels close to those that may cause adverse effects and levels in two adults (16 and 37 mg/kg) may be sufficient to result in adverse effects. These levels may be associated with the proximity of coal burning power stations (Thompson 1996, Llacuna et al 1995).

Zinc Zinc deficiency in the chicken, turkey, and Japanese quail is characterized by low survival, reduced growth rate and food intake, poor feathering, shortening and thickening of long bones of legs and wings, reduced egg production and hatchability, skeletal deformities in embryos, an uncoordinated gait, reduced bone alkaline phosphatase activity, and increased susceptibility to infection (http://www.pwrc.usgs.gov/cgi-bin). Ohlendorf et al (1986) reported background levels in American coot of 110-120 mg/kg dry wt in liver (27-30 mg/kg wet weight).

In this study levels of zinc in nestling liver were 30-123 mg/kg wet weight and in adults were 35-71 mg/kg wet weight suggesting levels were not well below background. The apparently higher levels may be due to deposition from the local power stations (Llacuna et al 1995).

In summary some trace elements, mercury, lead, copper and selenium were close to or above background levels in the nestling or adult liver samples and this was probably related to the proximity of the power plants to the heronry (Llacuna et al 1995). The levels could not be directly related to the deformities in the herons but they may contribute to an effect.

PCBs/dioxins The profile of PCBs in the heron fat and egg samples are typical of those for fish -eating birds including other species of heron and seabirds such as cormorants (Aurigi et al 2000). Thus PCB153 is the dominant PCB congener and PCB77 and PCB126 are the dominant non-ortho congeners. The data cannot be readily compared with those from the National Monitoring Scheme supplied by CEH due to major differences in analytical techniques which suggested, in contrast to all other published data, that PCB169 is the CSG 15 (1/00) 14 Project DEFRA title project code

major non-ortho-congener. This may be due to inability to separate co-eluted isomers due to the methods used, e.g. electron capture detector rather than mass spectrometer.

The PCB/dioxin residue data show that the nestlings which were euthanised due to the severity of deformity had higher levels of PCBs/dioxins in their fat deposits than birds from the same colony that were not deformed; the levels were similar to those in adult birds from the same area. The total mass of PCBs in nestling herons is known to increase between 5 and 15 days post-hatching in black-crowned night herons (Erwin and Custer 2000), due to intake of contaminated prey whilst metabolism is presumably limited. However, the mean mass of the sampled nestlings (Group 1 (undeformed) 893g, Group 2 (dead) 1077g and Group 3 (deformed) 1014g, Group 4 (adult) 1396 g) does not explain the increases; this would suggest Group 3 levels should be lower than Group 2. Assuming the fat concentrations (whole weight basis) to be an average of the total body burden (a gross assumption that allows the data to be normalised) the total WHO-TEQ can be calculated on a body burden basis giving 134 ng for Group 1, 289 ng for Group 2, 416 ng for Group 3 and 557 ng for Group 4 birds.

The levels of PCBs/dioxins in eggs from nests which had contained nestlings with deformities in 2002 were also far higher than levels in other eggs from other nests in the colony and from the Tring control site. It is known that there is little variation in contaminant levels between eggs in the same clutch and there is also no loss of contaminant mass from night heron eggs to chicks (Erwin and Custer 2000). This suggests that the female birds in these nests had been exposed to significantly higher levels of non-ortho and ortho- PCBs than other females in the same heronry. There is even greater concern raised by the “control” egg sample from Cheshire which contained a total WHO-TEQ of 2233 ng/kg fat.

The significance of PCB/dioxin residues have not been well defined in birds (De Luca-Abbott et al 2001) as many studies report the total PCB content (which is dependent on which, and how many, of the 209 congeners are quantified). However, studies have detected effects of PCBs/dioxins on egg production and embryo mortality in birds (Table 5). Chickens have been shown to be amongst the most sensitive species with short bowed legs, clenched toes and neck deformities in chicks hatched from PCB exposed eggs (congeners not specified) (Hoffman et al 1996). There are wide interspecies variations in sensitivity to PCBs and dioxins in birds with chickens the most sensitive (Peterson et al 1993) whereas mallards and pheasants are less sensitive to reproductive effects, although eggshell thinning has been observed (Hoffman et al 1996). Sanderson and Bellward (1995) showed that the receptor binding affinity of TCDD to the hepatic cytosolic Ah receptor was 15 fold greater in the chicken but the pigeon, heron and cormorant were similar (and were similar to the human placenta) suggesting that the latter 3 species may be similar in sensitivity. Peakall and Peakall (1973) showed that PCBs may affect the behaviour of parent birds as well as hatchability of eggs. Ringed turtle doves fed with Arochlor 1254 (a PCB mixture which contains 54% chlorine by weight) showed increased embryonic mortality when eggs were incubated by the parents but not when they were artificially incubated, suggesting decreased parental attentiveness.

Comparison of the published data for the non-ortho-PCBs 77, 126 on a lipid basis with data from eggs collected from control and contaminated sites shows similar profiles (Table 5). The WHO-TEQ data are less readily compared as it depends on the numbers of congeners analysed. However, the data are in the same order apart from those reported by Aurigi et al (2000) for the Danube delta where the data are well below other reported data for control sites. The WHO WHO-TEQs are established for humans and are less reliable for birds as they are more sensitive than mammals to PCB77 (Hoffman et al 1996) and PCB138 makes a greater contribution in birds than in mammals due to a combination of its abundance and its potency (Kennedy et al 1996) (PCB138 is one of the most abundant ortho-PCBs after PCB153, which is found in many fish-eating birds). The induction equivalency factors on which the WHO WHO-TEQ is based (together with metabolism data) are shown in Table 6. This shows there are a number of PCB congeners which induce P4501A in chick hepatocyte cultures at levels well below those for rat hepatocytes, i.e. they have far greater potency. The levels of PCB77 in the nestling fat are well above those in eggs, which are similar in profile to the adult fat sample and are deposited in eggs from in adult liver, it appears that the nestlings at Besthorpe have levels of accumulated PCBs which may be associated with effects reported in other studies.

Some of the best documented studies linking PCBs to avian reproductive failure have been documented in studies relating to the Great Lakes embryo mortality, edema, and deformity syndrome (GLEMEDS) (Gilbertson et al 1991, Grasman et al 1998). Hatching success in Lake Ontario herring gulls was 60% less than controls and subcutaneous oedema, impaired bone growth and congenital anomalies were present in hatchlings. The residues were also correlated with the total time eggs were unattended at the nest by the parent.

The differences between the PCB/dioxin levels detected in fat from nestlings which had no deformities, those found dead in the nest with deformities and those euthanised due the severity of their deformities suggest that these contaminants may be related to the deformities detected. The additional differences between these contaminants in eggs collected from control nests and from nests unaffected in 2002 when compared with eggs collected from those with deformed chicks in 2002 further supports the theory. The very high levels of PCBs/dioxins in the pooled control sample from Cheshire is difficult to interpret as there is no evidence of deformities in this colony but the levels are above those reported to result in embryo death due to deposition in the egg (Table 7).

Table 5 Comparison of residue data from Besthorpe and Tring (control) heronry with published data for heron species

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Thomas and Anthony 1999 Aurigi et Sanderson Custer et al 1997 al 2000 et al 1994 Besthorpe

ng/kg Heron* Heron Egg Control Contaminated Control Contaminat Contaminated egg lipid fat egg control egg egg egg ed egg 4306- 819- 667- 800-4400 1000-11800 78 2400-70000 PCB77 6730 2099 1118 1344- 1694- 1861- 2400- 6400-34200 54 5860 ± 620-42000 PCB126 2960 5397 1911 6800 1080 PCB169 167-714292-724182-321400-1200 600-3400 2.8 960 ± 120 0-4400 Dioxin WHO- 66-340 108-720 0-4180 TEQ 55-156 56-97 45-58 PCB WHO- 240-480 300-6760 12.5 480-38060 TEQ 205-463263-870278-280 Total WHO- 340-680 900-6880 5560 ± TEQ 260-619320-945323-338 1040 * nestling only

Table 6 Differences between the chick and rat hepatocyte derived induction equivalency factors (IEF) (2,3,7,8 TCDD =1) for PCB congeners and effect on potency (after Kennedy et al 1996) Congener IEF Chick IEF Rat Potency hepatocytes chick/rat 77 0.03 0.0009 33 81 0.2 0.00004 5000 126 0.3 0.3 1 169 0.02 0.003 7 105 0.005 0.0007 7 118 0.001 0.000009 111 156 0.001 0.00009 11 157 0.002 0.00006 33

Table 7 Summary of PCB and TCDD effect levels (Hoffman et al 1996). Concentration Effect 20-50 ng/kg TCDD in eggs Embryo mortality and teratogenesis in chickens, decreased productivity and teratogenesis in wood ducks 90-399 ng/kg TCDD equivalent in egg Embryotoxicity in Forster’s tern 150-250 ng/kg TCDD in eggs Decreased embryonic growth, oedema in herons 618-7366 ng/kg TCDD equivalent in eggs Embryotoxicity in Forster’s tern 1000 ng/kg TCDD in eggs Embryo mortality in pheasants <10,000 ng/kg TCDD in eggs Embryo mortality in bluebirds 1-5 mg/kg total PCBs in eggs Decreased hatching success for chickens 8-25 mg/kg total PCBs in eggs Decreased hatching success for terns., cormorants, doves, eagles 75-300 mg/kg total PCBs in brain Lethality in great cormorants, gulls, passerines, pheasants

The link between TCDD WHO-TEQs and bone deformities may be suggested by studies reported by Hart et al (1991) and Sanderson et al (1994). They determined the residues of PCDDs and PCDFs in great blue heron (Ardea herodias) eggs near a Kraft pulp mill in British Columbia, Canada after a colony failed to fledge young. They showed a negative correlation between TCDD equivalents (WHO-TEQ) in eggs from the same clutch with plasma calcium, wet, dry and ash weight and length of the tibia and beak length in the hatched chicks. Unfortunately in all these studies chicks were sacrificed for morphology measurements at 24hrs old and therefore the longer term effects, e.g. of decreased plasma calcium were not determined. The observation of depressed plasma calcium was suggested to be linked to decreased skeletal growth due to insufficient calcium for mineralization resulting from decreased calcium mobilisation from the shell across the chorioallantoic membrane. Calcium levels in avian plasma are modulated by peptide and

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steroid hormones. In the short term (minutes) parathyroid hormone stimulates bone resorption (increasing mobilisation from bone to plasma) and calcitonin inhibits bone resorption. In the longer term changes in intestinal calcium absorption and bone balance are affected by 1,25-dihydrovitamin D, the active form of vitamin D, modulated by parathyroid hormone (Hurwitz 1996). Thus during egg production female birds increase synthesis of 1,25-dihydrovitamin D and calcium absorption increases to accommodate the needs for eggshell deposition (Williams et al 1991). Shell calcium mobilisation is also Vitamin D dependent. The consistent observation in PCDD exposed birds of hepatic EROD (a cytochrome P450 dependent enzyme) activity induction (Hart et al 1991, Rattner et al 1994, Sanderson et al 1994) may reduce activation of vitamin D to 1,25-dihydrovitamin D by two other P450 dependent enzyme systems and thus affect the levels of plasma calcium (Hart et al 1991). TCDD dosing has been shown to delay ossification in mammalian embryos (Sparschu et al 1971, cited by Hart et al 1991) and reduce calcium deposition in bone (Allen and Leamy 2001). Lead has also been shown to affect vitamin D and calcium metabolism in some cases (see above).

The link between the PCB/dioxin residues in eggs and nestlings and changes in calcium deposition is supported by the plasma calcium measurements (Figure 5) and the eggshell thickness data for the Besthorpe heronry (Figure 6). These showed that plasma calcium levels (both raw data and adjusted for protein levels) were significantly lower in plasma sampled from deformed birds than from non-deformed birds at Besthorpe. The increased ALP activity in deformed birds may be an indication of reduced ossification of bone but should be treated with caution as ALP activity is highly age-dependent (Kertesz and Hlubik 2002) and the exact age of the sampled nestlings is unknown. The eggshell thickness measurements show that eggshells sampled from Besthorpe were significantly thinner (p<0.05) than those from the control sites (the four thinnest eggshells were from addled eggs collected from the Besthorpe colony). It is known that eggshell thickness does not change with stage of incubation in herons (Erwin and Custer 2000). However, the eggshells from the control Cheshire colony which showed the highest levels of total PCBs/dioxins did not show thinning suggesting the relationship is not straightforward and may be related to levels of particular congeners. Additional analysis is being undertaken to confirm that the thinning is not due to DDE levels.

Figure 5 Plasma chemistry results for deformed and non-deformed (control) birds from Besthorpe and for two control sites (Greenwood and Great Budworth). Asterisks show data significantly different to control data from Besthorpe (p<0.05)

20.00

18.00

16.00

14.00

12.00 Besthorpe control Besthorpe deformed 10.00 Greenwood control Gt Budworth control 8.00 *

6.00 *

4.00 * *

2.00

0.00 K Ca Ptn/10 adj Ca AP/100 AST/100 CK/1000

Figure 6 Eggshell thickness measurements for the eggs collected from Besthorpe and from control sites (measurements are the mean of 4 measurements around the mid-point of the egg).

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0.64

0.62

0.60

0.58

Besthorpe control

0.56

0.54

0.52

0.50

Possible sources of contamination The presence of fly-ash lagoons which were used until the 1970s (the site is in an area which formerly had many coal-fired power stations, e.g. Cottam, High Marnham) may be a source of dioxins and PCBs leaching into areas where the herons feed. However, the area of the affected heronry at Besthorpe is also within the flood plain of the river Trent and its tributaries. Higher levels of PCB/dioxins were found in magpies (Pica pica) feeding on the flood plains of major rivers in the Netherlands than in those feeding elsewhere (Canters and DeSnoo 1993). The need of the grey heron to find small prey for nestlings, and their diverse diet, e.g. earthworms, frogs, eels, rodents, ducklings and fish, means that they are likely to forage in marginal shallower water (Moser 1986). As the majority of grey herons are territorial in their feeding and have a single feeding site (Marion 1989) it is possible that either some of these are contaminated or the affected birds are those that are non-territorial and are feeding further from the colony or on poorer sites. PCBs/dioxins may not only be accumulated in fish but have also been shown to accumulate in earthworms from arable land (Matcheko et al 2002). This may explain the relatively high levels of PCB77 in nestling fat as earthworms accumulate PCB126 and PCB169 less than other congeners (Matcheko et al 2002). Further work is required to identify where the parent birds of the affected nestlings are feeding and identify the source of the pollution, e.g. by sampling food sources such as fish, earthworms, (directly from areas and as regurgitates from nestlings), soil and water in the vicinity of the colony and further sampling to determine individual variations in residue levels and correlation with deformities.

Conclusions

There are a number of observations in this study that support the hypothesis that the deformities in the heron nestlings are due to reduced calcium deposition in bone and suggests that PCBs/dioxins are associated with these effects.

1. The levels of calcium in plasma were lower in deformed than in apparently normal nestlings in the same colony and there was significant eggshell thinning in eggs from the Besthorpe colony when compared to eggs from other sites. 2. Concentrations of PCBs/dioxins expressed as total WHO-TEQ (TCDD equivalents) were higher in deformed nestlings and in nestlings found dead than those apparently unaffected. 3. The WHO-TEQ was also higher in eggs from nests that contained deformed nestlings in 2002 (this assumes birds are nest- loyal from year to year) than from other nests in the Besthorpe colony or the control Tring colony. 4. TCDD may affect vitamin D dependent calcium mobilisation from the shell across the chorioallantoic membrane thus result in insufficient calcium for mineralization and TCDD has also been observed to impact on calcium deposition in bone. A negative correlation has been reported between TCDD levels in eggs with plasma calcium, tibia length, wet, dry and ash weight in the hatched great blue heron chicks. Lead has also been shown to affect vitamin D and calcium metabolism in some cases. Lead levels in nestlings were higher in the deformed than in apparently unaffected nestlings. 5. The levels of PCBs/dioxins in heron nestlings were sufficiently high to suggest that this is the underlying cause of the deformities. The eggs from Cheshire colonies also raise concerns due to their very high residue levels of PCBs/dioxins

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suggesting the problem is not localised. Further work is required to identify the source of the pollution, e.g. sampling fish and other food sources (the diet of grey herons is diverse) in the vicinity of the colony and further sampling to determine individual variations in residue levels and correlation with deformities. The diverse diet of herons means that a number of food sources should be considered, e.g. earthworms due to the location of the site and other species using similar food sources may be affected.

References Allen, G.T., Blackford, S.H., Welsh, D. (1998) Arsenic, mercury, selenium and organochlorines and reproduction of interior least terns in the northern great plains, 1992-1994. Colonial Waterbirds. 21 (3) 356-366 Allen D.E., Leamy, L.J. (2001) 2,3,7,8-tetrachlorodibenzo-p-dioxin affects size and shape but not asymmetry of mandibles in mice Ecotoxicology 10 167-176 Ambidge, P.F., Cox, E.A., Creaser, C.S., Greenberg, M., Gem, M.G. deM., Gilbert, J., Jones, P.W., Kibblewhite, M.G., Levey, J., Lisseter, S.G., Meredith, T.J., Smith, L., Smith, P., Startin, J.R., Stenhouse, I., Whitworth, M. (1990) Acceptance criteria for analytical data on polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans Chemosphere 21 999-1006 Aurigi, S., Focardi, S., Hulea, D., Renzoni, A. (2000) Organochlorine contamination in bird’s eggs from the Danube delta Environ Poll 109 61-67 Blackburn, A., Kent, J. (1999) Problems at a heron colony BTO News 224 2-3 Boumphrey, R.S., Harrad, S.J., Jones, K.C., Osborn, D. (1993) Polychlorinated biphenyl congeners in tissues from a selection of British birds Arch. Environ. Contam. Toxicol 25 346-352 Burger, J., (2002) Food chain differences affect heavy metals in bird eggs in Barnegat bay, New Jersey. Elsevier Science, Environmental Research Section. A 90 33-39 Canters, K.J., DeSnoo, G.R. (1993) Chemical threat to birds and mammals in the Netherlands Rev Environ Contam Toxicol 130 1- 29 Conners, P.G., Anderlini, V.C., Risebrough, R.W., Gilbertson, M.J., Hays, H. (1975) Investigations of heavy metals in common tern populations. Canadian Field-Naturalist. 89 157-162. Custer, T.W., Hines, R.K., Melancon, M.J., Hoffman, D.J., Wickcliffe, J.K., Bickman, J.W., Martin, J.W., Henshel, D.S., (1997) Contaminant concentrations and biomarker responses in great blue heron eggs from 10 colonies in the upper Mississippi river USA Environ Contam Toxicol 16 260-271 De Luca-Abbott, S.B., Wong, B.S.F., Peakall, D.B., Lam, K.S., Young, L., Lam, M.H.W., Richardson, B. (2001) Review of effects of water pollution on the breeding success of waterbirds, with particular reference to Ardeids in Hong Kong. Ecotoxicology 10 327-350 Doganoc, D. (1996) Distribution of lead, cadmium and zinc in tissues of hens and chickens from Slovenia. Bull. Environ. Contam. Toxicol. 57 932-937. Eisler, R., (1987) Mercury hazards to fish, wildlife and invertebrates: a synoptic review. US Department of the Interior, Biological Report. 85 (1.10) Washington, DC Erwin, R.M., Custer, T.W., (2000) Herons as indicators. In. Heron Conservation. ed Kushlan. J. A., Hafner, H., Academic press. 312-330. Fairbrother, A., Fix, M., Ohara, T., Ribic, CA., (1994) Impairment of growth and immune function of Avocet chicks from sites with elevated selenium, arsenic and boron. J. Wildl. Manage. 30 (2) 222-233. Fairbrother, A., Brix, K.V., Toll, J.E., McKay, S., Adams, W.J. (1999) Egg selenium concentrations as predictors of avian toxicity Human and Ecol Risk Assessment 5 1229-1253 Fernandes, A., Foxall, C., Lovett, A., Rose, M., White, S. (2002) The effects of river flooding on PCDD/F and PCB levels in cows milk Organohalogen Compounds 58 37-40 Fimreeite, N. (1971) Effects of dietary methylmercury on ring-necked pheaseants. can Wildl. serv. Occ. Pap. 9. Franson, J.C. (1996) Interpretation of tissue lead residues in birds other than waterfowl In: Environmental contaminants in wildlife: Interpreting tissue concentrations (eds W.N. Beyer, G.H. Heinz, A.W. Redmon-Norwood) Lewis Publishers, Boca Raton, USA, 265-279 Furness, R.W. (1996) Cadmium in birds In: Environmental contaminants in wildlife: Interpreting tissue concentrations (eds W.N. Beyer, G.H. Heinz, A.W. Redmon-Norwood) Lewis Publishers, Boca Raton, USA, 389-404 Gilbertson, M., Kubiak, T., Ludwig, J., Fox, G. (1991) Great Lakes Embryo Mortality, Edema and Deformities Syndrome (GLEMEDS) in colonial fish-eating birds: Similarity to chick-edema disease J Toxicol Environ Health 33 455-520 Grantham, M. (2000) Increased mortality at a North Nottinghamshire Grey Heron Colony, Part 1 Analysis of historical data. Report for the Nottinghamshire Wildlife Trust Grasman, K.A., Scanlon, P.F., Fox, G.A. (1998) Reproductive and physiological effects of environmental contaminants in fish- eating birds of the Great Lakes: A review of historical trends Environ Monitoring and Assessment 53 117-145 Hart, L.E., Cheng, K.M., Whitehead, P.E., Shah, R.M., Lewis, R.J., Ruschkowski, S.R., Blair, R.W., Bennett, D.C., Bandiera, S.M., Norstrom, R.J., Bellward, G.D., (1991) Dioxin contamination and growth and development in great blue heron embryos. J. Toxicol and Env. Health. 32 331-344. Heinz, G. H. (1979) Methylmercury: Reproduction and behavioral effects on three generations of mallard ducks. J.Wildl. Manage. 43 394-401. Heinz, G.H., Hoffman, D.J., Gold, L.G. (1989). Impaired reproduction of mallards fed an organic form of selenium. J.Wildl. Manage. 53 (2). 418-428 Henny, C. J., Blus, L.J., Hoffman, D. J.,K Grove, R.A., Hatfield, J.S. (1991) Lead accumulation and osprey production near a CSG 15 (1/00) 19 Project DEFRA title project code

mining site on the Coeur d'Alene River, Idaho. Archives Environmental Contamination Toxicology. 21 415-424 Hernandez, L.M., Gomara, B., Fernandez, M., Jimenez, B. et al (1999) Accumulation of heavy metals and As in wetland birds in the area around Donana National Park affected by the Aznalcollar toxic spill Sci Total Environ. 242 293-308 Hoffman, D.J., Rice, C.P., Kubiak, T.J. (1996) PCBs and dioxins in birds In: Environmental contaminants in wildlife: Interpreting tissue concentrations (eds W.N. Beyer, G.H. Heinz, A.W. Redmon-Norwood) Lewis Publishers, Boca Raton, USA, 165- 207 Hoffman, D. J. (2002) Role of Selenum Toxicity and Oxidative Stress in Aquatic Birds. Aquatic Tocicology 57 11-26 Hontelez, L.C.M.P., van den Dungen, H.M., Baars, A.J. (1992) Lead and cadmium in birds in the Netherlands: A preliminary survey Arch. Environ Contam Toxicol 23 453-456 Hui, C. A., Takekawa, J. Y., Warnock, S.E., (2001) Contaminant profiles of two species of shorebirds foraging together at two neighbouring sites in south San Francisco Bay, California. Environmental Monitoring and Assessment. 71. 107-121 Hurwitz, S. (1996) Homeostatic control of plasma calcium concentration Crit. Rev in Biochem and Mol Biol 31 (1) 41-100 Kennedy, S.W., Lorenzen, A., Norstrom, R.J. (1996) Chick embryo hepatocyte bioassay for measuring cytochrome P4501A-based 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent concentrations in environmental samples Environ. Sci. Technol. 30 706- 715 Kertesz, J., Hlubik, I. (2002) Plasma ALP activity and blood PCV value changes in chick fetuses due to exposure of the egg to different xenobiotics Environ Poll 117 323-327 King, K.A., Custer, T.W., Quinn, J.S. (1991) Effects of mercury, selenium, and organochlorine contaminants on reproduction on Fosters terns and black skimmers nesting in a contaminated Texas bay. Arch. Contam. Toxicol. 20. 32-40. Krokos, F., Creaser, C.S., Wright, C. Startin, J.R. (1997) Congener specific method for the determination of ortho and non-ortho polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in food by carbon column fractionation and gas chromatography-isotope dilution mass spectrometry Fresenius Journal of Analytical Chemistry 357 732-742 Llacuna, S., Gorriz, A., Sanpera, C., Nadal, J. (1995) Metal accumulation in 3 species of passerine birds (Emberiza cia, Parus major and Turdus merula) subjected to air pollution from a coal fired power plant Arch. Environ Contam Toxicol 28 298- 303 Marion, L. (1989) Terrestorial feeding and colonial breeding are not mutually exclusive: the case of the grey heron (Ardea cinerea) 58 693-710 Matcheko, N., Tysklind, M., de Wit, C., Bergek, S., Andersson, R., Sellstrom, U. (2002) Application of sewage sludge to arable land – soil concentrations of polybrominated diphenyl ethers and polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls, and their accumulation in earthworms Environ Toxicol and Chem 21 (12) 2515-2525 Moser, M.E. (1986) Prey profitability for adult grey herons Ardea cinerea and constraints on prey size when feeding nestlings Ibis 128 392-405 Nicholson, J., Osborn, D. (1993) Kidney lesions in pelagic seabirds with high tissue levels of cadmium and mercury. J. Zool. London 200 99-118 Ohlendorf, H.M., Hoffman, D.J., Saiki, M.K., Aldrich, T.W. (1986) Embryonic mortality and abnormalities of aquatic birds - Apparent impacts of selenium from irrigation drainwater. Sci.Tot. Environ. 52 (1-2) 49-63. Ohlendorf, H.M., Kilness, A.W., Simmons, J.L., Stroud, R.K., Hoffman, D.J., Moore, J.F. (1988) Selenium toxicosis in wildaquatic birds. J. Toxicol. Environ. Health. 24 67-92 Ohlendorf, H.M. (1999) Selenium was a time bomb Human and Ecol Risk Assessment 5 1181-1185 Pain, D.J. (1996) Lead in waterfowl In: Environmental contaminants in wildlife: Interpreting tissue concentrations (eds W.N. Beyer, G.H. Heinz, A.W. Redmon-Norwood) Lewis Publishers, Boca Raton, USA, 251-264 Parslow, J. L. F., Jefferies, D. J., French, M. C. (1972) Ingested pollutants in puffins and their eggs. Bird Study 19 18-33 Peakall , D.B., Peakall, M.L. (1973) Effects of a polychlorinated biphenyl on the reproduction of artificially and naturally incubated dove eggs J. Appl. Ecol. 10 863-868 Peterson, R.E., Theobald, H.M., Kimmel, G.L. (1993) Developmental and reproductive toxicity of dioxins and related compounds: Cross-species comparisons Crit Rev. Toxicol 23 (3) 283-335 Rattner, B.A., Hatfield, J.S., Melancon, M.J. (1994) Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping blck-0crowned hight-heron embryos Environ Toxicol Chem 13 1805-1812 Rodriguez, M.P. (2001) A study of mortality and bone disease in chicks at Besthorpe Grey Heron (Ardea cinerea) Colony. MSc Project Report University of London Sanderson, J.T., Bellward, G.D. (1995) Hepatic microsomal ethoxyresorufin O-deethylase-indusing potenct in ovo and cytosolic Ah receptor-binding affinity of 2,3,7,8-tetrachlorodibenzo-p-dioxin – comparison of 4 avian species Toxicol Appl. Pharmacol 132 131-145 Sanderson, J.T., Elliott, J.E., Norstrom, R.J., Whitehead, P.E., Hart, L.E., Cheng, K.M., Bellward, G.D. (1994) Monitoring biological effects of polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in great blue heron chicks (Ardea herodias) in British Columbia J. Toxicol. Environm. Health 41 435-450 Smith G. J., Heinz, G. H., Hoffman, D. J., Spann, J. W., Krynitsky, A. J., (1988) Reproduction in black-crowned night-herons fed selenium. Lake Reservoir Manage. 4, 175-180 Spahn, S.A., Sherry, T.W. (1999) Cadmium and lead exposure associated with reduced growth rates, poorer fledging success of little blue heron chicks (Egretta caerulea) in south Louisiana wetlands Arch Environ Contam Toxicol 37 377-384 Spalding, M.G., Bjork, R.D., Powell, G.V.N., Sundlof, S.F., (1994) Mercury and the cause of death in great white herons. J. Wildl. Manage. 58 (4) 735-739

CSG 15 (1/00) 20 Project DEFRA title project code

Spallholz, J.E., Hoffman, D.J. (2002) Selenium toxicity: causes and effects in aquatic birds Aquatic Toxicol 57 27-37 Thomas, C.M., Anthony, R.G. (1999) Environmental contaminants in great blue herons (Ardea herodias)from the lower Columbia and Willamette rivers, Oregon and Washington, USA Environ. Toxicol and Chem 18 2804-2816 Thompson, D.R. (1996) Mercury in birds and terrestrial mammals In Environmental contaminants in wildlife: Interpreting tissue concentrations (eds W.N. Beyer, G.H. Heinz, A.W. Redmon-Norwood) Lewis Publishers, Boca Raton, USA, 341-356

Van Leeuwen, F.X.R., Younes, M.M. (2000) Assessment of the health risk of dioxins: re-evaluation of the tolerable daily intake (TDI) Food Additives and Contaminants 17 (4) 223-369

White, D. H., King, K. A, Prouty, F. M., (1980) Significance of organochlorine and heavy metal residues in wintering shorebirds at Corpus Christi, Texas, 1976-77. Pestic. Monitor. J. 14 58-63.

Williams, D.C., Paul, D.C., Herring, J.R. (1991) Effects of antiestrogenic compounds on avian medullary bone formation J. Bone and Mineral Res. 6 (11) 1249-1256

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