Influence of anthropogenic stress on fitness and behaviour of a key-species of estuarine ecosystems, the ragworm Nereis diversicolor

Environmental Pollution 158 (2010) 121–128

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Influence of anthropogenic stress on fitness and behaviour of a key-species of estuarine ecosystems, the ragworm Nereis diversicolor C. Mouneyrac a, b, *, H. Perrein-Ettajani a, b, C. Amiard-Triquet c a

MMS, EA2160, Faculte´ de pharmacie, 1 rue G. Veil, BP 53508, 44035 Nantes Cedex 1, France Institut de Biologie et Ecologie Applique´e, CEREA, Universite´ Catholique de l’Ouest, 3 Place Andre´ Leroy, Angers, 44 rue Rabelais, 49008 Angers Cedex 01, France c CNRS, Universite´ de Nantes, MMS, EA2160, Faculte´ de pharmacie, 1 rue G. Veil, BP 53508, 44035 Nantes Cedex 1, France b

Fitness, and behaviour in Nereis diversicolor are affected by anthropogenic pressure.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 January 2009 Received in revised form 21 July 2009 Accepted 23 July 2009

Fitness, (biometric measurements, reproduction) and behaviour that are ecologically relevant biomarkers in assessing the quality of estuarine sediments were studied by comparing the responses of the polychaete worm Nereis diversicolor – a key species in estuaries – along a pollution gradient. Intersite differences were shown for all the measured parameters: size–weight relationships, energy reserves as glycogen and lipids, sexual maturation patterns, total number of oocytes per female, total and relative fecundity, burrowing behaviour. The physiological and behavioural status of N. diversicolor was consistently disturbed in the larger, most contaminated estuaries (Loire and Seine, Fr.) compared to reference sites (Bay of Bourgneuf, Goyen estuary, Fr.). Many classes of potentially toxic chemicals present in these estuaries most likely contribute to these impairments but food availability may act as a confounding factor, interfering with the potential impact of contaminants. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Nereis diversicolor Fitness Reproduction Behaviour Ecologically relevant biomarkers

1. Introduction Estuaries are crucial in the life histories of many fish, invertebrates, birds, etc., and the sustainability of estuarine biodiversity is vital to the ecological and economic health of coastal regions. River transport is responsible for the influx of both nutrientsdunderlying the biological wealth of estuarine areas as well as the high productivity of nearby coastal areasdand toxic anthropogenic effluents from the whole river basin. Water quality in estuaries has also been decreasing as a consequence of inputs of chemicals associated with industrial and domestic activities, pesticides and fertilizers originating from agriculture historically, and estuaries have been areas of settlement for many human populations. Thus complex mixtures are present in estuarine areas, including many classes of compounds which are not yet accessible to analysis or are extremely expensive to analyse. In addition, natural factors such as salinity, temperature, hypoxia, are highly fluctuating is estuaries and can also influence biological responses. Even in a well-adapted coastal and estuarine species such as the ragworm Nereis

* Corresponding author at: MMS, EA2160, Faculte´ de pharmacie, 1 rue G. Veil, BP 53508, 44035 Nantes Cedex 1, France. Tel.: þ33 02 41 81 66 45; fax: þ33 02 41 81 66 74. E-mail address: [email protected] (C. Mouneyrac). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.07.028

diversicolor, it remains a challenge to assess the impact of natural and chemical environmental stresses. A biomarker is defined by Depledge (1993) as ‘‘A biochemical, cellular, physiological or behavioural variation that can be measured in tissue or body fluid samples or at the level of whole organisms that provides evidence of exposure to and/or effects of, one or more chemical pollutants (and/or radiations).’’ According to Lam and Gray (2003), biomarkers are relatively effective in revealing overall toxicities of complex mixtures, particularly those which are at a high level of biological organization such as physiological biomarkers relating to the growth or reproduction of individual organisms. This is also relevant in order to take into account non-chemical stress. Biochemical or physiological biomarkers are often suspected of being inefficient in predicting effects at higher levels of biological organization (Forbes et al., 2006) thus hampering ecological risk assessment. However, many recent studies have demonstrated that physiological biomarkers can be used to predict populational- and community-level parameters (Maltby et al., 2001; De Coen and Janssen, 2003; Baird et al., 2007; Durou et al., 2007b) as well as behavioural biomarkers (Dell’Omo, 2002; Amiard-Triquet, 2009). To interpret ecotoxicological data, it is needed to know the natural factors which can influence the physiology and the biology of the species studied. The abundant literature on energetics in polychaetes (Blackstock et al., 1982; Olive et al., 1985; Cammen, 1987; Durou et al., 2007b) provides a convenient basis.

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Behavioural endpoints are particularly useful to study the impact of contaminants in aquatic ecosystems because they are relatively sensitive, showing disturbances at concentrations far below those inducing mortality. This sensitivity is well-documented in the polychaete N. diversicolor (Moreira et al., 2006; Bonnard et al., 2009). Because sediments are the main sink and cycling center for most of the chemicals entering the environment (Gagnon and Fisher, 1997; Bernes, 2000), endobenthic species such as N. diversicolor appear as good models to assess the quality of sediments. Many reports exist about the use of this species for biomonitoring purposes (Scaps, 2002; Ait-Alla et al., 2006; Galloway et al., 2006; Moreira et al., 2006; Poirier et al., 2006; Durou et al., 2007a and literature cited therein). The ecological relevance of ecotoxicological studies may be improved by the choice of sentinel species among those which are key species in the functioning of the ecosystem of interest. In the case of N. diversicolor, bioturbation caused by the worm greatly affects the biogeochemical cycles of both nutrients and contaminants (Davey and Watson, 1995; Gunnarsson et al., 1999; Banta and Andersen, 2003 and literature cited therein, Gerino et al., 2003). N. diversicolor is also a main food source for many estuarine species such as fish and wading birds (Mc Lusky, 1989; Masero et al., 1999; Moreira, 1999). The aim of the present study is to investigate the potential disturbances experienced by N. diversicolor living in large, multistressed estuaries (Loire and Seine, Fr) by comparison with less contaminated sites such as a little estuary with low anthropogenic pressure (Goyen estuary, Fr) and a coastal area mainly devoted to oyster culture (Bay of Bourgneuf, Fr.). Impairments at the suborganismal (energy reserves) and individual levels (biometric measurements, sexual maturity, total number of oocytes per female, fecundity, burrowing kinetics) were examined in order to forecast potential effects at higher levels of biological organization through cascading effects. 2. Materials and methods 2.1. Choice of sampling sites Because it is impossible to analyse all the chemicals able to impair biological responses, the sampling areas of interest in the present study have been selected taking into account informations about i) the anthropogenic pressure both locally and at the scale of the river basin; ii) the data available for contaminants in the framework of the French ‘‘Mussel Watch’’; iii) the data provided by the regulation authorities for the implementation of the European Community Water Framework Directive (ECWFD, 2000). The Seine valley and its estuary are of major economic importance for France, notably due to the presence of two maritime ports of international importance associated with major river settlementsdRouen with 400,000 inhabitants and Le Havre with 200,000 inhabitants. The Seine estuary lies at the discharge point of a watershed area covering 79,000 km2. This area is home to 16 million people, and accounts for 50% of the river traffic in France, 40% of the country’s economic activity, and 30% of its agricultural activities. In addition, the Greater Paris area with its more than 10 million inhabitants contributes heavily to the Seine estuary’s upstream inputs. The Loire estuary is also exposed to a large range of chemical contaminants but at a lesser level than in the Seine. The Bay of Bourgneuf is mainly devoted to oyster culture and is a comparatively clean reference area. The ecological value of the Goyen, as well as the inputs of pollutants and nutrients in this river, are in agreement with the environmental aims defined in the ECWFD (Agence de l’Eau Loire-Bretagne en, 2004). All of these sites are near sampling sites monitored in the framework of the French ‘‘Mussel Watch’’ Programme (Claisse et al., 2006). Mussels originating from the mouth of the Seine river exhibit very high levels of silver, CB 153 (representative of PCB contamination) and fluoranthene (representative of PAH contamination) which are one order of magnitude higher than in the mouth of the Loire river. Levels of chromium, mercury, lead and S DDT (DDT and its metabolites DDD and DDE) were higher in the former whereas no consistent differences were shown for cadmium, copper, nickel and zinc. The Bay of Bourgneuf and the Goyen estuary were used as references since they are comparatively clean areas. The precise locations, salinity and temperature are shown in Table 1. Salinity was measured in the field on each sampling occasion in the water remaining at the surface of the

mudflat at low tide whereas mean air temperature was calculated from daily values available at www.meteociel.fr. 2.2. Collection N. diversicolor were collected gently by hand, at low tide, at the upper level of the intertidal mudflats. Three samplings were carried out according to the different objectives of the study. For the investigation of biometric measurements, sexual maturity and oocyte number (total number of oocytes, fecundity), fifty individuals were collected at random, on each site, in January, March, June and September 2006. An additional sampling was carried out in April 2007 for oocyte quantification. Worms were transported to the laboratory in cool boxes filled with sediment covered with approximately 2 cm of seawater and green algae from the site of origin. To allow them to eliminate their gut contents, worms were then transferred to aquaria containing filtered (10 mm) natural seawater used in oyster farming adjusted to the salinity of the sites of origin for 1 day. Starvation took place in a controlled room in which the temperature was nearing the field temperature at each sampling date. Then, each specimen was wiped for 1 min on absorbent paper, and then weighed using a precision balance. Further on, worms were fixed with 10% formalized seawater for storing them until analyses (length measurements, reproduction endpoints). Concerning quantification of energy reserves and sexual steroids, after collection, worms were immediately frozen in liquid nitrogen and transported to the laboratory, then stored at 80  C until analysis. For worms devoted to burrowing tests; individuals of N. diversicolor, during spring 2007 (Mid-April–early June), were handpicked at low tide from intertidal mudflats and transported with fresh algae in a cold container to the laboratory. 2.3. Biometric measurement and reproduction endpoints In N. diversicolor, the major difficulty in the calculation of a condition index is that they have a soft body and hard parts are scarce. The size–weight relationships have been proposed as tools for assessing the health-status of N. diversicolor (Durou et al., 2008). Because individuals can accidentally loose parts of their soft body and also have the ability to autotomize in response to stress like handling, the use of the length of the first three segments named L3 (peristomium, prostomium and first chaetiger) was recommended as a measure of size and is used in population studies (Gillet and Torresani, 2003). The L3 length was measured under a binocular magnifying glass. In N. diversicolor, the sex ratio (80%) is greatly in favour of females (Dales, 1950; Smith, 1976; Olive and Garwood, 1981; Mettam et al., 1982), thus the sexual maturity stages were determined only in female worms. In order to determine the sex and the sexual maturity stage of worms, an incision of individuals using a scalpel, approximately 30 segments behind the head, was realized. The segment content was deposited on a glass side and the coelomic fluid was examined under microscope. The worm’s sex and sexual maturity stages of females were determined according to Durou and Mouneyrac (2007) indifferent stage: presence of germ cells in the

Table 1 Precise locations of the sampling sites and temporal fluctuations of ecological parameters. Sites

Ecological parameters

Bourgneuf 2 040 40.6000 W 46 560 23.0800 N

January 2006

March 2006

June 2006

September 2006

April 2007

Temperature ( C)a Minimum 7.5 Maximum 1.5 Salinity 35.9

11.4 4.3 27.8

24.5 11.9 35.4

23.4 11.1 29.1

20.4 8.7 33

Loire 2 080 52.3800 W 47 160 09.8800 N

Temperature ( C)a Minimum 7.5 Maximum 1.5 Salinity 20.3

11.4 4.3 19.6

24.5 11.9 21.3

23.4 11.1 24.8

20.4 8.7 16.9

Seine 0 050 57.7200 W 49 280 44.8900 N

Temperature ( C)a Minimum 5 Maximum 0.5 Salinity 18.7

8.7 3.4 18.9

21.1 12.5 22.2

21.5 14.8 19.8

19.8 7.2 23.3

Goyen 4 290 14.6200 W 48 020 28.7100 N

Temperature ( C)a Minimum Maximum Salinity

18.9 8.8 11.4

a Mean of air temperature registered daily during the month of sampling at Saint Nazaire (11 km from the sampling site in the Loire estuary; 49 km from the sampling place in the Bay of Bourgneuf), Le Havre (3 km from the sampling site in the Seine estuary) and Quimper (36 km from the sampling site in the Goyen estuary).

C. Mouneyrac et al. / Environmental Pollution 158 (2010) 121–128 coelomic fluid (beginning of gametogenesis); development stage: small oocytes (20–65 mm) and oogonial cluster are present in the coelomic cavity; growth stage: growing oocytes (65–190 mm) freely suspended in the coelomic fluid, mature stage: oocytes large and spherical (190–225 mm). Sexually undifferentiated (no sexual products in the coelomic cavity) worms were also noted. For oocyte quantification, females were opened from the anterior to the posterior end in a Petri dish. The content was released, then shifted at first on a sieve of 300 mm mesh size to eliminate larger fragments resulting from dissection and then shifted on a sieve of 25 mm mesh size to collect oocytes. Next, oocytes were re-suspended in seawater filtered at 10 mm and adjusted to a salinity of 8 (V ¼ 25 mL). This solution was centrifuged for 10 min at 12,000 g and stored at 4  C during 36 h until the complete settlement of oocytes allowing to eliminate the major part (80%, V ¼ 20 mL) of the supernatant. Oocytes were re-suspended (V ¼ 5 mL) and three aliquots were sampled from this suspension. The number of oocytes was counted under a binocular microscope using a Nageotte hematocytometer (Durou et al., 2008). Total fecundity was estimated in mature females by the total number of oocytes >190 mm diameter. Relative fecundity corresponded to the ratio between the total number of oocytes >190 mm diameter and the wet weight of each mature female. 2.4. Biochemical parameters As an influence of weight on lipid concentrations has been previously reported in N. diversicolor (Mouneyrac et al., 2006; Durou et al., 2007a), only worms within the same wet weight range were selected for statistical analysis, according to the different objectives of the study: i) influence of sampling date on energy reserves in worms originating from the three studied sites, ii) influence of site on the pattern of energy reserves. Energy reserves (glycogen and lipids) were quantified in worms according to Durou and Mouneyrac (2007). 2.5. Burrowing tests The burrowing tests were described previously by Bonnard et al. (2009). Briefly, the burrowing behaviour of N. diversicolor was observed in animals placed in individual plastic units of 100 mL, filled with 5 cm of natural (collected from the sampling sites) sediment. Three experiments were conducted; A: ragworms allowed to burrow in their sediment of origin (collected from the same site as worms), B: ragworms from a reference site (Goyen) allowed to burrow either in their sediment of origin or sediments from other sites (Bay of Bourgneuf or Loire estuary or Seine estuary), C: ragworms from the contaminated Seine estuary allowed to burrow in their sediment of origin or sediment from the reference Goyen estuary. When worms were placed on the sediment, their positions were recorded every 2 min. The time when each organism was totally burrowed in the sediment was considered in the analysis of results. Twenty specimens were tested for each experimental condition. Burlinson and Lawrence (2007) have shown that worms were not affected by consecutive bioassays over one week. Thus in the present study, all the tests were carried out within a delay less than 1 week after collection. 2.6. Statistical analyses Statistical analyses were performed using SPSSÒv12.0 and XLSTATÒ v7.5. The curves showing the relationship between L3 length and wet weight as well as the burrowing kinetic curves of organisms were ln-transformed for linearization. Then, they were compared by using analysis of covariance (ANCOVA) between regression coefficients of the least-square regression lines. Normality of data was checked using Shapiro and Wilk’s W-test. To assess comparisons of means, a variance analysis (ANOVA) and then a Tukey’s test were performed.

3. Results 3.1. Relationships between L3 and wet weight The relationships between L3 length and wet weight was investigated in worms originating from three studied sites (Bay of Bourgneuf: N ¼ 157; Loire: N ¼ 141; Seine: N ¼ 107) and collected on four occasions (January, March, June and September 2006). In all cases (for each site), the relationships followed an exponential pattern (Fig. 1). It must be duly noted that the L3 length for the Bay of Bourgneuf and Seine specimens never reached the maximum values registered for Loire specimens. For each sampling month, linear regressions between L3 length and wet weight obtained after ln-transformation and the analyses of covariance using the site of origin as covariate are illustrated in Table 2. Except in two cases, significant intersite differences were observed. For an identical size assessed by L3 length, the body weight was generally lower in specimens from the Loire estuary.

123

3.2. Sexual maturity The distribution of N. diversicolor from the three studied sites according to sexual maturity stages is shown in Fig. 2. For worms from the reference site (Bay of Bourgneuf), sexually undifferentiated worms or females in indifferent stage were observed at each sampling period. In January and March 2006, 51% and 66% of worms were in development stage whereas in June and September only 22% and 10% of organisms were found in these stages. The highest percentage of females in growth stage was observed in September (31%) in comparison with the other sampling dates (January: 11%, March and June: 7%). Concerning worms from the Loire contaminated site, females were predominantly (between 75% and 93%) in development and growth stages for the whole sampling periods. A small percentage (5%) of undifferentiated worms was observed only in January. For worms from the Seine estuary, no sexually undifferentiated specimens or females in indifferent stage were observed in January whereas in June and September, the five sexual maturity stages were identified with higher percentages of undifferentiated animals or mature females in September (undifferentiated: 32%, mature: 11%) than in June (undifferentiated: 14%, mature: 2%). Mature females were observed in worms from the three studied sites in June and September (Bay of Bourgneuf: 4% and 3%, Loire: 15% and 3%, Seine: 2% and 11%, respectively). Concerning intersite differences, in January, the percentage of sexually undifferentiated worms or females in indifferent stage was higher in the Bay of Bourgneuf (11% and 26%, respectively) in comparison with the Loire estuary (5% and 3%, respectively). In contrast, no worms in these latter stages were observed in the Seine estuary. Concerning the other sampling periods (March, June and September 2006), no sexually undifferentiated worms were observed in the Loire estuary and the percentage of females in indifferent stage was always lower (March: 7%, June: 11%, September: 13%) in comparison with Bay of Bourgneuf (March: 24% June: 48%, September: 24%) or the Seine estuary (June: 38%, September: 14%). Worms from the Loire estuary were proportionally more in active gametogenesis (development, growth and mature stages) comparing to those from the Bay of Bourgneuf or the Seine estuary. 3.3. Oocyte production The production of oocytes by females from three studied sites is depicted in Table 3. For worms from the reference site (Bay of Bourgneuf), minimum values of the total number of oocytes per female were observed in January, March and September and maximum in June 2006 and April 2007. The total production was significantly (p < 0.005) lower in September in females from the Loire estuary. Minimum and maximum values of the total fecundity were observed in January 2006 and April 2007, respectively in specimens from the Seine estuary. The total number of oocytes was significantly (<0.05) higher in worms from both contaminated sites (the Loire and Seine estuaries) in comparison with those from the reference site excepted in June 2006 where no significant difference was observed between specimens from the Seine and the Bay of Bourgneuf. Even if the total oocyte number was higher in females from the Loire than in the Seine in January and June 2006, with regard to relative oocyte number (ratio between the total number of oocytes and the weight of each female) no differences were observed between both contaminated sites whereas significantly lower values were determined in worms from the reference site (Bay of Bourgneuf). The total fecundity estimated by the number of oocytes with a diameter >190 mm in mature females and the relative fecundity

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C. Mouneyrac et al. / Environmental Pollution 158 (2010) 121–128 1,2

1,3087x

Bay of Bourgneuf : y = 0,0171e R2 = 0,5391

Loire : y = 0,0177e1,1375x R2 = 0,7129

Seine : y = 0,0061e1,7539x 2 R = 0,578

Wet weight (g)

1

0,8

0,6 Bourgneuf Loire

0,4

Seine

0,2

0 0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

Length ofr L3 (en mm) Fig. 1. Relationships between the L3 length and the wet weight in Nereis diversicolor originating from the Bay of Bourgneuf (; N ¼ 157), Loire (,; N ¼ 241) and Seine (:; N ¼ 108) estuaries.

Mean values of energy reserves (glycogen, lipids) in N. diversicolor collected in three studied sites in January, March, June and September 2006 are depicted in Fig. 4. The most striking feature is the higher values of energy reserves as glycogen and lipids in worms from the Loire estuary. Intersite differences in energy reserves in worms according to their size classes (not shown) indicated that glycogen and lipid concentrations were, in the majority of the cases (11/12 for glycogen; 9/12 for lipids), significantly higher (p < 0.05) in worms from the Loire estuary in comparison with the Bay of Bourgneuf and the Seine estuary. No differences were observed in glycogen and lipid concentrations between specimens from the Bay of Bourgneuf and the Seine estuary. 3.5. Burrowing behaviour

Bay of Bourgneuf Sexual maturity stage (%)

3.4. Biochemical parameters

burrowing rate was determined for specimens from the Seine and the highest for those originating from the Goyen (reference site added for behavioural tests). Worms from the Bay of Bourgneuf and from the Loire estuary showed intermediate values. The behaviour of worms from the Goyen differed significantly from all three of the

100% 80% 60% 40% 20% 0% January

Date

Site

a

b

R

Comparison

p value

January

B L S

1.186 2.132 2.586

1.184 1.435 1.713

0.287 0.797 0.807

B vs L L vs S B vs S

<0.0001 0.023 <0.0001

March

B L S

2.190 2.443 –

1.278 1.507 –

0.528 0.794 –

B vs L L vs S B vs S

<0.0001 – –

June

B L S

2.730 1.947 1.710

1.403 1.202 1.205

0.729 0.665 0.389

B vs L L vs S B vs S

0.037 0.052 0.922

September

B L S

1.793 2.283 3.355

1.162 1.352 1.664

0.562 0.513 0.704

B vs L L vs S B vs S

0.006 0.005 <0.0001

September

80% 60% 40% 20% 0% January

March

June

September

Seine estuary Sexual maturity stage (%)

2

June

100%

The burrowing kinetic curves of worms in the sediments collected from their site of origin are shown in Fig. 5A. The lowest Table 2 Intersite comparison of size (L3 length) to wet weight linear regressions after logarithm transformation. (B ¼ Bay of Bourgneuf, L ¼ Loire, S ¼ Seine).

March

Loire estuary Sexual maturity stage (%)

(ratio between total number of oocytes >190 mm diameter and the weight of each female) in the three studied sites are depicted in spring 2006 and 2007 and autumn 2006 (Fig. 3A). Total and relative fecundity were higher (not significant) in specimens from both contaminated sites (Loire and Seine estuaries) in comparison with the reference site (Bay of Bourgneuf). The L3 length and the wet weight of mature females are shown in Fig. 3.B. The L3 length was significantly higher in worms from the reference site (Bay of Bourgneuf) compared with contaminated sites, whereas no significant differences were observed for the weight.

100% 80% 60% 40% 20% 0% January

March

June

September

Fig. 2. Sexual maturation in Nereis diversicolor collected from the Bay of Bourgneuf, Loire and Seine estuaries in January (Bay of Bourgneuf: N ¼ 35, Loire: N ¼ 40, Seine: N ¼ 24), March (Bay of Bourgneuf: N ¼ 29, Loire: N ¼ 29), June (Bay of Bourgneuf: N ¼ 28, Loire: N ¼ 28, Seine: N ¼ 46) and September (Bay of Bourgneuf: N ¼ 21, Loire: N ¼ 21, Seine: N ¼ 28) 2006 (percentage of each development stage in the whole sample examined). : sexually undifferentiated, : Indifferent, : Development, : Growth, : Mature.

Date

Site

January 2006

March 2006 June 2006

N

Total number of oocytes

Relative number of oocytes

Mean

Mean

Min.

Max.

Min.

Max.

B L S

17 37 22

2322 11,414 4321

67 67 67

16,067 55,667 26,667

15 48 53

0.7 1.5 3.4

83 161 389

B L

11 21

3430 11,800

67 67

3800 83,600

15 47

0.3 1.2

51 152

B L S

8 24 20

7008 10,600 7063

20 1067 200

15,333 66,067 18,000

19 34 32

0.6 6.1 1.0

41 178 75

Sept. 2006

B L S

17 24 15

3169 6143 6053

133 333 333

15,667 31,867 11,000

17 38 37

1.2 6.6 8.3

58 95 81

April 2007

B L S

15 25 26

7969 16,744 13,692

67 333 3600

22,867 103,600 27,647

25 55 47

0.8 3.2 14.6

72 250 116

other sites (Table 4). When specimens from the Goyen were allowed to burrow in sediments from the other sites, their burrowing rate was significantly higher in the sediment from their site of origin (Fig. 5B and Table 4). When worms from the multipolluted Seine estuary were allowed to burrow in the sediment from the Goyen, their burrowing rate was significantly higher in this comparatively clean sediment than in their sediment of origin (Fig. 5C and Table 4).

A

40000

Number of oocytes

35000

Total fecundity Relative Fecundity

30000 25000 20000 15000 10000 5000 0

B

Loire

2.50

Seine

L3 w.w

L3 (mm)

2.00 1.50 1.00 0.50 0.00 Bourgneuf

Loire

0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

Wet Weight (g)

Bourgneuf

Seine

Fig. 3. Total and relative fecundity (means  S.D, A) and L3 and wet weight (means  S.D, B) of mature females of N. diversicolor from the three sites (Bay of Bourgneuf, Loire, Seine).

125

30

Bay of Bourgneuf

25

Loire estuary

20

Seine estuary

15 10 5 0 January

March

June

September

14 Lipid concentrations (mg.g-1 w.w.)

Table 3 Oocyte production in N. diversicolor originating from the Bay of Bourgneuf (B), Loire (L) and Seine (S) estuaries: Total number of oocytes as the total number of oocytes per female and Relative number of oocytes as the ratio between total number of oocytes and the weight of each female.

Glycogen concentrations (mg.g-1 w.w.)

C. Mouneyrac et al. / Environmental Pollution 158 (2010) 121–128

Bay of Bourgneuf

12

Loire estuary

10

Seine estuary

8 6 4 2 0 January

March

June

September

Fig. 4. Glycogen and lipid concentrations (means  S.D) in Nereis diversicolor collected in the bay of Bourgneuf (), Loire (,) and Seine (:) estuaries in January (Bay of Bourgneuf: N ¼ 20, Loire: N ¼ 18), March (Bay of Bourgneuf: N ¼ 20, Loire: N ¼ 16), June (Bay of Bourgneuf: N ¼ 20, Loire: N ¼ 18, Seine: N ¼ 12) and September (Bay of Bourgneuf: N ¼ 17, Loire: N ¼ 19, Seine: N ¼ 15) 2006.

4. Discussion This study was designed to compare the physiological condition, the reproduction status and the burrowing behaviour of the endobenthic worm N. diversicolor living in estuaries corresponding to a gradient of anthropogenic pressure according to official sources of environmental monitoring (Goyen estuary z Bay of Bourgneuf < Loire < Seine). In addition, natural factors can also influence the biological responses of interest in this comparison. In estuarine and coastal area, salinity is one of these potentially confounding factors. However, N. diversicolor is well known as a highly euryhaline species (Oglesby, 1978), inhabiting both coastal and estuarine mudflats with salinities ranging from 2 to 65 (Smith, 1955; Mason, 1986; Arias and Drake, 1995). More specifically, no influence of salinity has been recognized on different aspects of physiology (water content, energy reserves as glycogen, lipids and protein concentrations) and growth of N. diversicolor (Durou et al., 2007b). Burrowing behaviour was also compared in N. diversicolor collected in April 2008 from the Goyen estuary at two stations with different salinities (13 and 30) and no significant differences were observed (Kalman et al. in prep.). The literature reports a large variation in the breeding season and the age of maturity of N. diversicolor populations over their geographical range. Reproduction period may focus on a single period per year (spring or summer), an extended breeding season with one or two spawning peaks, or spawning throughout the year (Scaps, 2002). In the present study, intersite differences of temperatures were limited. In addition, the South/North gradient of temperature is not the alone factor influencing the recruitment period as demonstrated in a previous study including the contaminated Seine site compared to another reference site, the Authie estuary (Gillet et al., 2008). Differences in sediment quality are also a source of questioning but in the present case, these differences tend to re-inforce the interpretation that contamination affects burrowing behaviour (detailed below). In order to diagnose individual health of annelids, as recommended by Durou et al. (2008), we have compared the size–weight relationships (size being assessed by L3 length) for N. diversicolor

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C. Mouneyrac et al. / Environmental Pollution 158 (2010) 121–128

Goyen estuary Bay of Bourgneuf Loire estuary Seine estuary

50

0 60

0

0

54

48

0 42

0 36

0 30

0

0

24

0

18

12

60

0

0

% of un-burrowed organisms

A 100

Time (seconds)

% of un-burrowed organisms

B 100 Goyen estuary Bay of Bourgneuf Loire estuary Seine estuary

50

0 0

20

40

60

90

0 0 12 15 Time (seconds)

0

18

% of un-burrowed organisms

C 100 Goyen estuary Seine estuary 50

0 60

0 54

0 48

0

0

42

0

36

30

0

0

24

18

0 12

60

0

0 Time (seconds) Fig. 5. Burrowing kinetics of Nereis diversicolor (N ¼ 210). A. Ragworms from four sites allowed to burrow in their sediment of origin; B. Ragworms from a reference site (Goyen) allowed to burrow either in sediment of origin or in sediments from other sites; C. Ragworms from the contaminated Seine estuary allowed to burrow in sediment of origin or sediment from the reference Goyen estuary.

Table 4 Slopes and regression coefficients of the best-fit regression lines obtained after lntransformation of the raw data shown in Fig. 5. Slopes with different superscripts differed significantly at the 95% level. Origin of ragworms

Origin of sediment

Figure

Slope

Regression coefficient R2

Goyen estuary Bay of Bourgneuf Loire estuary Seine estuary

Goyen estuary Bay of Bourgneuf Loire estuary Seine estuary

Fig. 5A

0.0346a 0.0078b 0.0165b 0.0060b

0.9629 0.7682 0.6948 0.7554

Goyen estuary

Goyen estuary Bay of Bourgneuf Loire estuary Seine estuary

Fig. 5B

0.0749c 0.0217d 0.0201d 0.0190d

0.8680 0.8682 0.8835 0.9203

Seine estuary

Seine estuary Goyen estuary

Fig. 5C

0.0060e 0.0176f

0.7541 0.9075

according to their site of origin. These relationships following an exponential pattern illustrate allometric growth in annelids with two stages of development/growth: stage 1 corresponding to segment proliferation and stage 2 to segment enlargement. Clearly, for specimens from the reference site (Bay of Bourgneuf), the weight increase was more important than the length growth in comparison with specimens from the Loire contaminated site. Concerning worms from the Seine contaminated site, the length growth seems to stop more quickly than the weight increase. This latter result is in accordance with previous population studies showing that the Seine worms were generally smaller (Gillet et al., 2008). Since the population status is partly dependent upon the recruitment of the new generation, the reproductive status of adults and the success of reproduction is of great importance. In a field population, individuals of N. diversicolor belong to one or more cohorts of different age or size. In the present study, comparison of sexual maturity status was carried out in females of N. diversicolor populations according to their sites of origin (Bay of Bourgneuf, the Loire and Seine estuaries). In the reference site (Bay of Bourgneuf), for the whole sampling dates, between 30 and 60% of worms were sexually undifferentiated or at the beginning of gametogenesis (females in indifferent stage) whereas in the Loire contaminated site less than 10% of individuals were observed in these stages. Indeed, the majority of females (90%) from this contaminated site (Loire) were in active gametogenesis (proliferation, growth, mature) for the whole sampling periods. For worms from the contaminated Seine estuary, no individuals were sexually undifferentiated or at the beginning of gametogenesis in January showing an early active gametogenesis undergoing. This last pattern has been already observed when comparing worms from the Seine estuary with those from another reference site, the Authie estuary (Durou and Mouneyrac, 2007). However, because samplings were not frequent enough, it seems difficult to conclude definitely about the precise breeding period of N. diversicolor populations according to their sites of origin. The estimation of size at maturity as a fitness component (Olive et al., 2000) reveals differences according to the site of origin with worms breeding smaller (L3 measurements) in higher stress environments (Seine and Loire estuaries). Moreover, a lower proportion of reference worms (Bay of Bourgneuf) were undergoing gametogenesis for the whole sampling periods (Fig. 2). These data seem to indicate that worms from the reference site were growing slower and reproducing later compared with worms from both contaminated sites. In N. diversicolor, like in most nereid species, reproduction effort is high with 70% of energy invested in reproductive tissues (Olive, 1983a,b; Gre´mare and Olive, 1986). The life cycle is a pattern of allocation resources between reproductive and somatic functions, body resources being transferred from somatic to germinal tissues prior to reproduction (Barnes et al., 1988; Olive et al., 2000). Since oocytes are freely suspended in the body cavity, it is well known, in such species, that the total number of oocytes per female depends strongly upon body volume (Olive, 1983a). Clearly, results from the present study showed that the total number of oocytes per female such as fecundity were generally higher in worms from both contaminated sites (the Loire and Seine estuaries) than the reference site (Bay of Bourgneuf). Large specimens (L3 > 2.7 mm) were observed in the Loire estuary but were absent in the Bay of Bourgneuf or in the Seine estuary. In this last site, the growth in weight seems to overcome the growth in length. It may be hypothesized that this feature is due to a reproductive effort particularly elevated in Seine individuals under stress. In reproductive processes, energy reserves are of considerable importance. Both glycogen and lipid concentrations were higher in

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the contaminated Loire estuary compared to the reference site (Bay of Bourgneuf) and to the contaminated Seine estuary. Previous studies (Durou et al., 2007a) have shown depletion in energy reserves in N. diversicolor from the Seine estuary in comparison with a reference site (the Authie estuary). This energy depletion could correspond to the cost of tolerance to contaminants present in this contaminated Seine estuary. Pook et al. (2009) demonstrated fitness costs associated with metal-resistance in N. diversicolor populations from the metal-rich Restronguet Creek (U.K.). Scope for growth, energy reserves (lipids, carbohydrates) and mass-specific fecundity were significantly lower in these metal-resistant populations compared with two non-resistant populations. The ability to resist a toxicant may be expensive in terms of energy, involving a re-allocation of the energy available for other processes (Calow, 1991), namely basal metabolism, growth and reproduction. Besides, it cannot be overlooked that food availability may also influence the level of energy reserves and the condition of worms. Even if N. diversicolor as an opportunistic species is considered able to fulfil its energy needs with different diets (Olivier et al., 1995; Meziane and Retie`re, 2002; Scaps, 2002) it is important to note in the present study the contrast in food availability between the Bay of Bourgneuf and the Loire estuary. A peculiar abundance of microphytobenthos has been observed at our station in the Loire estuary (personal observations). On the other hand, in the Bay of Bourgneuf, an important biomass of primary consumers such as the cultivated oyster (Crassostrea gigas) and the invasive species (Crepidula fornicata) can compete for the use of primary producers as food (Decottignies et al., 2007). Behavioural tests showed a clear contrast between worms originating from sites exposed to different anthropogenic pressures, particularly considering the specimens from the multipolluted Seine estuary and the Goyen (reference estuary) whereas those from the Bay of Bourgneuf and the Loire estuary showed intermediate responses. This is in agreement with the biomonitoring data showing that bioavailable contaminants were less abundant in the Loire than in the Seine estuary (Claisse et al., 2006). In the Bay of Bourgneuf, sediment was more sandy than in the other sites (about 50% in the fraction 50–200 mm), a fact which is known as unfavourable to a fast burrowing rate (Bonnard et al., 2009). On the other hand, sediments from the three estuarine mudflats were typically characterized by a high proportion of clay and silts. Among the possible causes responsible for behavioural disturbances, the influence of physiological damages or avoidance responses is well-documented (see reviews by Dell’Omo, 2002; Amiard-Triquet, 2009). Cross tests with worms from the Seine and the Goyen estuaries allowed burrowing in sediments from other sites have been used to distinguish between these two causes of behavioural impairments. The reduced burrowing speed of worms from the Goyen estuary exposed to sediments from the three other sites may be interpreted as an avoidance response (with a possible effect of a coarser sediment in the case of the Bay of Bourgneuf). On the other hand, specimens from the Seine showed a significant increase of their burrowing speed in the comparatively clean sediment from the Goyen, with similar grain size, an observation which indicates that avoidance rather than a physiological damage is most probably at the origin of their hypoactivity in the presence of their sediment of origin. 5. Conclusions In conclusion, results of the present study showed fitness (biometric measurements, reproduction status, fecundity, energy reserves) and burrowing behaviour changes in N. diversicolor according to the degree of anthropogenic pressure of their site of origin. It is likely that specimens from the Bay of Bourgneuf

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(reference site) are growing slower and reproducing later (favouring weight increase instead of reproduction) since for the whole sampling periods, 30–60% of worms were sexually undifferentiated or beginning gametogenesis; the size at maturity of females was higher and the fecundity (total and relative) was lower in comparison with worms from both large estuaries (the Loire and Seine estuaries). N. diversicolor from the Loire estuary (intermediate level of anthropogenic pressure) exhibited high levels of energy reserves and fecundity (total and relative). Moreover, these females were in advanced sexual maturity stages and large specimens were present. If we try to link condition, energy reserves and reproduction, it can be hypothesized that for worms from the Loire estuary, energy reserves are allocated to gametogenesis and length growth. With regard to worms from the most contaminated Seine site, energy reserves could be devoted to tolerance to chemical stress and to reproductive processes. The changes in the whole set of endpoints at the individual level investigated in this study may have consequences at higher levels of biological organization. Since the input of individuals within a population in terms of growth (biomass) and reproductive output (persistence) governs the maintenance of a population, energy disturbances may have consequences at the population level (Calow, 1991; Maltby et al., 2001). At the community level, alteration in burrowing behaviour may be responsible for an increased vulnerability of endobenthic species to predation as suggested for different species (Olla et al., 1983, 1984; Pearson et al., 1981). Anyway, it is needed to interpret the results of biochemical, physiological and behavioural biomarkers taking into account not only the levels of toxic chemical but also the trophic status of the system since at low levels of nutrients or food availability, the ability of organisms to cope with pollutants is generally decreased (Heugens et al., 2001). Acknowledgments Thanks are due to Françoise Gourdon, Guillaume Demenier and Pierre Cahagnier for their technical help. This study has been partly funded by the French Ministry of Environment in the framework of the PNRPE (National Research Programme on Endocrine disruptors). References Agence de l’Eau Loire-Bretagne en, 2004. Mise en place d’un diagnostic de surveillance du milieu littoral conforme a` la directive cadre. http://dev. memoris.fr/dcelittolb/iso_album/creocean_rapport_final_dec_2004.pdf. Ait-Alla, A., Mouneyrac, C., Durou, C., Moukrim, A., Pellerin, J., 2006. Tolerance and biomarkers as useful tools for assessing environmental quality in the Oued Souss estuary (Bay of Agadir, Morocco). Comp. Biochem. Physiol. 143C, 23–29. Amiard-Triquet, C., 2009. Behavioural disturbances: the missing link between suborganismal and supra-organismal responses to stress? Prospects based on aquatic research. Hum. Ecol. Risk Assess. 15, 87–110. Arias, A.M., Drake, P., 1995. Distribution and production of the polychaete Nereis diversicolor in a shallow coastal lagoon in the Bay of Cadiz (SW Spain). Cah. Biol. Mar. 36, 201–210. Banta, G.T., Andersen, O., 2003. Bioturbation and the fate of sediment pollutants. Experimental case studies of selected in fauna species. Vie Milieu 53, 233–248. Baird, D.J., Brown, S.S., Lagadic, L., Liess, M., Maltby, L., Moreira-Santos, M., Schulz, R., Scott, G.I., 2007. In situ-based effects measures: determining the ecological relevance of measured responses. Integr. Environ. Assess. Manag. 3, 259–267. Barnes, R.S.K., Calow, P., Olive, P.J.W., Golding, D.W., 1988. The Invertebrates. A New Synthesis. Blackwell Science, Oxford. Bernes, C., 2000. Persistent Organic Pollutants. A Swedish View of an International Problem. Swedish Environmental Protection Agency, Stockholm, Sweden, 152 pp. Blackstock, J., Barnes, M., Barnes, H., 1982. The Loch Eil project: biochemical composition of the polychaete, Glycera alba (Mu¨ller), from Loch Eil. J. Exp. Mar. Biol. Ecol. 57, 85–92. Bonnard, M., Rome´o, M., Amiard-Triquet, C., 2009. Effects of copper on the burrowing behaviour of estuarine and coastal invertebrates, the polychaete Nereis

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