Mercury (Hg) concentrations and stable isotope analysis
Hg concentrations measured in the dorsal muscles of perch varied notably in all three stations (Table 2), thus mean concentrations and standard deviations (± SD) were 188.2±42.0, 154.2±71.3 and 110.8±65.1 µg kg-1 of wet weight in Station 1, 2 and 3, respectively. The ranges of nitrogen stable isotope ratios (δ15N) measured in perch were obviously similar (between approximately 14 and 18 δ15N ‰) at all three sites. At the same time, stable isotope ratios of carbon (δ13C), which can be used as an indicator of distance between the feeding ground and marine environments, showed wide ranges (approximately 12 ‰ units) for perch individuals caught in Stations 1 and 2, covering also isotopic signals associated with the coastal sampling station, and relatively narrow ranges (approximately 4 ‰ units) for the individuals from Station 3. Similarly, to perch, also ruffe (Gymnocephalus cernua) and roach (Rutilus rutilus) exhibited high variations of Hg concentration and stable isotope ratios.
Table 2. List of collected organisms and measured mercury (Hg) concentration, carbon (δ13C ) and nitrogen (δ15N) isotopic ratios.
Species
|
Type
|
Hg µg kg-1 dw (Min÷Max)
|
Hg µg kg-1 ww (Min÷Max)
|
δ13C±SD ‰ (Min÷Max)
|
δ15N±SD ‰ (Min÷Max)
|
|
Station 1
|
SPM
|
-
|
38.5±18.2 (18.5÷54.3)
|
NA
|
-31.5±0.8 (-32.3÷-30.6)
|
4.2±3.0 (1.6÷7.6)
|
|
Amphipoda
|
Crustacean
|
57.7±0
|
NA
|
-27.9±0 (-27.9÷-27.9)
|
10.3±0 (10.3÷10.3)
|
|
Hydrachna
|
Arachnid
|
NA
|
NA
|
-33.4±0.1 (-33.5÷-33.3)
|
17.0±1.6 (12.0÷18.8)
|
|
Ephemeroptera
|
Insect
|
NA
|
NA
|
-33.3±0.0 (-33.3÷-33.3)
|
13.4±0.1 (13.3÷13.5)
|
|
Trichoptera
|
Insect
|
NA
|
NA
|
-31.4±0 (-31.4÷-31.4)
|
13.2±0.2 (13.0÷13.4)
|
|
Dreissena polymorpha
|
Bivalva
|
141.0±0
|
15.6±0
|
-32.3±0.7 (-33.0÷-31.6)
|
12.3±0.3 (12.0÷12.6)
|
|
Oligochaeta
|
Annelida
|
272.0±0
|
NA
|
-29.8±0.0 (-29.8÷-29.8)
|
13.8±0.0 (13.8÷13.8)
|
|
Chironomidae larva
|
Insect
|
253.0±0
|
2.3±0
|
-29.7±0.0 (-29.8÷-29.7)
|
13.4±0.0 (13.4÷13.4)
|
|
Orconectes limosus
|
Crustacean (crayfish)
|
223.0±0
|
33.8±0
|
-30.8±0.2 (-30.9÷-30.6)
|
15.0±0.1 (14.9÷15.0)
|
|
Gymnocephalus cernua
|
Fish
|
1539.0±516.2.0 (1904.0÷1174.0)
|
264.0±100.9 (182.8÷377.1)
|
-28.5±2.4 (-30.2÷-23.9)
|
17.4±0.9 (15.9÷18.5)
|
|
Alburnus alburnus
|
Fish
|
504.0±0
|
91.0±0
|
-30.2±0.5 (-30.8÷-29.7)
|
15.6±0.2 (15.4÷15.8)
|
|
Vimba vimba
|
Fish
|
NA
|
NA
|
-28.6±0.0 (-28.6÷-28.6)
|
18.1±0.0 (18.1÷18.1)
|
|
Rutilus rutilus
|
Fish
|
825.0±0
|
166±9.8 (159÷173)
|
-30.7±0.6 (-31.6÷-29.6)
|
16.1±0.5 (15.4÷17)
|
|
Silurus glanis
|
Fish
|
1003.0±0
|
187.8±0
|
-30.6±0.0 (-30.7÷-30.6)
|
17.9±0.0 (17.8÷17.9)
|
|
Sander lucioperca
|
Fish
|
1082.0±0
|
220.1±0
|
-30.1±0.0 (-30.1÷-30.1)
|
19.0±0.0 (18.9÷19.0)
|
|
Perca fulviatilis
|
Fish
|
1035.8±218.0 (456.0÷1308.0)
|
188.2±42.0 (82.2÷249.6)
|
-26.6±3.7 (-32.5÷-20.7)
|
17.3±1.1 (13.6÷18.8)
|
|
Station 2
|
SPM
|
-
|
52.8±22.7 (76.1÷30.6)
|
NA
|
-31.4±0.6 (-32.1÷-30.9)
|
4.6±2.8 (2.6÷7.8)
|
|
Amphipoda
|
Crustacean
|
10.1±0
|
NA
|
-26.1±1.0 (-26.8÷-25.4)
|
6.9±0.7 (6.4÷7.4)
|
|
Dreissena polymorpha
|
Bivalva
|
60.7±0
|
6.6±0
|
-31.5±0.1 (-31.5÷-31.4)
|
10.1±0.1 (10÷10.2)
|
|
Oligochaeta
|
Annelid
|
162.0±0
|
NA
|
-31.6±0 (-31.6÷-31.6)
|
10.6±0.1 (10.5÷10.6)
|
|
Chironomidae larva
|
Insect
|
145.0±0
|
NA
|
-35.6±0 (-35.6÷-35.6)
|
8.3±1.0 (8.1÷9.8)
|
|
Vimba vimba juv.
|
Fish
|
61.0±0
|
25.4±0
|
-27.4±0 (-27.4÷-27.4)
|
12.0±0 (12.0÷12.0)
|
|
Perca fulviatilis juv.
|
Fish
|
45.5±0
|
21.9±0
|
-28.7±0 (-28.7÷-28.7)
|
10.8±0 (10.8÷10.8)
|
|
Leuciscus cephalus juv.
|
Fish
|
36.9±0
|
15.6±0
|
-27.4±0 (-27.4÷-27.4)
|
11.8±0 (11.8÷11.8)
|
|
Rutilus rutilus juv.
|
Fish
|
47.4±0
|
23.1±0
|
-28.5±0 (-28.5÷-28.5)
|
10.9±0 (10.9÷10.9)
|
|
Gymnocephalus cernua
|
Fish
|
487.0±0
|
92.4±0
|
-29.6±0 (-29.6÷-29.6)
|
16.6±0.1 (16.5÷16.6)
|
|
Alburnus alburnus
|
Fish
|
427/0±0
|
85.1±0
|
-31.4±0.1 (-31.5÷-31.3)
|
14.8±0.1 (14.7÷14.8)
|
|
Sander lucioperca
|
Fish
|
887.1±502.8 (359.5÷1603.0)
|
175.0±97.8 (70.0÷337.5)
|
-28.9±2.1 (-31.1÷-24.5)
|
17.7±0.2 (17.3÷18.0)
|
|
Rutilus. rutilus
|
Fish
|
559.3±155.3 (364÷719)
|
112.6±33.4 (70.6÷146.9)
|
-29.67±0.8 (-58.2)
|
14.72±0.21 (14.4÷15.1)
|
|
Perca fulviatilis
|
Fish
|
813.3±367.4 (289.0÷1752.0)
|
154.2±71.3 (57.3÷351.2)
|
-24.8±3.4 (-32.9÷-20.9)
|
16.6±0.7 (14.4÷18.1)
|
|
Station 3
|
SPM
|
-
|
14.4±14.5 (2.58÷42.7)
|
NA
|
-26.5±1.8 (-28.9÷-24.2)
|
6.0±1.5 (4.2÷9.1)
|
|
Neomysis integer
|
Crustacean
|
28.8±0
|
NA
|
-22.6±0 (-22.6÷-22.6)
|
11.4±0 (11.4÷11.4)
|
|
Crangon crangon
|
Crustacean
|
300.7±0
|
NA
|
-19.6±0 (-19.6÷-19.6)
|
12.9±0 (12.9÷12.9)
|
|
Saduria entomon
|
Crustacean
|
103±0
|
20.4
|
-16.9±1.4 (-18.3÷-15.6)
|
12.8±3.6 (9.67÷16.0)
|
|
Amphipoda
|
Crustacean
|
36±0
|
NA
|
-20.1±1.5 (-21.0÷-16.7)
|
10.2±1.6 (6.6÷11.2)
|
|
Copepoda
|
Crustacean
|
NA
|
NA
|
-17.5±3.3 (-19.9÷-15.2)
|
7.3±0.5 (6.9÷7.7)
|
|
Cladocera
|
Crustacean
|
NA
|
NA
|
-24.3±0.2 (-24.5÷-24.1)
|
5.6±0.0 (5.6÷5.7)
|
|
Polychaeta
|
Annelid
|
NA
|
NA
|
-21.0±0.1 (-21.1÷-20.9)
|
14.6±0.1 (14.5÷14.8)
|
|
Limecola balthica
|
Bivalva
|
NA
|
NA
|
-21.7±0.0 (-21.8÷-21.7)
|
11.2±0.1 (10.9÷11.5)
|
|
Gymnocephalus cernua
|
Fish
|
343.0±0
|
68.7±0
|
-24.5±4.2 (-29.2÷-21.2)
|
16.1±0.2 (15.7÷16.4)
|
|
Neogobius melanostomus
|
Fish
|
134.0±8.1 (127.0÷141.0)
|
26.7±0.8
|
-21.0±0.3 (-21.3÷-20.4)
|
15.2±0.7 (14.6÷16.4)
|
|
Ammodytes tobianus
|
Fish
|
NA
|
NA
|
-23.3±0.0 (-23.3÷-23.3)
|
14.2±0.0 (14.1÷14.2)
|
|
Zoarces viviparus
|
Fish
|
NA
|
NA
|
-21.0±0.0 (-21.1÷-21.0)
|
17.4±0.0 (17.3÷17.4)
|
|
Sprattus sprattus balticus
|
Fish
|
NA
|
NA
|
-22.1±0.1 (-22.1÷-22.0)
|
14.0±0.0 (14.0÷14.1)
|
|
Platichthys. flesus
|
Fish
|
193.0±0
|
37.4±0
|
-21.9±1.9 (-24.6÷-20.6)
|
15.5±0.7 (14.6÷16.2)
|
|
Clipea harengus membras
|
Fish
|
182.8±59.1 (140.0÷320.0)
|
33.7±12.5 (24.7÷60.2)
|
-21.2±1.1 (-23.1÷-18.4)
|
13.5±1.2 (10.2÷15.2)
|
|
Osmerus eperlanus
|
Fish
|
227.0±87.6 (121.0÷331.0)
|
51.9±17.5 (29.3÷64.9)
|
-22.1±1.4 (-24.2÷-20.6)
|
16.6±1.0 (14.7÷17.6)
|
|
Sander lucioperca
|
Fish
|
223.0±0
|
45.5±0
|
-22.4±0.9 (-23.3÷-21.6)
|
16.6±0.3 (16.2÷16.9)
|
|
Rutilus. rutilus
|
Fish
|
363.4±224.2 (179.0÷687.0)
|
70.0±39.1 (37.8÷126.2)
|
-26.1±3.6 (-30.2÷-21.1)
|
15.0±0.6 (13.4÷16.0)
|
|
Perca fulviatilis
|
Fish
|
579.2±343.5 (210.0÷1794.0)
|
110.8±65.1 (44.5÷362.7)
|
-21.7±1.0 (-24.2÷-20.0)
|
16.6±0.9 (14.9÷18.6)
|
|
Table 2. List of collected organisms and measured mercury (Hg) concentration, carbon (δ13C ) and nitrogen (δ15N) isotopic ratios.
(IS LARGER THAN ONE A4 OR LETTER PAGE IN LENGTH, PLACED AT THE END OF THE DOCUMENT TEXT FILE)
Stomach content analysis
The analysis of stomach content showed that dietary preferences of perch significantly differ between fresh water and brackish water habitats (Figure 2). At sampling Stations 1 and 2, the crustaceans (found in 56% and 42% of the analyzed stomachs, respectively) were the predominant prey. Juvenile perch (22% at Station 1 and 25% at Station 2) and Chironomidae larva (11% at Station 1 and 21% at Station 2) were second favorite prey organisms while O. limosus and G. cernua were found mainly only in the digestive tract of perch from Station 1. At the same time, N. integer was the most preferred prey in Station 3 (found in 78% of stomachs). N. integer was also found in 25% of perch stomachs from freshwater Station 2. The N. melanostomus was the second most common prey in Station 3, where it was found in 29% of perch stomachs. The A. tobianus and C. harengus membras were represented only in 12% and 6% of stomachs from Station 3.
Cluster analysis
Scatterplot of the calculated stable isotope ratios δ13C and δ15N demonstrated clear evidence that perch specimens migrate between the sampling stations (Figure 3). Substantial proportion of specimens sampled in Stations 1 and 2 had isotopic signals consistent with feeding in Station 3 (Figure 3A). Consequently, we divided the dataset into four subgroups, according to the three characteristics: sampling place, stable isotope ratios δ13C, and stable isotope ratios δ15N related to a trophic position of organism (Figure 3B and 3C).
The division was done as a cluster analysis based on the linear model criterion of least squares. Three of the subgroups were clearly representing respective sampling stations, while the fourth subgroup was well positioned as the mixed group with overlapping isotope ratio signals, which cannot be associated to any of the three sampling stations.
Exploration of the identified groups
The data was re-examined comparing Hg concentrations and distribution of individual’s length among the new groups designated via cluster analysis. Group 1 exhibited the highest Hg concentrations (Figure 4A) while lowest mean concentrations of Hg was found in group 3. Opposite to concentration levels, the highest mean length of perch was found in group 3 while the lowest one in group 2. The groups 1 and 4 exhibited the middle values (Figure 4B). Although the calculated bioaccumulation slopes were quite similar among the groups (coefficient values from 0.015 to 0.029), the intercepts differed noticeably (coefficient values from 1.4 for group 3 to 2.1 for group 1), thus indicating high variation of background Hg concentrations (Figure 4C).
Quantification of feeding preferences
Feeding preferences of the assigned subgroups were defined by means of Bayesian mixing model SIAR (Figure 5) based on results of the stomach content analysis of every perch individual representing respective subgroup. The different types of crustaceans, G. cernua, Chironomidae larva and O. limosus were defined as the main food sources for perch in the Station 1. The feeding base in the Station 2 mainly consisted of crustaceans, G. cernua, Chironomidae larva and juvenile perch while the N. integer, N. melanostomus, A. tobianus, C. harengus membras and crustaceans were main food items of perch in the Station 3. The most preferred prey of the newly formed mixed group was O. limosus from Station 1, juvenile perch from the Station 2, and N. integer and N. melanostomus from the Station 3.
The food items’ contribution ratios were extracted from the model and used for the further analysis of influence of dietary preferences. The data tables are published in the supplementary materials.
Biomagnification Factor (BMF)
Biomagnification factor was calculated for specific food chains, reflecting the localized diet of perch for each identified subgroup. The absolute values of the calculated biomagnification factors were quite similar for the station-assigned groups (1.45, 1.40, 1.46, respectively), but substantially higher for the mixed group (1.76).
The biomagnification curve of the mixed group (group 4) was significantly different from the others by a steeper slope (Table 3, M2) and by higher intercept compared to group 3 (Table 3, M1). Meanwhile, groups 1 to 3 had statistically similar slopes, thus indicating similar biomagnification patterns (Table 3, M2). At the same time, significantly lower intercept of group 3, compared to groups 1 and 2 (Table 3, M1) probably denotes again lower Hg background concentrations found at the station.
Table 3 Comparison of biomagnification curves. Bold italic text indicates significant differences between the curves.
|
M1: without interaction1
|
M2: with interaction2
|
ANOVA: M1 vs M23
|
Compared groups
|
F value
|
p-value
|
F value
|
p-value
|
F value
|
p-value
|
1. vs 2.
|
0.009
|
0.926
|
0.187
|
0.669
|
0.187
|
0.669
|
1. vs 3.
|
8.569
|
0.005
|
0.011
|
0.916
|
0.011
|
0.916
|
1. vs 4.
|
0.000
|
0.990
|
13.480
|
<0.001
|
13.484
|
<0.001
|
2. vs 3.
|
9.943
|
0.002
|
0.771
|
0.383
|
0.771
|
0.382
|
2. vs 4.
|
0.785
|
0.381
|
10.410
|
0.003
|
10.414
|
0.003
|
3. vs 4.
|
6.859
|
0.011
|
15.110
|
<0.001
|
15.110
|
<0.001
|
1The regression model M1 includes independent variable δ15N, independent factor Group and dependent variable LOG mercury concentrations.
2The regression model M2 includes interaction between independent variable δ15N and independent factor Group and dependent variable LOG mercury concentrations.
3One-way analysis of variance of the regression models M1 and M2
Influence of dietary preferences
Generalized Additive Modelling was implemented to understand how dietary preferences of perch in different feeding grounds affect the Hg uptake. To avoid covariance of food source variables three validated models with different combination of food items were selected and interpreted (Table 4). The obtained results indicated seasonality (spring and autumn sampling) as a significant factor affecting measured Hg LOG-concentrations, for example, samples collected in spring had higher levels of Hg concentration than the autumn samples (demonstrated by a positive intercept correction for the spring season from 0.106 up to 0.120). The stable isotope ratio δ15N showed a significant relationship with the Hg concentration, however the positive slope coefficient was only 0.09 in the all three models.
The models let us to establish that the food item C. harengus membras had the most significant mitigating effect on Hg concentration, with negative slopes ranging from -0.349 to -0.501. Another food item with significant Hg-lowering properties was Crustacea with a negative slope coefficient of -0.460. The rest of the food items were not significant at the α = 0.05 level, although had different directions of the influence and slope values. A highly positive effect was observed for Chironomidae larva (slope values from 0.233 to 0.460). O. limosus (slope values from 0.143 to 0.184), perch juvenile (slope values from 0.010 to 0.186) and G. cernua (slope value 0.120) were other food items that contributed to the uptake of Hg by perch. N. melanostomus and N. integer exhibited a neutral influence on Hg concentration measured in consumer perch, indicating slightly positive slope coefficients of 0.080 and 0.023, respectively.
Table 4. Estimated regression parameters (intercept and slope values), standard errors, t-values and P-values for the Gaussian GAM presented in equations 1, 2 and 3.
(IS LARGER THAN ONE A4 OR LETTER PAGE IN LENGTH, PLACED AT THE END OF THE DOCUMENT TEXT FILE)
Table 4. Estimated regression parameters (intercept and slope values), standard errors, t-values and P-values for the Gaussian GAM presented in eqn (1, 2, 3).
Model A
|
|
log (µij) =
|
|
|
0.536 + 0.092 × δ15Nij - 0.460 × Crustaceaij + 0.460 × Chironomidae larvaij + 0.098 × Perch juvenileij + 0.120 × G.cernuaij + 0.142 × O.limosusij - 0.349 × C.harengusij + s(Length)ij AUTUMN
0.642+ 0.092 × δ15Nij - 0.460 × Crustaceaij + 0.460 × Chironomidae larvaij + 0.098 × Perch juvenileij + 0.120 × G.cernuaij + 0.142 × O.limosusij - 0.349 × C.harengusij + s(Length)ij SPRING
|
|
Estimate
|
Std. Error
|
t value
|
Pr(>|t|)
|
|
|
(Intercept)
|
0.53618
|
0.31742
|
1.689
|
0.095569
|
|
|
SeasonS
|
0.10594
|
0.02712
|
3.907
|
0.000211
|
|
|
δ15N
|
0.09255
|
0.01916
|
4.83
|
7.60E-06
|
|
|
Crustacea
|
-0.4601
|
0.19332
|
-2.38
|
0.019996
|
|
|
Chironomidae larva
|
0.45997
|
0.23451
|
1.961
|
0.053751
|
|
|
Perch juvinile
|
0.09828
|
0.13144
|
0.748
|
0.457114
|
|
|
G. cernua
|
0.11981
|
0.12699
|
0.943
|
0.348636
|
|
|
O. limosus
|
0.14249
|
0.13139
|
1.084
|
0.281832
|
|
|
C. harengus
|
-0.34935
|
0.16865
|
-2.071
|
0.041948
|
|
|
Approximate significance of smooth terms:
|
|
|
edf
|
Ref.df
|
F
|
p-value
|
|
|
s(Length)
|
2.901
|
3.649
|
18.3
|
<2e-16
|
|
|
R-sq.(adj) = 0.759 Deviance explained = 79.1%
|
|
|
|
|
|
|
|
|
Model B
|
log (µij) =
|
|
|
0.514 + 0.090 × δ15Nij + 0.299 × Chironomidae larvaij + 0.186 × Perch juvenileij + 0.174 × O.limosusij - 0.484× C.harengusij + 0.079 × N.melanostomusij+ s(Length)ij AUTUMN
0.628 + 0.090 × δ15Nij + 0.299 × Chironomidae larvaij + 0.186 × Perch juvenileij + 0.174 × O.limosusij - 0.484 × C.harengusij + 0.079 × N.melanostomusij + s(Length)ij SPRING
|
|
|
|
|
|
Estimate
|
Std. Error
|
t value
|
Pr(>|t|)
|
|
|
(Intercept)
|
0.51438
|
0.32367
|
1.589
|
0.11639
|
|
|
SeasonS
|
0.11453
|
0.02798
|
4.093
|
0.00011
|
|
|
δ15N
|
0.09064
|
0.01931
|
4.694
|
1.24E-05
|
|
|
Chironomidae larva
|
0.29922
|
0.21468
|
1.394
|
0.16766
|
|
|
Perch juvinile
|
0.18633
|
0.12887
|
1.446
|
0.15255
|
|
|
O. limosus
|
0.17368
|
0.12915
|
1.345
|
0.18291
|
|
|
C. harengus
|
-0.484
|
0.16458
|
-2.941
|
0.0044
|
|
|
N. melanostomus
|
0.0792
|
0.10274
|
0.771
|
0.44329
|
|
|
Approximate significance of smooth terms:
|
|
|
edf
|
Ref.df
|
F
|
p-value
|
|
|
s(Length)
|
2.814
|
3.545
|
17.62
|
<2e-16
|
|
|
R-sq.(adj) = 0.745 Deviance explained = 77.5%
|
|
|
|
|
|
|
|
|
Model C
|
log (µij) =
|
|
|
0.529 + 0.091 × δ15Nij + 0.233 × Chironomidae larvaij + 0.167 × Perch juvenileij + 0.184 × O.limosusij - 0.501 × C.harengusij + 0.023 × N.integerij+ s(Length)ij AUTUMN
0.649 + 0.091 × δ15Nij + 0.233 × Chironomidae larvaij + 0.167 × Perch juvenileij + 0.184 × O.limosusij - 0.501 × C.harengusij + 0.023 × N.integerij+ s(Length)ij SPRING
|
|
|
|
|
|
Estimate
|
Std. Error
|
t value
|
Pr(>|t|)
|
|
|
(Intercept)
|
0.52858
|
0.34508
|
1.532
|
0.1299
|
|
|
SeasonS
|
0.12052
|
0.02698
|
4.467
|
2.87E-05
|
|
|
δ15N
|
0.09056
|
0.01975
|
4.584
|
1.87E-05
|
|
|
Chironomidae larva
|
0.23255
|
0.24345
|
0.955
|
0.3426
|
|
|
Perch juvinile
|
0.16704
|
0.15511
|
1.077
|
0.2851
|
|
|
O. limosus
|
0.18396
|
0.13431
|
1.37
|
0.175
|
|
|
C. harengus
|
-0.50118
|
0.22139
|
-2.264
|
0.0266
|
|
|
N. integer
|
0.02286
|
0.13537
|
0.169
|
0.8664
|
|
|
Approximate significance of smooth terms:
|
|
|
edf
|
Ref.df
|
F
|
p-value
|
|
|
s(Length)
|
2.747
|
3.467
|
19.01
|
<2e-16
|
|
|
R-sq.(adj) = 0.742 Deviance explained = 77.3%
|
|