ICES ASC 2008/D:03

Theme Session D: New trends in diseases of marine organisms: causes and effects

- Not to be cited without prior reference to the authors -

Hyperpigmentation in dab ( limanda): spatial and temporal patterns, host effects and possible causes

Felix Baumgart 1,2, Thomas Lang 2, Stephen Feist 3, David Bruno 4, Patricia Noguera 4 and Werner Wosniok 5

1 Department of Biological Sciences, Zoology, University of Rostock, Universitätsplatz 2, 18055 Rostock, Germany, E-mail: [email protected]

2 Federal Research Centre for Rural Areas, Forestry and Fisheries (vTI), Institute of Fishery Ecology, Deichstraße 12, 27472 Cuxhaven, Germany, E-mail: [email protected]

3 Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK, E-mail: [email protected]

4 Fisheries Research Services, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen AB11 9DB, UK, E-mails: [email protected]; [email protected]

5 University of Bremen, Institute of Statistics, PO Box 330440, 28334 Bremen, Germany, E-mail: [email protected]

Abstract

Hyperpigmentation is a term describing a pigment anomaly affecting dab (Limanda limanda) in the North Sea and, less frequently, dab in adjacent areas and other North Sea flatfish species. The condition is characterised by the occurrence of green to black patchy pigment spots in the skin of the upper body side and pearly-white pigment spots in the skin of the lower body side, caused by a hyperplasia of pigment cells (melanophores and iridiphores, respectively). In heavily affected fish, the condition is sometimes associated with inflammatory reactions in the integument. The most interesting aspect is the significant increase in prevalence that has been recorded in almost all North Sea areas in the past approximately 20 years. Prevalences recorded in hot spot areas for this condition increased from approximately 5 % to more than 50 %. There is a pronounced spatial pattern of hyperpigmentation in the North Sea, with higher prevalences in areas closer to the coast compared to areas in the more central part of North Sea. In the dab stock from the western , hyperpigmentation is virtually absent. There is indication that hyperpigmentation is related to size and age and that it affects growth and condition of affected fish. Some of the potential causes of hyperpigmentation have been studied (involvement of pathogens) and these are highlighted in the present paper others still need to be studied.

Keywords: pigment anomaly, hyperpigmentation, North Sea dab, spatial and temporal patterns ICES ASC 2008 1

Introduction

The present study focuses on the occurrence of hyperpigmentation, a specific pigment anomaly of the skin known since the 1980s to occur in North Sea dab (Limanda limanda). In flatfish species, pigment anomalies are well documented and have been described in the literature to affect wild populations (De Veen 1969, Gartner 1986, Macieira et al. 2006) as well as fish in aquaculture (Ottesen & Strand 1996, Venizelos & Benetti 1999, Bolker & Hill 2000). However, there is indication that both the prevalence and the types of pigment anomalies described in the literature are different from the hyperpigmentation recorded in North Sea dab. Hyperpigmentation is one of the externally visible diseases of dab recommended as target disease for fish diseases surveys in the North Sea and adjacent areas and is included in the methodological guidelines for general biological effects monitoring under the OSPAR Joint Assessment and Monitoring Programme (JAMP) (OSPAR 1997). Consequently, it has been incorporated in the range of diseases recorded in the German fish disease monitoring programme and long-term data are available for the North Sea and the western Baltic Sea. Additional, but more scattered data (in terms of regional and temporal coverage), are available from reference areas, e.g. the English Channel, Irish Sea, Celtic Sea and Icelandic waters.

The common dab is characterised by a wide distribution in the North Sea and adjacent areas in connection with a high abundance (Bohl 1957, Daan et al. 1990, Rijnsdorp et al. 1992, Knijn et al. 1993, Zühlke 2001). It is a relatively stationary species (Bohl 1957, Lozan 1989, Damm et al. 1991) and it is considered to react sensitively to environmental stressors (Dethlefsen et al. 1987, Lang et al. 2003). Because of these reasons, dab is widely used as bioindicator in national and international programmes monitoring and assessing effects of anthropogenic stressors on fish health largely in the North Sea and methodological guidelines for various parameters are available (McVicar et al. 1988, OSPAR 1997, 2003, Bucke et al. 1996, Feist et al. 2004).

The study the results of which are presented aimed at providing information on:

• macroscopic and microscopic characteristics of hyperpigmentation in dab • spatial and temporal patterns in the prevalence of hyperpigmentation • the impact of host-specific factors (e.g. size, age, sex) on the prevalence of hyperpigmentation • effects of hyperpigmentation on the host • possible causes of hyperpigmentation ICES ASC 2008 2

Materials and methods

Sampling and disease examination

The German long-term fish disease monitoring has been carried out on board RV Walther Herwig II and III since the early 1980s during winter cruises (December) and summer cruises (August/September) (Tab. 1). At present, diseases are recorded in 12 main sampling areas in the North Sea and at least 3 main sampling areas in the Baltic Sea (for location, see Tab. 2 and Fig. 3). More limited studies were also carried out in adjacent waters such as the English Channel, Irish Sea, Celtic Sea and at reference areas at the south coast of Iceland. Fishing is carried out by means of bottom trawling with standard gears (GOV or 140 ft bottom trawl), with a towing time of 1 hour and a speed of 3 - 4 knots. Per sampling area, usually 3 - 5 hauls are taken.

Tissue samples for histology and samples for virology and bacteriology were taken during the Research Cruise 194 (30.05. - 03.06.2007) of RV Scotia (UK) from the Scottish areas of the North Sea (Marr Bank, Moray Firth, Bell Rock, Wee Bankie, St. Abbs and Fair Isle).

Table 1: Research cruises of RV Walther Herwig II and III in 12 North Sea areas (N) and 3 Baltic Sea areas (B), S = Summer cruise, W = Winter cruise, blank = no cruise; each sampling campaign takes 17 - 22 days. Date Cruise Sampling area (dd/mm/yy) ID N01 N02 N03 N04 N05 N06 N07 N10 N11 N22 P01 P02 B01 B12 B11 07/06/88 WH090 S S S S S S S S S 04/01/89 WH094 W W W W W W W W 20/05/89 WH098 S S S S S S S S S 05/01/90 WH103 W W W W W W W W 05/05/90 WH107 S S S S S S S S S 03/01/91 WH112 W W W W W W W W W W 26/06/91 WH116 S S S S S S S 04/01/92 WH120 W W W W W W W W W W W W 18/06/92 WH125 S S S S S S S S 07/01/93 WH130 W W W W W W W W 24/06/93 WH135 S S S S S S S 11/01/94 WH142 W W W W W W W W W W W 22/06/94 WH148 S S S S S S 06/01/95 WH155 W W W W W W W 20/06/95 WH161 S S S S S S S S 03/01/96 WH167 W W W W W W W W W W W W W 08/05/96 WH172 S S S S S S S S 20/12/96 WH178 W W W 17/05/97 WH185 S S S S S S S S S 21/12/97 WH191 W W W W W W W 11/05/98 WH195 S S S S S S S S S 14/12/98 WH200 W W W W W W W W W W W 15/06/99 10/12/99 WH212 W W W W W W W W W W W 15/06/00 10/12/00 WH223 W W W W W W W W W W 15/08/01 14/12/01 WH234 W W W W W W W W 24/08/02 WH242 S S S S S S S 13/12/02 WH245 W W W W W W W 30/08/03 WH255 S S S S S S S S S S 17/12/03 WH258 W W W W W W 16/09/04 WH267 S S S S S S S S S S S S S S 07/12/04 WH269 W W W W W W W W W W W 26/08/05 WH278 S S S S S S S S 19/12/05 WH281 W W W W 02/09/06 WH291 S S S S S S S S S 12/12/06 WH294 W W W W W W W W W ICES ASC 2008 3

Fish for disease examination are sorted from the catches immediately and are kept alive in seawater. For the Scotia samples fish are not kept alive but sorted and checked immediately. The catch com- position is recorded and catch data are used to calculate the catch per unit effort (CPUE) for dab (number/weight of specimens per one hour of trawling).

The total length (to the cm below) and wet weight are measured in specimens taken for disease wetweight[g]·100 examination, facilitating the calculation of condition factors (CF = length[cm]3 ). Sex of dab is identified visually by the shape of the gonad. Dab were inspected externally for signs of diseases and parasites and internally for the presence of liver anomalies (including tumours), using standardised ICES methodologies (Bucke et al. 1996, Feist et al. 2004) as well as guidelines provided through the fish disease component of the BEQUALM programme (http://www.bequalm.org). Otoliths for subsequent age determination are taken from all fish inspected for liver anomalies.

The presence of hyperpigmentation is recorded according to 3 severity grades (grade 1: Less than/equal to 10 % of the skin surface covered by pigment anomalies; grade 2: between 10 % and up to twice the area of the spread-out caudal fin covered by pigment anomalies; grade 3: more than twice the area of the spread-out caudal fin covered by pigment anomalies) (for examples, see Fig. 1).

Fish disease database

All single fish data generated on board the research vessel are entered to a PC interface in real time and are incorporated into the fish disease database of the vTI Institute of Fishery Ecology. The database analysed in the present study comprises individual fish data from in total 70 research cruises carried out in the period 1981 to 2008. For the present study, data from 35 research cruises covering the period from summer 1988 to winter 2006 were analysed (see Tab. 1). The database is structured in a descriptive part with variables providing temporal and spatial information, host-specific individual information (sex, length, wet weight, age) and a binary part with variables providing information on the presence/absence of diseases and their severity grades assigned. In total, the database contains individual data from more than 400.000 specimens of dab ≥ 10 cm, out of which 176.207 fulfilling the criteria defined in terms of spatial and temporal coverage were used for the present analysis (see Tab. 2).

Table 2: Location of the sampling areas in the North Sea and the Baltic Sea, total number of dab examined in each of the 12 North Sea (N) and 3 Baltic Sea (B) areas from summer 1988 to winter 2006 Area Geographical coordinates Individuals N01 54◦15’ - 54◦26’N, 07◦25’ - 07◦39’E 21.670 N02 54◦06’ - 54◦29’N, 03◦21’ - 04◦08’E 10.052 N03 52◦46’ - 53◦10’N, 03◦28’ - 04◦10’E 8.771 N04 54◦25’ - 54◦50’N, 02◦00’ - 02◦32’E 22.364 N05 55◦15’- 55◦29’N, 00◦00’ - 00◦25’W 13.938 N06 56◦15’ - 56◦26’N, 01◦44’ - 02◦14’W 19.700 N07 57◦45’ - 57◦59’N, 00◦46’ - 01◦20’W 9.021 N10 56◦43’ - 56◦56’N, 03◦30’ - 03◦55’E 10.643 N11 55◦30’ - 55◦41’N, 06◦49’ - 07◦24’E 17.128 N22 53◦30’ - 53◦44’N, 01◦18’ - 01◦47’E 14.412 P01 55◦22’ - 55◦47’N, 04◦40’ - 05◦14’E 7.361 P02 56◦16’ - 56◦41’N, 02◦40’ - 03◦24’E 4.278 B01 54◦28’ - 54◦45’N, 10◦12’ - 10◦59’E 8.901 B11 54◦40’ - 54◦55’N, 13◦00’ - 13◦55’E 7011 B12 54◦12’ - 54◦27’N, 11◦22’ - 11◦51’E 957 Total 12 North Sea boxes, 3 Baltic Sea boxes 176.207 ICES ASC 2008 4

Histology

Tissue samples from different organs (skin, liver, spleen, kidney, gonad, gill, brain, heart, stomach and intestine) were taken from 10 dab each without and with hyperpigmentation grade 1, 2, 3, respectively. Samples were fixed in 10 % neutral buffered formalin and were processed subsequently applying routine histology, involving embedding in paraffin, sectioning into 3 µm sections (rotation microtome Shandon Finesse) and haematoxylin and eosin (H & E) staining. The histology processing was completed at the Centre for Environment, Fisheries and Aquaculture Science (Cefas) in Weymouth, UK. Details to the histology process can be found in Feist et al. (2004). The analysis of processed material was done using light microscope.

Virology and bacteriology

Virology and bacteriology were donecarried out at the Fisheries Research Services (FRS), Marine Laboratory, in Aberdeen, UK. Three organs (liver, muscle/skin, gill) from 54 hyperpigmented fish grade 1, 2, 3 as well as from 18 control fish (female > 20 cm) were sampled for laboratory analysis by virus culture. Tissues were sampled individually and either snap frozen in liquid nitrogen or sampled into transport media on the final day of sampling. Tissues were homogenised with 9 ml DMEM/20 % serum, stored at 4 ◦C and centrifuged for 15 minutes at 3,000 rpm. 20 µl of fluid homogenised organs were inoculated in 2 ml of EMEM-10 % Foetal Bovine Serum, 1 % 200 mM L-Glutamine and 6 different cell lines (TV-I, tail fin; FFN, flounder fin; TO, salmon head kidney leucocytes; BF-2, bluegill fibroblast; CHSE-214, Chinook salmon embryo; SBL, sea bass larvae).

Samples of 6 areas, 3 grades of hyperpigmentation, controls and 6 cell lines resulted in a total number of 1.296 samples analysed for the presence of viral pathogens, mainly belonging to 5 types of viruses (Infectious Anaemia Virus (IAV), Viral Haemorrhagic Septicaemia Virus (VHSV), Infectious Pancre- atic Necrosis Virus (IPNV), Infectious Haematopoietic Necrosis Virus (IHNV), Spring Viraemia of Carp Virus (SVC)). Incubation was performed at 15 ◦C in a dark environment. Each sample was processed individually and passed two or three times on 6 cell lines and examined regularly for up to 10 weeks. Bacteriological samples (from the same fish, see above) from skin and kidney were inoculated onto tryptic soy agar with 2 % NaCl and colonies examined biochemically.

Statistics

The trend of temporal patterns was tested with the Spearman rank correlation test. To determine the significance of differences between groups, data were compared using analysis of variance (one-way ANOVA) with F-test. If F-test showed significants Post-hoc test (Tukey HSD test, Honest Significant Difference) was used to show the grad of differences between groups. The condition factor was tested of normal distribution with Kolmogosov-Smirnow test. The confidence interval for the prevalence was calculated with 95 %, limiting quantile 0,975 for P = 0.05, double sided α = 2,5 %.

(x+1)F x πtop = n−x+(x+1)F and πbottom = x+(n−x+1)F

Correlations were considered significants at a probability of error P < 0.05. All calculations were done using the computer program Statistica (Software-System for Data-analysis, Version 6, StatSoft, Inc. 2003). ICES ASC 2008 5

Results

Macroscopic and microscopic characteristics of hyperpigmentation

The normal pigmentation of dab is uniformly sandy brown (light brown to grey), rarely with scattered dark or coloured spots on the upper body side. The lower body side is uniformly grey to white and appears translucent. It is recognised that dab do vary slightly from this uniform colour to reflect different sediments. Hyperpigmentation is characterised by the occurrence of patchy green to black spots in the skin on the upper pigmented body side (Fig. 1; 1A, 2A) and pearly-white or green to black spots on the lower body side (Fig. 1; 1B, 2B) at varying degree of discolouration. The condition is different from olive to brown ’normal’ pigment spots (sometimes associated with healing processes related to skin ulcers or mechanical damage) commonly to be seen in dab and other flatfish species such as flounder (Platichthys flesus).

Figure 1: Hyperpigmentation in dab (Limanda limanda) with different severity grades. 1A: hyperpig- mentation grade 1 of the upper body side. 1B: grade 1, lower body side; 2A: hyperpigmentation grade 3 of the upper body side; 2B: grade 3, lower body side (grading according to BEQUALM guidelines; http://www.bequalm.org )

Primarily North Sea dab (Limanda limanda) is affected by hyperpigmentation. The condition has, however, been also observed at a much lower prevalence in other flatfish species from the same habitats, e.g. in long rough dab (Hippoglossoides platessoides), (Buglossidium luteum), lemon (Microstomus kitt) and flounder (Platichthys flesus). ICES ASC 2008 6

A E B E SSp SSp S S

SC

SC

Subc Subc

M M C D

Infiltration

Figure 2: Histology of hyperpigmentation in North Sea dab (Limanda limanda). A, C Section through normal skin of control fish, upper body side (magnification 40x and 100x). E, F normal skin of control fish, lower body side (40x and 400x). Normal skin is showing intact epithelium and no obvious pigmentation (arrow). E, F. Section showing tightly packed iridophores containing platelets of guanine (arrow) between the epidermis and Stratum compactum. B, D melanocytes below the epidermis (40x-100x). Note the presence of lymphocytic infiltration in the connective tissues of the dermis (arrow). (E: Epidermis, SSp: Stratum spongiosum, S: Scale, SC: Stratum compactum, Subc: Subcutis (Hypodermis), M: Muscle). ICES ASC 2008 7

Microscopically, the normal dab skin consists of an epidermis and an underlying dermis (Fig. 2; A, C, upper body side; E, F lower side). The epidermis is a semi-stratified epithelium with the outermost cells becoming flattened and eventually sloughed from the surface of the skin. Occasionally, some of these cells appear rounded and clearly losing attachment from adjacent cells. Immediately below the epidermis is the eosinophilic basal lamina overlying the dermis, which consists of a thin layer containing a network of capillaries and connective tissue within which occasional pigment cells (both melanocytes and iridophores in different proportions, depending on the location on the fish), lymphocytes and phagocytes may be present in the Stratum spongiosum. The scale pockets are located underneath this layer and above the Stratum compactum which defines the lower limit of the dermis. Below this is the adipose tissue and somatic muscle. In the upper surface of dab exhibiting hyperpigmentation (Fig. 2 B, D) there is a conspicuous hyperplasia of melanocytes arising within the uppermost layer of the dermis, which usually appear as discrete dendritic cells containing numerous melanin granules. The melanocyte layer of dab with hyperpigmentation grade 3 is significantly thicker as in control fish without hyperpigmentation (Tukey HSD test, p < 0.05). In grade 3 fish, lymphocytic infiltration within this layer was sometimes observed (Fig. 2 D), suggesting the onset of an active immune response. Hyperpigmentation occurring on the lower body side of dab usually represents a different histopathological picture. In most cases, there is a hyperplasia of iridophores rather than melanocytes, although the latter are occasionally seen and, in severe cases, produce spots or patches of macroscopically visible melanisation amongst the pearly-white areas of iridophore cell hyperplasia. Iridophores characteristically contain numerous guanine platelets which appear as stacks of light olive brown cytoplasmic inclusions (Fig. 2 E, F), which are birefringent in polarised light. In the specimens examined during the current investigation, lymphocytic infiltration amongst the layer of hyperplastic iridophore cells was not observed.

Virology and bacteriology

No evidence of micro-organisms or parasites was seen by light or electron microscopy. No evidence of virus was seen by reading the cell-lines of the dab samples from the liver, muscle/skin and gill.

Bacteriological examination showed three organisms that were isolated both in normal and pigmented fish from the skin and the kidney. All colonies were Gram negative rods, with growth recorded at 4 ◦C, 15 ◦C and 22 ◦C but no growth at 37 ◦C. Colony type ’B’, showed motility, an oxidase and catalase positive and a fermentative reaction and was identified as Vibrio vulnificus (API 20E). All three isolates were freeze dried. Overall there was no obvious differences between hyperpigmented and control fish.

Spatial and temporal patterns in the prevalence of hyperpigmentation

Hyperpigmentation has been recorded at stations in the entire North Sea since the beginning of the fish disease monitoring programme at the beginning of the 1980s with a pronounced regional pattern. Data from adjacent waters (English Channel, Irish Sea, Celtic Sea) reveal that hyperpigmentation in dab is present, too, but is much less prevalent. In dab from the Baltic Sea, the condition has only been recorded occasionally at a low prevalence. ICES ASC 2008 8

The prevalence in North Sea dab differs markedly between regions and develop overall spatial patterns (Fig. 3). In the period 2001 - 2006, the mean prevalence ranged from < 10 % in areas in the northern central and in the southernmost North Sea up to > 50 % in the German Bight, at the Dogger Bank and in the Firth of Forth area. Other areas (P01, P02, N03) were generally characterised by a low prevalence. Areas closer to the coast have generally higher prevalences than more central areas, except for area N03.

Figure 3: Spatial pattern of hyperpigmentation in the North Sea, prevalence of hyperpigmentation (%) (factor of circle) in the period 2001 - 2006 (arithmetic means), males and females and all size classes (> 10 cm) combined. (Software: Ocean Data View)

As clearly can be seen from Fig. 4 and 5, the prevalence of hyperpigmentation in the North Sea has increased since 1988, especially within the last decade. The temporal pattern of the long-term data show a general positive trend in all 12 North Sea sampling areas by Spearman rank correlation which is significant (p < 0.05) in 8 of the 12 areas (N01, N02, N04, N05, N06, N07, N11, P01). ICES ASC 2008 9

Figure 4: Time-series pattern; prevalence (with 95 % confidence intervals) of hyperpigmentation in dab (Limanda limanda); males and females and all size classes (> 10 cm) combined; North Sea areas N01, N02, N03, N04, N05, N06; from summer 1988 to winter 2006.

The areas N01, N04 and N06 show a particularly strong increase and the area N22 a constantly high prevalence over the whole time series. Hence, these areas were considered as ’high prevalence areas (HPA)’ and data from these areas were combined for some of the analyses performed. The starting prevalences in the HPA in 1988 were < 5 %, except for areas N04 and N22, and increased to maximum levels in the range of > 50 % in 2006 (all size classes and both sexes combined). ICES ASC 2008 10

Figure 5: Time-series pattern; prevalence (with 95 % confidence intervals) of hyperpigmentation in dab (Limanda limanda); males and females and all size classes (> 10 cm) combined; North Sea areas N07, N11, N22, N10 from summer 1988 to winter 2006.

Impact of host-specific factors on the prevalence of hyperpigmentation

In the following, the effects of selected host-specific factors (sex, length, age, severity grade) on the prevalence of hyperpigmentation are described. For the analysis, only data from the 4 high prevalence areas (HPA) N01, N04, N06, N22 were used.

Sex: The impact of the sex of the fish on the prevalence varied between areas. In two (N06, N22) of the four areas there was a statistically significant difference between male and female dab (one-way ANOVA, N06: p < 0.001, N04: p = 0.255, N01: p = 0.601, N22: p < 0.05). Areas N04 and N01 did not indicate significant differences between males and females. In area N22, the prevalence in males was significantly lower than in females. This is in contrast to area N06 where the prevalence in females was significantly lower than in males. When combining the data from the four areas (HPA) there was no statistically significant difference in prevalence between males and females (one-way ANOVA, HPA: p = 0.852). This change are not directed linked to the sex. The effect sex is bonded with growth differences between sex of dab (Bohl 1957, Lozan 1988, 1989, 1992, Knust 1990, Rijnsdorp et al. 1992).

Body length: To test the relationship between the prevalence of hyperpigmentation and the total body length, data from female and male dab were used separately, because sex-specific differences in growth are well documented in the literature (Bohl 1957, Lozan 1988, 1989, 1992, Knust 1990, Rijnsdorp et al. 1992). From Fig. 6 it can be seen that the prevalence of hyperpigmentation (all severity grades combined) in female dab steeply increased with length, especially in the size range 17 ICES ASC 2008 11

- 25 cm. Thereafter, it levelled of and remained at a more or less constant niveau of around 40 - 45 % up to the largest size class 32 cm. There was a slight tendency of a decrease in prevalence in the largest size classes. However, due to the relatively low number of large specimens in the samples, some uncertainties remain. Length class 31 cm showed the highest overall prevalence of > 50 %. The total length of the smallest specimens recorded displaying hyperpigmentation was 12 cm, the smallest dab with hyperpigmentation severity grade 3 were 13 cm (male) and 14 cm (female), respectively. The prevalence of hyperpigmentation grade 1 was generally higher than that of severity grades 2 and 3. However, with increasing length, there was a shift towards higher prevalences of severity grades 2 and 3. In the largest specimens (31 - 32 cm), grade 1 was less prevalent than grade 2.

Similar patterns were recorded in male dab (not shown). The overall prevalence in all length classes of males was similar to females. However, the prevalence in males of lower length classes was higher than in females. As in females, there was a shift in prevalence towards higher severity grades with increasing length.

Figure 6: Prevalence of hyperpigmentation according to length class (10 - 32 cm) in female dab (Limanda limanda) from the high prevalence areas (HPA); cumulation of severity grades 1, 2, 3; sampling campaigns 1988 - 2006.

Age: The analysis of age data was mainly based on female dab because male dab were less abundant and age data from males did not cover all age groups sufficiently. Furthermore, it has to be noted that age data were only available for females ≥ 20 cm total length (those examined for liver anomalies; see Material and methods). Fig. 7 shows that the prevalence of hyperpigmentation in age classes 2 - 4 years was similar (17 % - 19 %), but that there was a decreasing trend with increasing age in the age groups 5 - 7 years, corresponding to the tendency observed in the length data. With respect to the proportions in prevalence of the 3 severity grades, the data are in line with the length data and reveal an increasing proportion of severity grades 2 and 3 with higher age.

Severity grade: Hyperpigmentation is classified into three different severity grades coded as 1, 2 or 3 (see Materials and Methods). The difference in prevalence of the severity grades 1, 2 and 3 was statistically significant (one-way ANOVA, p < 0.001) in all four areas: grade 1 had a significantly higher prevalence than grades 2 and 3 (Tukey test HSD, p < 0.001) and the prevalence of grades 2 ICES ASC 2008 12

Figure 7: Prevalence of hyperpigmentation according to age class (2 - 7 years) in female dab (Limanda limanda) from the high prevalence areas (HPA); cumulation of severity grades 1, 2, 3; sampling campaigns 1994, ’95, ’96, ’97, ’98, ’99, 2001, ’03, ’06). and 3 was similar without any significant differences (Tukey test HSD, p > 0.05). Spearman’s rank correlation test showed the strongest positive temporal trend in fish with severity grade 1 (Tab. 3). The trend of grade 2 had the lowest increasing rate in all four HPAs in relation to grade 1 and 3. In the case of area N22, the prevalence trend curve for grade 2 was negative (Tab. 3), reflecting a decrease over time. N22 showed the smallest increase in absolute prevalence but was characterised by a generally high prevalence at the beginning of the observation period as well a high average prevalence over time. In all four areas grade 3 showed a slight but continuous increase of prevalence over time. The trend of grade 3 in area N22 was slightly but non-significantly positive. However, grade 1 ’lifted off’ from the higher grades 2 and 3 in all areas at a nearly identical time around 1993 - 1995. In this time the prevalence of grade 1 was steeply increasing in all four areas, a development that continued especially after the years 1999 - 2000. Areas N22 and N04 showed signals indicating a temporal relationship between lower and higher severity grades. There was a time lag in the increase in prevalence of grade 3 in relation to the increase recorded for grade 1.

Table 3: Spearman’s rank test for the influence of the three severity grades of the trend curve of hyperpigmentation in dab; areas N01, N04, N06, N22 (HPA); all size classes; males and females combined Variable Spearman’s Area Hyp. p < 0.05 rank test grade Prevalence/ 0,92605 N01 1 significant time 0,586275 N01 2 significant 0,821584 N01 3 significant 0,883431 N04 1 significant 0,223974 N04 2 0,648827 N04 3 significant 0,949866 N06 1 significant 0,723262 N06 2 significant 0,902244 N06 3 significant 0,664596 N22 1 significant -0,402597 N22 2 0,10559 N22 3 ICES ASC 2008 13

Effects of hyperpigmentation on the host

For the time being, only effects on growth and on condition factors were analysed.

Growth: From the growth parameters age and length there is indication for differences between the growth of controls and hyperpigmented fish (see Tab. 4). Fish with hyperpigmentation (severity grades 1 - 3 combined) were statistically significantly larger (difference: 0,34 cm) than fish without hyperpigmentation (one-way ANOVA p < 0.05). Especially fish with hyperpigmentation grade 3 were significantly larger compared to control fish and grade 1 fish (Tukey HSD test p < 0.05). There was no significant difference between control fish and grade 1 and 2 fish, resp. (Tukey HSD test p > 0.05).

Table 4: Tukey HSD test; effect of growth in controls and hyperpigmentation grade 1, 2, 3; 12 North Sea areas, female dab (Limanda limanda) (age 3 - 6 years); sampling campaigns 1994, 95, 96, 97, 98, 99, 2001, 03, 06) (significant relationships in bold) Factor Control Hyp. 1 Hyp. 2 Hyp. 3 Hyp. 1- 3 Mean total 24,004 24,133 24,500 25,040 24,344 length (cm) Control 0,838641 0,082127 0,000739 0,006434 Hyp. 1 0,838641 0,487723 0,013429 0,645493 Hyp. 2 0,082127 0,487723 0,453412 0,950072 Hyp. 3 0,000739 0,013429 0,453412 0,087048 Hyp. 1 - 3 0,006434 0,645493 0,950072 0,087048

Condition factor: The data shown in Fig. 8 indicate differences in mean condition factors (CF) between fish with and without hyperpigmentation (only females were taken into account for consistency reasons). In all areas represented in the figure, including their combinations, there was a tendency for lower CFs in hyperpigmented fish, particularly in fish with hyperpigmentation grade 3, possibly reflecting a worse nutritional status of hyperpigmented compared to non-affected fish. Taking data from all 12 areas together, this difference was statistically significant (ANOVA p < 0.001). The lower severity grades of hyperpigmentation (1, 2) were not significantly different (Tukey HSD test, p > 0.05) in condition factor. However, there was indication of lower condition factors in grade 2 and 1 fish compared to controls. Grade 3 fish showed significant differences (Tukey HSD test, p < 0.01) to the controls and to grade 1 fish. In sampling areas N04 and N06, the differences were significant, whereas they weren’t in areas N01 and N22 (Tukey HSD test, p > 0.05).

The finding of decreased condition factors in hyperpigmented fish seems to be in contrast to the growth data that indicate a faster growth of fish with hyperpigmentation grade 3 compared to control fish and fish with grades 1 or 2 (see Tab. 4). ICES ASC 2008 14

Figure 8: Condition factors (arithmetic mean, standard deviation, standard error) of dab (Limanda limanda) according to the severity grade of hyperpigmentation; sampling areas N01, N04, N06, N22 (see Fig. 3); high prevalence areas (HPA) and all 12 North Sea areas combined; females; all size classes combined; winter 2002 - winter 2006. ICES ASC 2008 15

Discussion

The occurrence of pigment anomalies in flatfish species has been described to occur in wild fish (De Veen 1969, Gartner 1986), Macieira et al. 2006) as well as in farmed fish (Ottesen & Strand 1996, McEvoy et al. 1998, Venizelos & Benetti 1999, Bolker & Hill 2000, Copeman & Parrish 2002, Bolker et al. 2005, Yamanome et al. 2005, 2007).

However, there are differences between the anomalies described in the literature compared to the phe- nomenon of hyperpigmentation reported in the present paper. Either the prevalence or the features of the pigment anomalies were different. Hyperpigmentation in dab is characterised by an increasing prevalence over the past 20 years, observed in almost all sampling sites in the North Sea, with current maximum prevalences of > 50 % in certain ’hot spot’ areas (see Fig. 4, 5). A similar increase has not been reported before for any of the other pigment anomalies. Furthermore, hyperpigmentation involves a hyperplasia of both melanocytes and iridophores (see Fig. 2), whilst pigment anomalies re- ported previously in wild and farmed flatfish merely consisted of an increase in number of melanocytes or melanosomes only or in a lack of pigmentation (albinisms). In fish with severe cases of hyperpig- mentation, an infiltration of lymphocytes was recorded, a finding different to some earlier reports of pigment anomalies in wild or farmed fish.

There are, however, certain similarities between hyperpigmentation and pigment anomalies associ- ated with exposure to UV-B radiation described in the literature. The pigment melanin can protect against UV-B radiation (Hessen 1996, De Lange 2000). In flatfish exposed to UV-B radiation, cellular changes of the integument occur, with an increasing number and hyperplasia of goblet cells, swelling of epidermis (Blazer et al. 1997, McFadzen et al. 2000, Schubert 2004), as well as an increasing melanocyte layer thickness (Fabacher et al. 1997), with more free melanosomes within the melanocytes compared to control fish (Fabacher & Little 1995). To some extent, lymphocyte infiltration in the relevant skin-layer was reported (Fabacher & Little 1995, Blazer et al. 1997), which show similarities to a ’sunburn’ (Bullock 1982, Berghahn et al. 1993). In the research of Jokinen et al. (2000), UV-B radiation provoked a stress response in fish with an increasing number of lymphocytes. In larvae of Coregonus sp., experimentally exposed to moderate UV-B radiation, 30 % more melanin was recorded in the skin compared to the non-exposed control group. At the end of the UV-experiment, exposed larvae were significantly larger than the non-exposed control larvae (Häkkinen et al. 2002). An increase in melanin (through hyperplasia of melanocytes) and a faster growth rate were also observed in hyperpigmented dab in the present study. Correspondingly, am- phibians exposed to UV-B radiation showed an increased melanin storage in the skin, although these represent effects that occurred during older life stages (Belden et al. 2003).

The general temporal increase in prevalence of hyperpigmentation in North Sea dab recorded in almost all North Sea sampling areas (see Fig. 4, 5) corresponds to the increase in effective UV-B radiation caused by the global ozone depletion in the stratosphere (Megie 1991, Crutzen 1992, Kerr & McElroy 1993, Zerefos et al. 1998). The scale of ozone depletion has been 4 - 5 % per decade, corresponding to an increase of UV-B radiation in average of 10 % per decade (Megie 1991, Kerr & McElroy 1993, Zerefos et al. 1998). Since 1980, the UV-B radiation has increased by approximately 30 %. In the upper water layer, aquatic organism like zooplankton (Hessen 1996, De Lange et al. 2000, Rhode et al. 2001), bacterial plankton (Herndl 1993), amphibians (Belden et al. 2003, Blaustein & Belden 2003), fish embryos and larvae (Lesser et al. 2001, McFadzen et al. 2000, Dethlefsen et al. 2001, Steeger et al. 2001, Häkkinen et al. 2002) and fish (Fabacher et al. 1997, Jokinen et al. 2000, Alemanni et al. 2003) are exposed to UV-B radiation and have been shown to be susceptible to increased UV-B radiation. Interestingly, the strongest decrease in the northern hemisphere in overall ozone with nearly 6 % per decade occurs in late winter and spring ICES ASC 2008 16

(Crutzen 1992), the same period as the main spawning time of dab in the North Sea (February - April) (Daan et al. 1990, Campos et al. 1994).

If UV-B radiation is causally involved in the development of hyperpigmentation, the impact is likely to be restricted to early life stages of dab, because only these are exposed to UV radiation. For instance, the embryos and young larvae of dab are pelagic and are drifting in surface water layers, particularly during calm weather in the period from early spring to summer where they may be exposed to UV-B radiation. Also young life stages that have already passed metamorphosis may be exposed in shallow waters, e. g. in the Wadden Sea area, especially during low tides. It is unlikely that older life stages with deeper waters as preferable habitats are at risk to get exposed to UV-B radiation due to limitations in the penetration of UV radiation into deeper water layers.

It is interesting to note that first macroscopic signs of hyperpigmentation only occur in dab of 12 cm total length and that both prevalence and severity are increasing as fish increase in total length (see Fig. 6). This implies that either (a) the effect is manifested early during ontogenesis (in embryos/larvae) but that the effects are only visible macroscopically after a certain period of time or (b) that the effect is manifested in small dab (approx. 12 cm), possibly linked to UV-B exposure in shallow coastal waters or (c) that there is a chronic impact leading to an increase in prevalence and severity. Particularly the latter possibility would speak against the hypothesis of UV-B exposure as a cause of hyperpigmentation (see above). It has to be taken in account, however, that catches of small dab were not representative in the study because of the type of gear used and possibly also because of the areas sampled (more offshore). Further studies on young life stages and the metamorphosis from pelagic, symmetrical larvae to benthic, asymmetrical juveniles are required for more conclusive information with respect to the influence of size on the occurrence of hyperpigmentation.

The age data also reveal that the higher severity grades are increasing in prevalence as fish get older (see Fig. 7). However, there is a signal for a decrease in general prevalence with increasing age, possibly indicating an increased mortality, particularly in dab severely affected by hyperpigmentation. This is in line with the data on condition factors (see Fig. 8), clearly revealing that dab with hyperpigmentation severity grade 3 are characterised by significantly lower condition factors than fish with severity grades 1 or 2 and those without hyperpigmentation. Furthermore, it has been observed that dab with severity grade 3 have to highest mortality when maintained in experimental facilities (Lang, unpublished data).

Some spatial differences were recorded in the North Sea as regards the absolute prevalence levels as well as the temporal trends of hyperpigmentation. The highest prevalences occurred in areas closer to the coast, while dab from areas in the more central North Sea were less affected (see Fig. 3). The steepest increase in prevalence was noted in the German Bight (area N01) (at least over the past approx. 10 years). An increase also occurred in most other North Sea areas, with the exception of two areas where the prevalence remained almost stable over the entire observation period (see Fig. 4 and 5). A decrease in prevalence was not observed in any of the areas. In many cases, the increase was more or less linear, indicating a constant and almost North Sea-wide change in ecosystem conditions responsible of hyperpigmentation. Only in the German Bight, the increase more resembled an exponential function, indicating that ecosystem changes leading to hyperpigmentation were most pronounced in this region in the past decade. It is interesting to note that the strongest increase in prevalence occurred in hyperpigmentation severity grade 1 whereas the increase was less pronounced in the higher severity grades. There was some indication of a delay effect in the increase of the three severity grades (the increase in prevalence of severity grades 2 and 3 started later in time than the increase in severity grade 1), revealing a temporal progression in the population from severity grade 1 towards severity grade 2 and 3. In areas adjacent to the North Sea (e.g., English Channel, Irish Sea, south coast of Iceland), hyperpigmentation has been recorded, too, however, at a significantly lower ICES ASC 2008 17 prevalence. Long-term data from the Western Baltic Sea reveal that dab have only very occasionally been affected. Reasons are so far unresolved; a hypothesis is that early life stages in the Baltic Sea are not affected by UV-B radiation because they are distributed in deeper water layers because of a lower buoyancy due to the lower salinity in the Baltic Sea and are, therefore, not exposed to UV-B radiation.

In some of the sampling areas, there were differences between males and females with respect to the prevalence of hyperpigmentation. However, there was no general and consistent statistically significant pattern. Differences in prevalence recorded can very likely be attributed to differences in growth between male and female dab (females grow faster than males; Bohl 1957, Lozan 1988, 1989, 1992, Knust 1990, Rijnsdorp et al. 1992). Female dab showed the highest prevalence of hyperpigmentation in higher length classes, whilst, in males, prevalence was highest in lower length classes. For instance, males of the length class 20 - 24 had a significantly higher prevalence than females of the same size. If combining data from all length classes, there are no significant difference between males and females. These findings indicate that there is no sex-specific difference in susceptibility to hyperpigmentation.

As with sex, there were no significant seasonal effects (summer vs. winter) on the prevalence of hyperpigmentation (data not shown), indicating that the condition can be regarded as a chronic condition, not significantly influenced by seasonal variations, e. g. in hydrographic factors. However, in three (N01, N04, N22) of the four high prevalence areas, there was a tendency since 2003 for elevated winter prevalences compared to summer data. The reasons are unknown; possibly, seasonal migration patterns play a role. During winter, dab is migrating from cold coastal waters towards warmer bottom water in offshore areas (Bohl 1957, Lozan 1989, Saborowski & Buchholz 1996, 1997). Changing climatic conditions in the North Sea might influence this migration pattern and thus the catch composition.

In the following, some potential causes of hyperpigmentation and its spatial and temporal patterns (other than UV-B radiation) will be discussed:

Pathogens: There is indication from the literature that pigment anomalies can be induced by para- sitic trematodes (De Veen 1969), but no parasites were detected in the skin by means of by light microscopy and electron microscopy. Pathogens known to cause a dark pigmentation in fish include Viral Haemorphagic Septicaemia Virus (VHSV) and Infectious Haematopoietic Necrosis Virus (IHN). However, the virology carried out in the present study did not indicate an involvement of these viruses in hyperpigmentation. Also other infective pathogens were not identified, neither other viruses nor bacteria. From the data it can be concluded that there is apparently no connection between pathogens and hyperpigmentation.

Population structure: Data on the catch composition (not shown) indicate that the catch per unit of effort (CPUE; number or weight of dab caught per one hour trawling with the standard gear) decreased since the early/mid 1990s in the four high prevalence areas. Furthermore, the mean length of the fish decreased slowly but significantly. According to the relationship between total length of dab with the prevalence of hyperpigmentation (see above), this would theoretically lead to a decrease in prevalence in females (highest prevalence in larger fish) and an increase in males (highest prevalence in smaller fish) because of the sex-specific growth.

Climate change: There is evidence of a significant increase in water temperature in the North Sea (Reid et al. 2001, Beaugrand 2004, Dulvy et al. 2008, especially in its southern part (BSH 2003, Hughes 2004, Wiltshire & Manly 2004, Mccip 2006). The increase in water temperature correlates with the increase in prevalence of hyperpigmentation and it can, thus, not be excluded ICES ASC 2008 18 that a causal link exists. However, possible mechanisms are at present unknown and more research is required to investigate the relationship.

Nutrition: One factor possibly linked to climate change and the associated rise in water temperature could be changes in nutrition. There is some indication that there have been changes in food compo- sition of North Sea dab over the past approx. two decades years, e.g. an increasing consumption of gastropods and echinoidea (Mintenbeck 2007). It is known that food composition and nutritional value are critical factors with respect to the development of pigmentation in young flatfish (Rainuzzo et al. 1994, Naess & Lie 1998, Estevez et al. 1999, Venizelos & Benetti 1999, Hamre et al. 2007). The finding of significantly lower condition factors in severely hyperpigmented dab reveals that food composition (or quantity) may be causally involved. It is noteworthy in this context that dab from the western Baltic Sea that are only very sporadically affected by hyperpigmentation are characterised by the fastest growth and the highest condition factors. However, for more conclusive statements on the role of nutrition, studies on food composition of life stages of dab with and without hyperpigmentation are needed.

Contaminants: It can not be excluded that exposure to water- or sediment-bound contaminants, either affecting early life stages in surface or shallow coastal water layers or older life stages in deeper water layers, have an impact on the pigmentation. In early pelagic planktonic life stages (embryos, early larvae), UV-associated phototoxicity of organic compounds (e.g. PAHs) in the surface microlayer (Lyons et al. 2002, 2006, Kirby et al. 2007) may play a role. However, more research is needed to clarify a potential connection.

Genetic shift: It cannot be excluded that a genetic shift has led to the increase in prevalence of dab with hyperpigmentation. However, the speed in the increase of the prevalence as well as the fact that there is no obvious selective advantage of hyperpigmentation (this would only be the case if early life stages exposed to UV-B radiation were characterised by higher pigmentation protecting them from UV-B-caused DNA damage, which is obviously not the case) speak against this hypothesis. Again, more research is needed to verify or reject a possible link.

In conclusion, it can be stated that hyperpigmentation is a condition increasing in prevalence in North Sea dab. There is an impact of host-specific factors on the prevalence and the condition which appears to show a negative impact on the general physiological state of specimens severely (grades 2 - 3) affected. Based on the lack of knowledge on the possible causes of hyperpigmentation and of the increase in prevalence of this condition, it is emphasised that there is an apparent need for more research. ICES ASC 2008 19

References

[1] Alemanni M.E., Lozada M., Zagarese H.E. (2003) Assessing sublethal effects of ultraviolet radiation in juvenile rainbow trout (Oncorhynchus mykiss). Photochemical and Photobiological Sciences, 2: 867-870

[2] Beaugrand G. (2004) The North Sea regime shift: evidence, causes, mechanisms and consequences. Progress in Oceanography, 60: 245-262

[3] Belden, L.K., Moore, I.T., Mason, R.T., Wingfield, J.C., Blaustein, A.R. (2003) Survival, the hormonal stress response and UV-B avoidance in Cascades Frog tadpoles (Rana cascadae) exposed to UV-B radiation. Functional Ecology, 17: 409-416

[4] Berghahn R., Bulluck A.M., Karakiri M. (1993) Effects of solar radiation on the population dy- namics of juvenile flatfish in the shallows of the Wadden Sea. Journal of Fish Biology, 42: 329-345

[5] Blaustein A.R. and Belden L.K. (2003) Amphibian defenses against ultraviolet-B radiation. Evolution and Development, 5: 89-97

[6] Blazer V.S., Fabacher D.L., Little E.E., Ewing M.S., Kocan K.M. (1997) Effects of Ultraviolet-B Radiation on Fish: Histologic Comparison of a UVB-Sensitive and a UVB-Tolerant Species. Journal of Aquatic Health, 9 (2): 132-143

[7] Bohl H. (1957) Die Biologie der Kliesche (Limanda limanda) in der Nordsee. Berichte der Deutschen Wissenschaftlichen Kommission für Meeresforschung, 15: 1-57

[8] Bolker J.A. and Hill C.R. (2000) Pigmentation development in hatchery-reared flatfishes. Journal of Fish Biology, 56: 1029-1052

[9] Bolker J.A., Hakala T.F., Quist J.E. (2005) Pigmentation development, defects, and patterning in summer flounder (Paralichthys dentatus). Zoology, 108: 183-193

[10] Bruno D.W. and Poppe, T.T. (Ed.) (1996) In: A Colour Atlas of Salmonid Diseases. Academic Press, London: 194pp.

[11] BSH (2003) Loewe P., Becker G., Brockmann U., Frohse A., Herklotz K., Klein H., Schultz A. (Ed.) In: Nordsee und Deutsche Bucht 2002 - Ozeanographischer Zustandsbericht. Hamburg - Germany, Berichte des BSH (Federal Maritime and Hydrographic Agency), 33: 89pp.

[12] Bucke D., Vethaak D., Lang T., Mellergaard S. (1996) Common diseases and parasites of fish in the North Atlantic: training guide for identification. ICES Techniques in Marine Environmental Sciences, 19: 27pp.

[13] Bullock A.M. (1982) The pathological effects of ultraviolet radiation on the epidermis of teleost fish with reference to the solar radiation in higher . The Royal Society of Edinburgh, 81: 199-210

[14] Campos W.L., Kloppmann M., Von Westernhagen H. (1994) Inferences from the horizontal dis- tribution of Dab (Limanda limanda) and (Platichthys flesus) larvae in the Southeastern North Sea. Netherlands Journal of Sea Research, 32: 277-286

[15] Copeman L.A. and Parrish C.C. (2002) Lipid composition of malpigmented and normally pigmented newly settled yellowtail flounder, Limanda ferruginea (Storer). Aquaculture Research, 33 (15): 1209-1219

[16] Crutzen P.J. (1992) Ultraviolet on the increase. Nature, 356: 104-105 ICES ASC 2008 20

[17] Daan N., Bromley P.J., Hislop J.R.G., Nielsen N.A. (1990) Ecology of North Sea Fish. Netherlands Journal of Sea Research, 26: 343-386

[18] Damm U., Lang T., Rijnsdorp A.D. (1991) Movements of Dab (Limanda limanda) in the German Bight and Southern Bight: Results of German and Dutch Tagging Experiments in 1988, 1989: ICES CM, 2: 18pp.

[19] De Lange H.J., Van Donk E., Hessen D.O. (2000) In situ effects of UV radiation on four species of phytoplankton and two morphs of Daphnia longispina in an alpine lake (Finse, Norway). Verh. Internat. Verein. Limnol., 27: 2008-2013

[20] De Veen J.F. (1969) Abnormal pigmentation as a possible tool in the study of the populations of the (Pleuronectes platessus L.). Conseil Permanent International Pour L‘Exploration De La Mer, 32: 344-383

[21] Dethlefsen V., Watermann B., Hoppenheit M. (1987) Disease of North Sea dab (Limanda limanda) in relation to biological and chemical parameters. Archiv für Fischereiwissenschaften, 37: 107-237

[22] Dethlefsen V., von Westernhagen H., Tüg H., Hansen P.D., Dizer H. (2001) Influence of solar ultraviolet-B on pelagic fish embryos: osmolality, mortality and viable hatch. Helgoland Marine Research, 55: 45-55

[23] Dulvy N.K, Rogers S.I., Jennings S., Stelzenmuller V., Dye S.R., Skjoldal H.R (2008) Climate change and deepening of the North Sea fish assemblage: a biotic indicator of warming seas. Journal of Applied Ecology, 45 (4): 1029-1039

[24] Estevez A., McEvoy L.A., Bell J.G., Sargent J.R. (1999) Growth, survival, lipid composition and pigmentation of turbot (Scophthalmus maximus) larvae fed live-prey enriched in Arachidonic and Eicosapentaenoic acids. Aquaculture, 180: 321-343

[25] Fabacher D.L., Little E.E., Ewing M.S., Kocan K.M. (1997) Effects of Ultraviolet-B Radiation on Fish: Histologic Comparison of a UVB-Sensitive and a UVB-Tolerant Species. Journal of Aquatic Animal Health, 9: 132-143

[26] Fabacher D.L. and Little E.E. (1995) Skin component may protect fishes from ultraviolet-B radiation. Environmental Science and Pollution Research, 2: 30-32

[27] Feist S.W., Lang T., Stentiford G.D., Köhler A. (2004) Biological effects of contaminants: The use of liver pathology of the European flatfish, dab (Limanda limanda L.) and flounder (Platichthys flesus L.) for monitoring biological effects of contaminants. ICES Techniques in Marine Environmental Sciences, 28: 47pp.

[28] Gartner J.V. (1986) Observations on anomalous conditions in some flatfishes (Pisces: Pleuronecti- formes), with a new record of partial allbinism. Environmental Biology of Fishes, 17: 141-152

[29] Haekkinen J., Vehniäinen E., Ylönen O., Heikkilä J., Soimasuo M., Kaurola J., Oikari A., Karjalainen J. (2002) The Effects of Increasing UV-B Radiation on Pigmentation, Growth and Sur- vival of Coregonid Embryos and Larvae. Environmental Biology of Fishes, 64: 451-459

[30] Hamre K., Holen E., Moren M. (2007) Pigmentation and eye migration in Atlantic (Hip- poglossus hippoglossus L.) larvae: new findings and hypotheses. Aquaculture Nutrition, 13 (1): 65-80

[31] Herndl G.J. (1993) Major role of Ultraviolet-B in controlling bacterioplankton growth in the surface layer of the ocean. Nature, 361: 717-719 ICES ASC 2008 21

[32] Hessen D.O. (1996) Competitive trade-off strategies in artic Daphnia linked to lelanism and UV-B stress. Polar Biology, 16: 573-579

[33] Hughes S.L. (Ed.) (2004) In: The Scottish ocean climate status report 2002 and 2003. Aberdeen: Fisheries Research Services. 50pp.

[34] Jokinen E.I., Salo H.M., Markkula S.E., Aaltonen T.M., Immonen A.K. (2000) Effects of ultra- violet light on immune parameters of the roach. Toxicology Letter, 15: 303-310

[35] Kerr J.B. and McElroy C.T. (1993) Evidence for large upward trends of Ultraviolet-B Radiation linked to Ozone Depletion. Science, 262: 1032-1034

[36] Kirby M.F., Lyons B.P., Barry J. and Law R.J. (2007). The toxicological impacts of oil and chemically dispersed oil: UV mediated phototoxicity and implications for environmental effects, statutory testing and response strategies. Marine Pollution Bulletin, 54(4): 472-475

[37] Knijn R.J., Boon T.W., Heessen H.J.L., Hislop J.R.G. (1993) Atlas of North Sea Fishes - Based on bottom-trawl survey data for the years 1985 - 1987. ICES Cooperative Research Report, No.194: 1-268

[38] Knust R. (1990) Food and condition of dab (Limanda limanda) from the Dogger Bank and the German Bight. ICES CM, 62: 1-7

[39] Lang T., Dethlefsen V., von Westernhagen H. (2003) Fischkrankheiten und embryonale Miss- bildungen. In: Lozan J.L., Rachor E., Reise K., Sündermann J., v. Westernhagen H. (Ed.); Warnsignale aus Nordsee & Wattenmeer: Eine aktuelle Umweltbilanz. Wissenschaftliche Auswertungen, Hamburg - Germany: 219-228

[40] Lesser M.P., Farrell J.H., Walker C.W. (2001) Oxidative stress, DNA damage and p53 expression in the larvae of (Gadus morhua) exposed to ultraviolet (290 - 400 nm) radiation. Journal of Experimental Biology, 204: 157-164

[41] Lyons B.P., Pascoe C.K., McFadzen I.R.B. (2002) Phototoxicity of pyrene and benzo[a]pyrene to embryo-larval stages of the Pacific oyster Crassostrea gigas. Marine Environmental Research 54, 627-631

[42] Lyons B.P., Goodsir F., Thain J.E., Wedderburn J., McFadzen I.R.B. (2006). Toxicity and pho- totoxicity of sea surface microlayer samples collected from the North Sea to embryo-larval stages of the pacific oyster Crassostrea gigas. In: Biological effects of contaminants in pelagic ecosystems. Hylland K., Lang T., Thain J., Vethaak A.D., Wosniok W. (Ed.): Society of Environmental Toxicology and Chemistry (SETAC), Brussels (2006), 367376

[43] Lozan J.L. (1988) Verbreitung, Dichte und Struktur der Population der Kliesche (Limanda limanda) in der Nordsee mit Vergleichen zu den Populationen um Island und in der Ostsee anhand meristischer Merkmale. Archiv für Fischwissenschaft, 38: 165-189

[44] Lozan J.L. (1989) Autoökologische Untersuchungen über die Kliesche (Limanda limanda) der Nordsee. Disseration: Bundesanstalt für Fischerei. Hamburg - Germany, Nr.101

[45] Lozan J.L. (1992) Sexual Differences in food intake, digestive tract size, and growth performance of dab, Limanda limanda. Netherlands Journal of Sea Research, 29: 223-227

[46] Macieira R.M., Joyeux J.C., Chagas L.P. (2006) Ambicoloration and morphological aberration in the sole Achirus declivis (Pleuronectiformes: Achiridae) and two other cases of color abnormalities in achirid soles from southeastern Brazil. Neotrop. Ichthyol., 4: 287-290 ICES ASC 2008 22

[47] McEvoy L.A., Naess T., Bell J.G., Lie O. (1998) Lipid and fatty acid composition of normal and malpigmented Atlantic halibut (Hippoglossus hippoglossus) fed enriched Artemia: a comparison with fry fed wild copepods. Aquaculture, 163: 237-250

[48] McFadzen I.R.B., Baynes S., Hallam J., Beesley A., Lowe D. (2000) Histopathology of the skin of UV-B irradiated sole (Solea solea) and turbot (Scophthalmus maximus) larvae. Marine Environmental Research, 50: 273-277

[49] MCCIP (2006) Annual Report Card 2006. Marine climate change impacts partnersship. www.mccip.org.uk/arc

[50] McVicar A.H., Bruno D.W., Fraser C.O. (1988) Fish disease in the North sea in relation to sewage sludge dumping. Marine Pollution Bulletin 19: 169-173

[51] Megie G. (Ed.) (1991) In: Ozon - Atmosphäre aus dem Gleichgewicht. Springer-Verlag, Berlin - Ger- many, 177pp.

[52] Mintenbeck K. (2007) Unpublished work. Durchführung von Mageninhaltsanalysen von Fischen zur Umsetzung der Ergebnisse des Forschungsprojektes: Identifizierung von organischen Schadstoffen in Nord- und Ostsee. Projektbericht für das Umweltbundesamt, FKZ 363 01 141, 31 pp.

[53] Naess T. and Lie O. (1998) A sensitive period during first feeding for the determination of pigmenta- tion pattern in Atlantic halibut, Hippoglossus hippoglossus L., juveniles: the role of diet. Aquaculture Research, 29: 925-934

[54] OSPAR (1997) JAMP Guidelines for general biological effects monitoring. OSPAR Commission Ref. No. 1997-7.

[55] OSPAR (2003) JAMP Guidelines for contaminant-specific biological effects monitoring. OSPAR Com- mission Ref. No. 2003-10.

[56] Ottesen O.H. and Strand H.K. (1996) Growth, development, and skin abnormalities of halibut (Hip- poglossus hippoglossus) juveniles kept on different bottom substrates. Aquaculture, 146: 17-25

[57] Rainuzzo J.R., Reitan K.I., Jorgensen L., Olsen Y. (1994) Lipid composition in turbot larvae fed live feed cultured by emulsions of different lipid classes. Comparative Biochemistry and Physiology, 107A (4): 699-710

[58] Reid P.C., Holliday N.P., Smyth T.J. (2001) Pulses in the eastern margin current and warmer water off the north west European shelf linked to North Sea ecosystem changes. Marine Ecology Progress Series, 15: 283-287

[59] Rhode S.C., Pawlowski M., Tollrian R. (2001) The impact of ultraviolet radiation on the vertical distribution of zooplankton of the genus Daphnia. Nature, 412: 69-72

[60] Rijnsdorp A.D., Vethaak A.D., Van Leeuwen P.I. (1992) Population biology of dab Limanda li- manda in the southeastern North Sea. Marine Ecology Progress Series, 91: 19-35

[61] Saborowski R. and Buchholz F. (1996) Annual changes in the nutritive state of North Sea dab. Journal of Fish Biology, 49: 173-194

[62] Schubert M. (2004) Auswirkung der UV-Strahlung auf Fischbrut. Rundgespräche der Kommission für Ökologie, Bayerische Akademie der Wissenschaft, 27: 117-123 ICES ASC 2008 23

[63] Steeger H.U., Freitag J.F., Michl S., Wiemer M., Paul R.J (2001) Effects of UV-B radiation on embryonic, larval and juvenile stages of North Sea plaice (Pleuronectes platessa) under simulated ozone-hole conditions. Helgoland Marine Research, 55: 56-66

[64] Venizelos A. and Benetti D.D. (1999) Pigment abnormalities in flatfish. Aquaculture, 176: 181-188

[65] Wiltshire K.H. and Manly B.F.J. (2004) The warming trend at Helgoland Roads, North Sea: phyto- plankton response. Helgoland Marine Research, 58: 269273

[66] Yamanome T., Chiba H., Takahashi A. (2007) Melanocyte-stimulating hormone facilitates hyper- melanosis on the non-eyed side of the barfin flounder, a pleuronectiform fish. Aquaculture, 270: 505-511

[67] Yamanome T., Amano M., Takahashi A. (2005) White background reduces the occurrence of staining, activates melanin-concentrating hormone and promotes somatic growth in barfin flounder. Aquaculture, 244: 323-329

[68] Zerefos C., Meleti C., Balis D., Tourpali K., Bais A.F. (1998) Quasi-biennial and longer-term changes in clear sky UV-B solar irradiance. Geophysical Research Letters, 25 (23): 4345-4348

[69] Zuehlke R.(2001) Monitoring Biodiversity of Epibenthos and Demersal Fish in the North Sea and Skagerrak. EC Project: 98/021. Monitoring Report 2001 to the Commission of the European Community. 98pp.