Developmental and subcellular effects of chronic exposure to sub-lethal concentrations of ammonia, PAH and PCP mixtures in brown trout (Salmo trutta f. fario L.) early life stages

Aquatic Toxicology 65 (2003) 39 /54 www.elsevier.com/locate/aquatox

Developmental and subcellular effects of chronic exposure to sub-lethal concentrations of ammonia, PAH and PCP mixtures in brown trout (Salmo trutta f. fario L.) early life stages Till Luckenbach a,*, Hermann Ferling b, Maike Gernho¨fer a, Heinz-R. Ko¨hler a, Rolf-Dieter Negele b, Eva Pfefferle c, Rita Triebskorn a,d a

Animal Physiological Ecology, University of Tu ¨ bingen, Konrad-Adenauer-Straße 20, D-72072 Tu ¨ bingen, Germany b Bavarian State Agency for Water Research, Demollstraße 31, D-82407 Wielenbach, Germany c Steinbeis-Transfer Center for Applied and Environmental Chemistry, Alteburgstrasse 150, D-72762 Reutlingen, Germany d Steinbeis-Transfer Center for Ecotoxicology and Ecophysiology, Kreuzlingerstrasse 1, D-72108 Rottenburg, Germany Received 7 January 2002; received in revised form 18 October 2002; accepted 15 April 2003

Abstract Brown trout (Salmo trutta f. fario L.) early life stages were studied for physiological effects caused by chronic exposure to sub-acute levels of unionised ammonia, a mixture of PCP and PAHs, and a combination of ammonia and the mixture of organics during the entire embryonic development. Nominal concentrations of tested compounds were based on field data. Accumulation data for PAHs and PCP in trout tissue reflected respective water concentrations of PCP and PAHs. Physiological responses were studied by early life stage tests (ELST) and by the analysis of the 70 kDa stress protein (hsp70). Endpoint responses in the ELST were: accelerated development, pre-hatching, and increased heart rates. For these endpoints, response levels were highest in the ammonia treatment, followed by the exposure to the PCP/PAH mixture. Weight was reduced in embryos treated with the PCP/PAH mixture, but not in the group treated with this mixture combined with ammonia. Induction of hsp70 by the test agents was found to be stage-specific with increased response levels at advanced developmental stages. In both the ELST and hsp70 analysis, response levels were lower in the combined ammonia/PCP/PAH treatment than in groups treated with either ammonia or the PCP/PAH mixture alone. # 2003 Elsevier B.V. All rights reserved. Keywords: Ammonia; PCP; PAHs; Chronic toxicity; Fish embryo; Brown trout; Early life stage test; Hsp70

1. Introduction * Corresponding author. Present address: Hopkins Marine Station of Stanford University, Oceanview Blvd, Pacific Grove, CA 93950-3094, USA. Tel.:
/1-831-6556227; fax:
/1-8313750793. E-mail address: [email protected] (T. Luckenbach).

Subsequent to the severe pollution of surface waters in the 1960s and 1970s which had drastically affected biota in lakes and streams of Europe, emphasis was put on restoration of waters and treating of waste waters. As a consequence, con-

0166-445X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-445X(03)00107-3

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T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

tamination was continuously reduced during the last decades as reported for example, for the Rhine river (Stigliani et al., 1993; Chovanec et al., 1996; Van Dijk et al., 1996; Malle, 1996). In parallel to reduced pollution, an increase of species numbers has been found, indicating recovery of biota (Engels et al., 1996; Malle, 1996). However, even though pollution generally may have decreased, pristine sites are rare and in many cases waters are still contaminated with a broad variety of pollutants in low concentrations. Thus, aquatic organisms are facing low but chronic background concentrations of xenobiotics and are affected by synergistic effects of xenobiotic mixtures (e.g. Triebskorn et al., 2001). Although organisms may tolerate this chronic background stress, they may be more vulnerable to additional stressors such as adverse temperatures or other confounding factors (Linton et al., 1998; Ko¨hler et al., 2001; Schwaiger, 2001). In 1998, we performed a semi-field experiment with brown trout early life stages exposed in flowthrough systems to evaluate embryotoxic potentials of two small streams in Germany which have pollution patterns that mirror many streams in central Europe (Luckenbach et al., 2001; Triebskorn et al., 1997, 2001). Early life stage tests (ELST) and limnochemical and chemical analyses indicated the presence of various toxic agents in sublethal concentrations which caused stress and impaired developmental success in trout embryos (Luckenbach et al., 2001). Therefore, the objective of the present laboratory based study was to investigate physiological effects in brown trout chronically exposed during the entire embryonic development to selected compounds (ammonia, PAHs, and PCP) at levels found in the two aforementioned streams. Effects of ammonia, a PCP/PAH mixture, and a mixture of ammonia and PCP/PAH were evaluated in separate test series. Net stress effects were studied by ELST and the level of the stress protein hsp70. These assays measure effects on the development and protein integrity, respectively. In addition, specific effects on gills and liver in advanced stages were analysed ultrastructurally (Gernho¨fer et al., 2003, in preparation).

2. Materials and methods 2.1. Experimental design Eggs from six 4-year-old females were pooled and artificially inseminated with sperm from six 4year-old males of brown trout (Salmo trutta f. fario L.). Parental animals were obtained from the fish breeding stock of the research institute of the Bavarian State Agency for Water Research, Wielenbach, Germany. Exposure to xenobiotics began after water-hardening of eggs approximately 15 h after fertilisation on 25 November 1999 (day 0) and was terminated at the end of the embryonic period after 79 days (staging nomenclature after Balon, 1975). The embryos were incubated in trays of perforated stainless steel (30 /25 /16.8 cm / length /breadth /height; diameter of perforation: 2 mm) which were inserted in glass tanks filled with water (volume 90 l). In each glass tank, embryos were incubated in two separate trays for either (1) monitoring development of trout embryos in different treatments (n /500) or (2) sampling specimens for staging and for studies on hsp70 and ultrastructure of liver and gills (n / 1000). Water was continuously replaced at a rate of 9 l h1 in a flow-through system resulting in an approximately 90% replacement of the total water volume per day (Sprague, 1969). Stock solutions of test substances which were stored in brown glass flasks and fresh well water (conductivity: 730 mS cm 1, total hardness: 21.28 dH) were delivered into a mixing chamber by a peristaltic pump (Minipuls 3, Abimed /Gilson, Langenfeld, Germany) and homogenised by mechanical stirring. The different treatment groups contained (1) ammonia (NH3: 25 mg l 1; referred to in this paper as ammonia treatment: AT), (2) an organic mixture containing polyaromatic hydrocarbons (total PAHs: 130 ng l1; acenaphthylene (ANY): 5 ng l 1, chrysene (CHR): 5 ng l1, fluoranthene (FLA): 12 ng l 1, fluorene (FLU): 12 ng l 1, naphthalene (NAP): 36 ng l1, pyrene (PYR): 6 ng l1, phenanthrene (PHE): 54 ng l 1) and pentachlorophenol (PCP: 20 ng l 1) (referred to in this paper as PCP/PAH mixture treatment: PT), or (3) a combination of ammonia and the PAH/PCP mixture in concentrations listed above (referred to

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

in this paper as ammonia/PCP/PAH mixture treatment: APT). All values given are nominal concentrations. Actual concentrations were measured as described below. PAHs and PCP were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). A stock solution of PT was made by solubilising PAHs and PCP in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany). This PT stock solution was diluted with water in a ratio 1:33.3, and, in a second step, 1:300 (final dilution 1:10 000). Pure solvent was diluted the same way for a solvent control (SC). The final [DMSO] of 0.1% (v/v) corresponded to the water concentration proposed by Rudolph and Boje (1986) for DMSO used as solvent for hydrophobic chemicals in toxicological tests. Ammonia was added as NH4Cl (Merck). The amount of NH4Cl which was required to obtain the desired concentration of NH3 was determined on the basis of water temperature and pH (Wade et al., 1998). In addition to the solvent control, a second control with untreated water (referred to in this paper as water control: WC) was set up. Each exposure group was replicated (n /2). At regular intervals, water parameters were controlled and coagulated eggs or dead embryos were removed and recorded. Embryos were kept in darkness in order to avoid light-induced effects (Hamdorf, 1960). When external feeding started, fish were fed twice a day to satiety with starter food (Biomar, Bra˚nde, Denmark). 2.2. Early life stage test (ELST) For early life stage studies, exposure started with 500 eggs per replicate. The examined endpoints of the ELST were (1) developmental rate, (2) time course of hatching, (3) heart rate, (4) growth, (5) malformation rate, and (6) mortality. The developmental rate was determined in specimens fixed in buffered formalin at days 39, 49, and 79 of exposure using a scoring scheme proposed by Killeen et al. (1999). This scoring scheme allows for very subtle staging of brown trout during embryonic development. Developmental advancement of embryos was quantified by attributing score points to certain stage-typical characteristics according to the method of Killeen et al. (1999).

41

To determine whether test substances caused acceleration or delay of development, scores were evaluated for specimens of the same age from the different treatments and compared. Heart rates (min 1) were calculated from the time needed for 20 heart beats. Percentage rates of hatched embryos during the hatching period were calculated based on final hatching numbers. Temporal aberrations in hatching between treatments were evaluated by comparing hatching rates at day 42. After hatching, embryos with morphological malformations (skeletal deformities, duplications of heads/tails, deformities of yolk, alterations to head) were removed and recorded. At the end of exposure (day 79), wet weight to the nearest 0.001 g and total length to the nearest 1 mm were measured in 50 fishes from each replicate. On the basis of these data the condition factor was calculated using Fulton’s equation: C /(W / 100) /TL 3 /1000; C /condition factor, W / weight (g), TL /total length (mm) (Fulton, 1904). Mortality rates were calculated from total numbers of fertilised eggs (500 eggs per replicate at the beginning of exposure). The fertilisation rate was determined by evaluating the percentage of eggs from WC which reached the eyed stage. Eggs which did not reach the eyed stage were classified as unfertilised. 2.3. Hsp70 analysis For hsp70 analysis, specimens were frozen in liquid nitrogen. Advanced stages were anaesthetised in 4-amino benzoic acid ethyl ester prior to freezing. Hsp70 was quantified on the basis of a standardised immunoblotting protocol developed by Ko¨hler et al. (1996) which was modified for brown trout early life stages. Pre-hatching stages and early eleutheroembryos were dissected to remove the yolk. Advanced eleutheroembryos/ early juveniles, which had nearly completely depleted their yolk reserves, were homogenised as a whole (n/10 per replicate group and sampling occasion). Specimens were homogenised in icecold extraction buffer (80 mM potassium acetate, 5 mM magnesium acetate, 20 mM Hepes, 1% (v/v) protease inhibitor cocktail (product number P8340, Sigma, Saint Louis, USA), pH 7.5) and

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T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

centrifuged at 4 8C at 20 000 /g for 5 min. Total protein concentration in the supernatants was determined according to the method of Bradford (1976). Constant protein weights (10 mg of total protein/lane) were analysed using minigel SDSPAGE (12% acrylamide, 0.12% bisacrylamide (w/ v)). Protein was transferred to nitrocellulose by semi-dry blotting and the filter blocked for 2 h in 50% horse serum in TBS (50 mM Tris pH 7.5, 150 mM NaCl). After washing in TBS, monoclonal antibody (mouse anti-human hsp70; Dianova GmbH, Hamburg, Germany, dilution 1:5000 in 10% horse serum/TBS) was added and the samples were then incubated at room temperature overnight. After repeated washing in TBS, the nitrocellulose filter was incubated in secondary antibody, goat anti-mouse IgG (H
/L) coupled to peroxidase (Dianova GmbH, dilution 1:1000 in 10% horse serum/TBS), at room temperature for 2 h. After further TBS washing, the antibody complex was detected by 1 mM 4-chloro(1)naphthol and 0.015% H2O2 in 30 mM Tris pH 8.5 containing 6% methanol. Grey scale value quantification of the Western blot protein bands was performed with a densitometric image analysis system (E.A.S.Y.; Herolab GmbH, Wiesloch, Germany) after background subtraction, taking both the staining intensity and the spatial extension of the bands into account. Replicates of control and exposed specimens were run on different gels to minimise the influence of methodological variation on mean grey value calculation. 2.4. Analytical procedures Temperature, pH, and [O2] were measured regularly in all tanks by using pH 96 and oxi 96 electrodes from WTW (Weilheim, Germany) (Table 2). Total ammonia was measured using the test-kit LCK 304 and the spectral photometer CADAS 100 (Lange, Du¨sseldorf, Germany). [NH3] was calculated from total ammonia, temperature, and pH by using the following equation: [NH3] /([NH3
/NH4
] 10(pH
0.032904 T10.0655)) (1
/10(pH
0.032904 T10.0655)) 1 (Wade et al., 1998). Water samples collected daily from treatments with the PCP/PAH mixture (PT, APT) were pooled into three groups according to exposure

Table 1 Names of the test series, abbreviations, and nominal concentrations of the various compounds applied in the present experiment Test name

Abbreviation Nominal concentration

Water control Solvent control Ammonia treatment PCP/PAH treatment Ammonia/PCP/ PAH treatment

WC SC AT PT APT

0.1% DMSO (v/v) 25 mg NH3 l 1 20 ng PCP l1, 130 ng PAHs l 1, 0.1% DMSO (v/v) 25 mg NH3 l 1, 20 ng PCP l 1, 130 ng PAHs l 1, 0.1% DMSO (v/v)

times: days 1 /22, 25 /43, and 68 /75 for 21, 18, and 7 days of exposure, respectively. These samples and two random samples (days 48 and 75) were analysed for those organic compounds used in the pollutant mixture. Water samples were collected in 2 l brown glass flasks and stored at 4 8C until analysis. As a control, one random sample of fresh well water was also analysed. To study accumulation of organic compounds in tissue over the entire exposure period, tissue samples from trout from the treatments with PCP/PAH mixture (PT 1, PT 2, APT 1, APT 2 (numbers refer to the different replicates)) and from the controls (SC 1, WC 1) were analysed for the organic compounds. For this analysis, approximately 300 individuals from each group were pooled to achieve a total mass of at least 40 g wet weight of tissue. Fish were anaesthetised and then frozen in dry ice and stored at /20 8C until analysis. Analyses of organic compounds in water and tissue samples were performed using GC /MS as described by Honnen et al. (2001). 2.5. Statistical analyses In the ELST, significant differences in the endpoints ‘‘mortality’’ and ‘‘hatching rates’’ were evaluated by the Chi-square (x2) test. P -values of multiple comparisons were Bonferroni-adapted. For analysing score values representing the developmental advancement according to Killeen et al. (1999) median, minima, and maxima values and 25

For abbreviations see Table 1. The numbers 1 and 2 in the codes for the exposure situation refer to replicate experiments. n : number of replicate samples taken from each experiment.

9.30 (9.20/ 9.30) n/12 7.83 (7.82/ 7.84) n/10 9.23 (7.86/ 9.56) n/7 9.30 (9.20/ 9.30) n/14 7.94 (7.93/ 7.96) n/11 8.34 (7.23/ 8.98) n/7 9.30 (9.30/ 9.30) n /6 7.94 (7.94/ 7.95) n /4 9.80 (7.66/ 9.85) n /5 9.30 (9.30/ 9.40) n/10 8.01 (8.00/ 88.02 n/8 9.81 (7.89/ 9.83) n/7 9.40 (9.40/ 9.60) n/10 7.93 (7.92/ 7.94) n/11 8.86 (7.15/ 9.43) n/8 9.50 (9.45/ 9.60) n/10 7.90 (7.89/ 7.91) n/10 8.94 (7.33/ 9.63) n/8 9.40 (9.40/ 9.40) n/8 7.88 (7.88/ 7.89) n/5 9.03 (7.58/ 9.46) n/7 T (8C)

9.30 (9.30/ 9.30) n/7 pH 8.00 (8.00/ 8.02) n/6 [O2] (mg 8.91 (8.33/ l 1) 9.46) n/6

9.02 (9.10/ 8.98) n/6 7.89 (7.88/ 7.89) n/6 9.00 (7.99/ 9.23) n/6

9.40 (9.40/ 9.50) n/8 7.92 (7.91/ 7.92) n/3 8.84 (7.89/ 9.48) n/7

AT 2 AT 1 SC 2 SC 1 WC 2 WC 1

Table 2 Median values and range (minimum/maximum) for temperature, pH, and [O2] in different exposures

PT 1

PT 2

APT 1

APT 2

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

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and 75% percentiles were determined and data sets of different treatment groups were compared using the Mann /Whitney U -test and subsequent Bonferroni-adaption of P -values. The data for heart rate and growth parameters (weight, length, condition factors) were tested for normal distribution using the Kolmogorov /Smirnov test. For the condition factor, normal distribution of data was found in all replicate groups. Heart rate data followed a normal distribution except in one group (WC 2), the same was also seen for the weight data where only group AT 1 failed to show a normal distribution. Therefore, this data was treated parametrically. Data was processed further by determining mean values and standard deviations (S.D.) and analysed using ANOVA and the Tukey /Kramer HSD test. Length data was not normally distributed and thus was analysed nonparametrically. For endpoint responses in the ELST, data from replicates of each control and treatment group were, respectively, pooled, since differences between corresponding replicates were not significant (P
/0.05). Data representing levels of hsp70 were normally distributed in each group, thus differences between groups were tested by ANOVA and the Tukey /Kramer HSD test. When statistically testing data of the replicate groups of each control or treatment group and each sampling occasion (three sampling occasions, five treatment groups with two replicates, n /10 per replicate), only two out of a total of 30 data sets were found to be weakly significant (P B/0.05 for differences between replicates of WC and PT from day 52), whereas the rest were found not to be significant. Therefore, data of the replicates of the different treatments were pooled for further analyses. For statistical analyses JMP version 3.2.6 (SAS Institute Inc., 1999) was used.

3. Results 3.1. Analytical data Mean [NH3] in ammonia treatments (AT, APT) differed only slightly from the nominal concentration of 25 mg NH3 l 1 and concentrations varied over a narrow range during exposure (Table 3).

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In treatment groups with the PCP/PAH mixture (PT, APT), [PAHs] in water samples collected during the periods from days 1 to 22, 25 to 43 and in single samples at days 48 and 75 were so low in each test series that it is doubtful that these substances remained dissolved in the water (compare with values for uncontaminated well water, Table 4). Only in the water sample collected during the period from 68 to 75 days were PAHs from the PCP/PAH mixture obviously present. In this sample, concentrations exceeded the nominal concentrations by approximately 100% (Table 4). Tissue concentrations of PT and APT embryos differed from controls by a factor 3/4, confirming that generally, [PAHs] in the water were very low in PT and APT groups leading to low accumulation in tissue (Table 5). In contrast to PAHs, PCP was found in most of the water samples. Only at day 48, PCP was not found in PT 1, PT 2, and APT 2 (Table 4). Analytical data for [PCP] differed from nominal concentrations in a range from 0.05 to 3.75-fold. PCP was not found in well water (Table 4) and tissue from control fish (Table 5). 3.2. Early life stage test Developmental advancement was evaluated using the scoring scheme for brown trout early life stages developed by Killeen et al. (1999) on specimens from day 39 (pre-hatching stage), day 49 (post-hatch stage), and day 79 (advanced embryonic stage). At day 39, developmental scores were determined by tips of pelvic fins which levelled with and extended past the ventral fin fold, by pigment between the back of the eye and

the operculum, and by a notch in the dorsal finfold behind the dorsal fin. Scores for embryos from day 49 were determined by the number of segments in the caudal fin, by the number of anal fin rays, and by a ridge developing behind the adipose fin. For scores at day 79, ventral procurrent rays and segments in the caudal fin, and branchiostegal rays in the operculum were counted. According to scores, embryos from day 39 (median: 537 points, min: 502, max: 552, n /189) were at step 31 and 32 (caudal fin ray phase, F5 and F6, respectively), specimens from day 49 (median: 573 points, min: 566, max: 581, n/185) were at step 33 (caudal fin ray phase, F7), and specimens from day 79 (median: 1006 points, min: 987, max: 1015, n / 225) were at step 40 (finfold resorption phase, R6). At day 39, median scores were significantly lower in WC than in all other groups (P B/0.05 for WC vs. SC, P B/0.01 for WC vs. APT, P B/0.001 for WC vs. AT/PT, Fig. 1). Scores were highest in PT which, as well as being higher than in WC, were also significantly higher than in SC (P B/0.05). Differences between the three groups PT, AT, and APT were not significant (P
/0.05; Fig. 1). At day 49, differences between WC and PT were significant (P B/0.05, Fig. 1). Scores at day 79 were significantly lower in WC than in AT and PT (P B/ 0.001, Fig. 1) and in SC than in AT (P B/0.05). Heart rates in AT were significantly higher than in WC and APT (P B/0.01; Fig. 2). In SC and PT, heart rates were between the values for WC/APT and AT, but differences compared with other groups were not significant (P
/0.05). In all groups hatching started nearly at the same time: after 40 days in SC (one hatched embryo), in AT (three hatched embryos), in PT (two hatched

Table 3 Median concentrations and range of total ammonia (NH3
/NH4
) and unionised ammonia (NH3) (mg l 1) in test series treated with ammonia AT 1, AT 2 (ammonia treatment) and APT 1 and APT 2 (ammonia/PCP/PAH mixture treatment) and in the water control (WC)

mg NH3
/NH4
l 1: median (minimum/maximum) mg NH3 l 1: median (minimum/maxim)

AT 1 n/7

AT 2 n/7

APT 1 n/7

APT 2 n/7

WC n/3

1800 (1750/ 1830) 24.9 (24.6/25.5)

1800 (1720/ 1860) 26.9 (25.3/27.3)

1880 (1780/ 1920) 27.9 (25.8/28.8)

2000 (1850/ 2050) 22.7 (21.5/23.8)

55 (35/58)

The numbers 1 and 2 in the codes for the exposure situation refer to replicate experiments.

1.0 (0.6/ 1.0)

0.002 n.d. n.d. 0.003 0.002 0.001 n.d. 0.009 0.062 0.010 0.020 0.109 0.024 0.018 0.010 0.254 0.005 n.d. n.d. 0.003 0.001 0.007 0.003 0.014 0.001 n.d. n.d. n.d. n.d. 0.004 n.d. 0.005 0.007 n.d. n.d. 0.008 0.009 0.006 n.d. 0.030 The numbers 1 and 2 in the codes for the exposure situation refer to replicate experiments. n.d.: not detected.

0.008 n.d. 0.002 0.026 0.007 0.014 0.004 0.014 0.059 0.009 0.019 0.103 0.022 0.017 0.009 0.239 0.006 n.d. n.d. 0.014 0.005 0.003 n.d. 0.028 n.d. n.d. n.d. n.d. n.d. 0.004 n.d. 0.004 0.003 n.d. n.d. 0.002 0.006 0.006 n.d. 0.016 0.060 0.009 0.019 0.107 0.024 0.019 0.009 0.247 0.012 n.d. n.d. 0.014 0.005 0.003 n.d. 0.027 0.001 n.d. n.d. n.d. n.d. 0.002 n.d. 0.003 0.007 n.d. n.d. 0.006 0.010 0.006 0.004 0.032 0.001 n.d. n.d. 0.003 0.001 0.007 0.003 0.014 0.055 0.009 0.017 0.094 0.021 0.016 0.008 0.220 0.01 n.d. n.d. 0.012 0.002 0.002 n.d. 0.025 0.001 n.d. n.d. n.d. n.d. n.d. n.d. 0.001 0.004 n.d. n.d. 0.004 0.004 0.004 n.d. 0.016 PAH 16 [NAP] [ANY] [FLU] [PHE] [FLA] [PYR] [CHR] [PAH 16] (sum)

68 /75 0.009 n.d. 68 /75 1 /22 25 /43 48 68 /75 75 1 /22 25 /43 48 0.012 0.017 0.075 0.046 0.012 0.009 0.014 0.052 n.d. 68 /75 75 1 /22 25 /43 48 0.006 0.001 0.056 0.035 n.d. Experiment time (d) 1 /22 25 /43 48 [PCP] 0.044 0.047 n.d.

well wat. APT 2 APT 1 PT 2 PT 1

Table 4 Concentrations of PCP and PAHs (mg l 1) in test series treated with organic cocktail PT 1, PT 2 (PCP/PAH treatment) and APT 1 and APT 2 (ammonia/PCP/PAH mixture treatment) measured in pooled and single random samples and in well water

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

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embryos), and in APT (three hatched embryos); and after 41 days in WC (one hatched embryo). However, after hatching had started, hatching did not proceed at the same rate in the different treatments. Hatching rates increased fastest in ammonia treated embryos, followed by PT and SC, which both showed similar hatching rates, then followed by the APT and finally by the WC. Thus, after 42 days, hatching rates were significantly higher in AT compared with the other treatments and the controls (P B/0.001 for AT vs. WC/APT, P B/0.01 for AT vs. SC/PT, Fig. 3). Differences between SC and APT, and WC and APT were weakly significant (P B/0.05). SC and PT, and PT and APT did not differ significantly (P
/0.05). When evaluating growth parameters of specimens at the end of exposure, no significant differences for length measurements were found among replicates of respective groups or in intergroup comparisons (P
/0.05). Median length of all specimens was 28 mm with minima of 26 mm and maxima of 31 mm (n/500). Mean weights were highest in AT (P B/0.05 for AT vs. WC and P B/0.01 for AT vs. PT; Fig. 4a). The weight of PT specimens was also significantly lower than in APT (P B/0.05). The mean condition factor was highest in APT which was significantly higher than in PT (P B/0.01), the group with the lowest condition factor (Fig. 4b). The fertilisation rate was 98.3%. Mortality rates differed in a very narrow range among different groups, and these differences were not significant (P
/0.05). Until hatching, mortality rates were 7.3% in WC, 6.5% in SC, 6.0% in AT, 7.4% in PT, and 7.3% in APT. After hatching, mortality did not further increase in any of the groups. Malformation rates were low in all groups with 1.4 and 1.5% in WC and SC, respectively, 3.1% in AT, and 2.3% in both PT and APT. Differences in malformation rates were not significant (P
/0.05). 3.3. Hsp70 level Expression of hsp70 was measured in embryos at three developmental stages (pre-hatching, day 41; post-hatch, day 51; end of embryonic period, day 78). Over the course of the exposure, hsp70 levels tended to decrease slightly in the water and

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T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

Table 5 Tissue concentrations of PCP and PAHs (mg kg1 dry weight) in trout embryos treated with the PCP/PAH mixture (PT 1, PT 2, APT 1, APT 2) and from the solvent control (SC) and water control (WC) PT 1

PT 2

APT 1

APT 2

SC 1

WC 1

[PCP]

122.8

105.3

125.3

77.1

n.d.

n.d.

PAH16 [NAP] [ANY] [FLU] [PHE] [FLA] [PYR] [CHR] [PAH 16] (sum)

9.6 3.9 n.d. 82.2 17.5 10.4 n.d. 123.6

14.9 3.4 n.d. 75.2 15.8 8.3 n.d. 117.6

14.3 2.5 n.d. 66.7 15.7 7.9 n.d. 107.1

13.3 3.9 n.d. 72.3 18.8 8.6 4.9 121.8

4.5 2.1 n.d. 20.3 8.0 3.8 3.0 41.7

10.1 1.4 n.d. 13.2 2.3 2.4 n.d. 29.3

The numbers 1 and 2 in the codes for the exposure situation refer to replicate experiments. n.d.: not detected.

solvent control groups, but to increase in PCP/ PAH- and ammonia-treated embryos (Fig. 5). In embryos from day 41, hsp70 levels were 17/36% higher in AT compared with the other groups, however, differences between groups were not significant (P
/0.05, n/20 for each group; Fig. 5). After hatching (day 51), hsp70 levels were significantly elevated in SC (P B/0.05), PT (P B/ 0.01), AT, and APT (P B/0.001 each) compared with WC (Fig. 5). At this stage, differences between non-WC groups were not significant (P
/0.05). At day 78, hsp70 levels remained low in WC, whereas values for AT, PT and APT were significantly higher (P B/0.001). These treatments also showed significantly higher hsp70 levels than SC (P B/0.01 for SC vs. APT; P B/0.001 for SC vs. AT/PT). Differences between WC and SC at day 78 were not significant (P
/0.05). There also were no significant differences when comparing hsp70 levels in AT, PT, and APT at this stage, even though levels in PT were 24/28% higher than in AT and APT, respectively (P
/0.05, Fig. 5). S.D. were high in pre-hatching embryos compared with post-hatching (day 51), where S.D. were 15/25% lower than before. In the treatment groups (AT, PT, APT) S.D. at the end of the embryonic period were similar to values immediately after hatching, however, in the controls (WC, SC) values were reduced by further 15 and 46% when comparing days 51 and 78 (Fig. 5).

4. Discussion

4.1. Early life stage test In the ELST, significant differences in endpoint responses were found in all treatment groups (AT, PT, APT) and in the SC, thus indicating stress caused by compounds of the respective groups. Effects by the solvent DMSO have to be taken into account when interpreting the responses found in PT and APT. For the time course of hatching and heart rates, response levels were similar in SC and PT suggesting that in PT, DMSO was also the main cause for responses of these endpoints. However, developmental rates were increased and growth was reduced in PT compared with SC which may have been either due to specific effects of the PCP/PAH mixture or combinatory effects of the PCP/PAH mixture and DMSO. The effects observed in the ELST were at sublethal levels. The tested agents did not increase mortality or induce malformations, which indicates that embryos were not acutely affected. Lethal concentrations of NH3 for stages exposed from eyeing (Solbe´ et al., 1989) and LC50 values (96 h, 12, 35 days) presented by Thurston and Russo (1983) for rainbow trout of similar size to the brown trout used in the present study were about 10/19-fold higher than the NH3 concentration tested in this study. However, Solbe´ et al.

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

47

Fig. 1. Developmental advancement of trout embryos from different exposure groups at different time points of exposure (39, 49, 79 d). Developmental advancement is represented by developmental scores according to Killeen et al. (1999). Data of replicates of each treatment were pooled. Box-plots show median, 10, 25, 75 and 90th percentiles. m represent out-liers. Significant differences were determined by the Wilcoxon/Kruskal /Wallis two-sample test. P -values were Bonferroni-adapted. *: P B/0.05, **: P B/0.01, ***: P B/ 0.001. n : number of specimens. For abbreviations see Table 1.

(1989) also report that, in contrast to the present study, NH3 concentrations similar to those applied in our study caused considerable mortality rates in rainbow trout early life stages when exposed from fertilisation (71.1% at 0.027 mg NH3 l 1). The nominal PCP concentration in the present study was approximately 2000/6500-fold below LC50 values (96 h) reported for various salmonids (Webb and Brett, 1973; Davis and Hoos, 1975). Accelerated development, premature hatching and increased heart rates all indicate that tested compounds including the solvent resulted in in-

creased metabolic rates in embryos. The responses are typical reactions to xenobiotics described by numerous workers (reviewed by Von Westernhagen, 1988). Premature hatching of embryos in SC, AT, PT, and APT compared with WC may have been partly due to an accelerated development in the treatment groups and the solvent control. However, differences in the time course of hatching among groups are not sufficiently explained by differences in developmental rates alone. Though developmental rates were obviously higher in

48

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

Fig. 2. Mean heart rates9/S.D. of trout embryos from different exposures at day 40. Data of replicates of each treatment were pooled. Significant differences were determined by ANOVA and Tukey /Kramer HSD test. **: P B/0.01. n/10. For abbreviations see Table 1.

Fig. 4. (a) Mean body weights and (b) mean condition factors9/S.D. of trout from different exposures at the end of exposure (day 79). Data of replicates of each treatment were pooled. Significant differences were determined by ANOVA and the Tukey /Kramer HSD test. *: P B/0.05, **: P B/0.01. n/100. For abbreviations see Table 1.

Fig. 3. Percentages of hatched trout embryos in different exposures at day 42. Data of replicates of each treatment were summarised. Significant differences were determined by x2 test. P -values were Bonferroni-adapted. *: P B/0.05, **: P B/0.01, ***: P B/0.001. n : total number of embryos (not hatched and hatched). For abbreviations see Table 1.

embryos treated with test agents compared with the control, developmental rates did not significantly differ among AT, PT, and APT. Thus, the hatching process itself was obviously affected differently by the tested compounds. It is known that the hatching time point can vary within the

developmental period in fish embryos, since it also depends on environmental conditions (Yamagami, 1988). Low O2 levels appear to be a specific trigger that initiate hatching in teleosts (Rombough, 1988). In conditions of low [O2] in the ambient water (and, thus, in the perivitelline fluid) (Hamdorf, 1961; DiMichele and Powers, 1984; Yamagami, 1988; Oppen-Berntsen et al., 1990), or of a high O2 demand following activated metabolism (Berezovsky et al., 1979 cited in Rombough, 1988), premature hatching is induced. Thus, differences in metabolic rates and oxygen demand caused by different test compounds in the present experiment may also have caused differences in the time course of hatching in different groups. Hatching

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

49

Fig. 5. Mean levels of the stress protein, hsp70, (arbitrary grey scale values, mean9/S.D.) of specimens from different exposures at days 41 (before hatch), 51 (hatch completed), and 78 (end of embryonic development). Data of replicates of each treatment were pooled. Significant differences were determined by ANOVA and Tukey /Kramer HSD test. Significance levels *: P B/0.05; **: P B/0.01; ***: P B/0.001. n/20. For comparisons with SC significance levels are in brackets, those not in brackets are for WC. For abbreviations see Table 1.

which was earliest in AT indicated that ammonia had probably induced the highest increase of metabolic rates among the tested agents. This also was shown by our results on heart rates which were highest in the AT groups. Toxicity of NH3 may result from interaction with several biochemical pathways, such as carbohydrate and fat metabolism and protein synthesis (reviewed by Cooper and Plum, 1987). Paley et al. (1993) observed disturbed ionic regulation in rainbow trout alevins due to increased external [NH3]. Furthermore, high external [NH3] interferes with NH3 excretion in fish (Cameron and Heisler, 1983; Wright et al., 1993; Wilson et al., 1994). Under conditions of high external [NH3] internal NH3 has to be excreted against a gradient in an energy demanding process (Cameron and Heisler, 1983; Wilson et al., 1994) which consequently causes activation of metabolism. Although length did not significantly differ among different groups, clear differences were found for weight. As discussed for hatching, this

also may have partly been due to different developmental rates in embryos treated with test compounds. Embryos from groups which showed accelerated development may have started feeding earlier than control embryos and higher metabolic rates may have led to higher feeding activities resulting in higher weight gains. This may explain why weights from WC embryos with lower metabolic activity than the other groups were comparably low. However, weight and condition factor were lowest in PT, even though in this group metabolism was obviously more active. This may reflect a specific effect of PCP since reduced growth in fish due to PCP exposure has been found in various studies (Holmberg et al., 1972; Webb and Brett, 1973; Samis et al., 1994). Toxic effects of PCP are mainly attributed to uncoupling of oxidative phosphorylation (Weinbach, 1954). In fish, this leads to metabolic stress indicated by increasing oxygen consumption (Crandall and Goodnight, 1962; Peer et al., 1983; Kim et al., 1996) and accelerated utilisation of tissue energy

50

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

reserves (Holmberg et al., 1972; Samis et al., 1994) which, in consequence, may lead to decreased growth. In contrast, embryos from ammonia treated groups (AT and APT) showed comparably high weights. Increased growth at low ammonia levels was also found in chinook salmon at [NH3] below 0.026 mg l 1 compared with an uncontaminated control (Robinson-Wilson and Seim, 1975). It is remarkable that in particular weight and the condition factor of embryos from APT were significantly higher than in PT despite APT also containing PCP. Though increased weights in AT and APT may be attributed to accelerated growth and to higher food intake due to higher metabolic rates, increased water uptake, an effect specific to ammonia, may have contributed to higher weight gains. Thus, Lloyd and Orr (1969) observed that sub-lethal concentrations of NH3 lead to increased urine excretion in rainbow trout and suggested that this is a consequence of increased permeability of membranes in fish exposed to NH3 enriched water. In APT, the responses indicated antagonistic effects of ammonia and the PCP/PAH mixture (in combination with the solvent) for all endpoints. Differences in endpoint responses from the control generally were lower for the combination of ammonia and the PCP/PAH mixture (APT) than for either ammonia (AT) or PCP/PAH mixture (PT) alone (Table 6). In this study, the staging scheme for brown trout developed by Killeen et al. (1999) was used for the first time to evaluate effects of xenobiotics on the developmental rate. Killeen et al. (1999) demonstrated the suitability of their scoring scheme for staging trout embryos reared at different temperatures. In the present study, the staging scheme allowed detection of slight differences in developmental rates which could not be displayed by established ELST criteria, such as the parameters ‘‘time until eyeing’’ or ‘‘depletion of yolk reserves’’ (data not shown). 4.2. Hsp70 level In advanced stages, the level of the stress protein, hsp70, was clearly induced by ammonia, the PCP/PAH mixture, and the combination of

both. As found in the ELST, the combination of ammonia and PCP/PAH mixture had antagonistic effects also for induction of hsp70. At day 51, hsp70 levels in embryos from APT were between PT and AT, and at day 78 these levels were slightly lower than in AT and considerably lower than in PT. In SC, expression levels of hsp70 were only slightly increased compared with the control, thus increased hsp70 levels in PT can be attributed to PCP and/or PAHs, although it cannot be excluded that DMSO, in combination with PCP and PAHs, has contributed to mixture toxicity effects. Response levels of expression of hsp70 increased with exposure time. This may reflect (1) that stress due to tested compounds increased in embryos in parallel with the exposure time, (2) that the more advanced developmental stages were increasingly susceptible to stressors, or (3) that the more advanced embryonic stages were more able to respond to toxic stress by increasing hsp70 expression. It has been shown previously in zebrafish embryos that induction of hsp70 depends on developmental stage (Krone et al., 1997; Yeh and Hsu, 2000). It is unlikely that pre-hatching stages which did not show any hsp70 response to exposure were less affected by toxicants than more advanced stages due to potential protection by the chorion since it was shown for rainbow trout that ammonia is capable of passing through the chorion (Rahman-Noronha et al., 1996). Furthermore, results from the ELST indicated stress responses in pre-hatching stages. The basic cause for increased expression of stress proteins (hsps) is increased amounts of misfolded or denatured intracellular proteins (Ananthan et al., 1986; McDuffee et al., 1997). In experiments with fish, hsp expression was induced mainly by temperature changes (e.g. Krone et al., 1997; Currie et al., 2000; see also reviews by Sanders, 1993; Iwama et al., 1998; Lewis et al., 1999), but also xenobiotic stress was found to increase tissue levels of hsp (reviewed by Iwama et al., 1998; Schramm et al., 1999; Ko¨hler et al., 2001). Ammonia has been shown to interfere with protein and amino acid metabolism (reviewed by Cooper and Plum, 1987). As a consequence, the amount of protein fragments may increase thereby inducing hsp expression. Thus, in AT, increased hsp70 expression may

T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

51

Table 6 Response levels in the different treatment groups and the solvent control found in ELST and hsp70 analyses and ultrastructure studies on liver and gills (Gernho¨fer et al., 2002, this issue)

ELST

hsp70

US liver US gills

Development rate Hatching Heart rate Growth Malformations Mortality General 41 days 51 days 78 days General General

AT

PT

APT

SC


/
/
/
/
/
/
/
/ / / /
/
/
/ /
/
/
/
/
/ /
/
/
/


/
/
/
/
/
/
/ / /
/
/ /
/
/
/
/
/
/
/
/
/
/
/
/


/
/ / / / /
/ /
/
/
/
/
/
/
/
/
/
/

/
/
/
/ / / /
/ /
/ /
/
/

Numbers of
/ represent relative response levels compared with the water control. /, no response.

indicate proteotoxic, ammonia-specific effects. On the other hand, increased hsp levels in treatment groups AT, PT, and APT could reflect, in a more non specific way, a general state of increased stress. Elevated amounts of damaged proteins could also be due to an increased protein turnover as a consequence of a generally activated metabolism which was indicated by results of the ELST. 4.3. General conclusions Results of the ELST and hsp70 analyses presented in this paper and cellular responses in liver and gills detected by ultrastructural studies (Gernho¨fer et al., 2003, in preparation) indicated effects in brown trout early life stages by sub-acute levels of the tested compounds. Depending on the method of examination relative response levels to agents were found to be different, which reflects the specific potentials of the different methods to show the way in which trout embryos were affected by the tested substances (Table 6). ELST and hsp70 analysis indicated general stress effects in the entire organism, with ELST endpoints and stress protein levels reflecting general metabolic responses and effects on the protein metabolism, respectively. The ultrastructural studies on liver and gills demonstrated specific reactions which

show they are particularly affected by xenobiotic exposure. The liver, responsible for metabolism of hydrophobic compounds, was affected mainly by organic compounds. The reaction of gill cells reflected the vulnerability of this sensitive organ, involved in gas and ion exchange, to the harmful effects of all of tested xenobiotics in the ambient environment (Table 6). All methods applied indicated slight effects of the solvent DMSO. However, response levels in PT generally were higher than in SC indicating specific effects by PCP/PAH, or by combined effects of PCP/PAH and solvent (Table 6). The fact that effects due to PCP/PAH were found is remarkable, since nominal and measured concentrations of both PCP and PAHs were considerably below the lowest effective concentrations for lethal and sublethal responses compared with other studies (see Webb and Brett, 1973; Van Brummelen et al., 1998). ELST, hsp70 analyses, and studies on the ultrastructure of gills all indicated less-than-additive or even antagonistic effects of ammonia and the PCP/PAH mixture (Table 6). The reason for antagonistic effects of ammonia and the organic mixture is not known. Possible explanations may be, that either (1) ammonia and organic compounds interacted chemically resulting in a less toxic potential of either compounds or (2) the

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T. Luckenbach et al. / Aquatic Toxicology 65 (2003) 39 /54

combination of ammonia and the PCP/PAH mixture exceeded a toxic threshold value above which detoxifying metabolic processes are induced and reduced toxic potentials of tested xenobiotics. In groups treated with either ammonia (AT) or the organic mixture (PT) these detoxification processes may not have been induced, because concentrations of toxins had remained beneath a critical threshold value of induction. Only in hepatocytes, ultrastructural effects caused by the combination of ammonia and the PCP/PAH mixture were found to be slightly higher than those induced by the organic mixture or ammonia alone (Table 6). Concentrations of compounds tested in this study were adapted to levels found in the field during a semi-field exposure experiment with brown trout early life stages (Luckenbach et al., 2001). In the semi-field experiment response levels of ELST endpoints were considerably higher than in the present laboratory study. Thus, in contrast to the present study, mortality rates were increased in trout embryos due to exposure to polluted water in the semi-field (Luckenbach et al., 2001). This shows that stress levels in the field obviously were considerably higher than in the laboratory treatments with ammonia, PCP/PAH or their mixture in this study. Furthermore, there were also differences in sublethal responses in both experiments. Whereas embryos from treatments AT, PT, and APT in the present study showed increased developmental rates and similar or higher weight gains compared with control groups, development was retarded and growth was reduced in stream waterexposed trout embryos (Luckenbach et al., 2001). However, even though the exposure situation in the present laboratory experiment apparently did not completely mirror the situation in the semifield study, ammonia and the combination of PCP and PAHs were shown to cause stress in trout embryos at very low concentrations. Although, the combined effects of ammonia and organic agents appeared to act in a less-than-additive or even antagonistic way in this study, they most likely contributed to detrimental effects in free-living fish exposed to more complex mixtures of environmentally relevant chemicals.

Acknowledgements This study was financially supported by a grant from the Landesbank Baden-Wu¨rttemberg to T.L. and R.-D.N. and a scholarship from the state of Baden-Wu¨rttemberg to T.L.. The authors would like to thank Thomas Hu¨lsmann, Maja Kilian, and Julia Schwaiger for technical help and Jaime Bainbridge and Cathy Thaler for proof-reading of the manuscript.

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