Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species

Comparative Biochemistry and Physiology, Part C 214 (2018) 61–67

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Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species

T

⁎

Kazuhiko Mochida , Katsutoshi Ito, Mana Ito, Takeshi Hano, Nobuyuki Ohkubo National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research and Education Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan

A R T I C LE I N FO

A B S T R A C T

Keywords: Acute toxicity test Dithiocarbamate fungicide Early-life stage toxicity test Embryo toxicity test Gene expression analysis

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ± 36 mg) were about 12 and 630 μg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 μg/L and a no-observed-effect concentration of 2.1 μg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 μg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

1. Introduction Restrictions on the use of organotin antifoulants enacted in the 1980s resulted in new antifouling biocides being used on fishing nets and ship hulls. In Japan, since the early 2000s fishing nets have been treated with polycarbamate (PC) and boron-based compounds such as triphenyl (octadecylamine) boron (TPB-18) and among these, the most used biocide has been PC, which accounts for > 90% of total biocide emissions in this decade. Boron-based biocides produced only 0.5–0.6% of total emissions (PRTR [Pollutant Release and Transfer Register], Japan; http://www.env.go.jp/en/chemi/prtr/prtr.html). PC is classified one of the dithiocarbamate fungicides (DTC), which are formed by dimethyldithiocarbamate (DMDC) and ethylenebisdithiocarbamate (EBDC), and is composed of two molecules of DMDC and one molecule of EBDC combined with zinc. PC along with EMDC and EBDC were originally developed for agricultural use to control diseases in a wide variety of crops (Davies and White, 1985). Because DTCs had agricultural uses, there are some data for toxicity of this compound to freshwater organisms (Van Leeuwen et al., 1985a, 1985b, 1986). As far as we know, there is only one study dealing with the toxicity of PC to marine organisms (Hano et al., 2017), and it demonstrated the acute toxicity of PC to three marine fish species. Thus, even though PC has been used for aquaculture nets, the data for the

toxicity of this compound to marine organisms were definitely insufficient, and such information is necessary for a primary risk assessment of this compound in the marine environment. The primary objective of the current study was to determine acute and chronic toxicity values of PC for a marine fish species, the mummichog (Fundulus heteroclitus), by conducting the acute toxicity and early life-stage (ELS) toxicity tests. Chronic toxicity values for PC were then predicted for three domestic marine fish species using the acute toxicity values obtained in the previous study (Hano et al., 2017) and the acute:chronic ratio (ACR) calculated from the toxicity values for mummichog. Kenaga (1982) has previously reported that the chronic toxicity value could be reliably predicted from the respective acute toxicity value for organic compounds using the ACR under some conditions. We also carried out the embryo toxicity test to examine the toxicity of PC to fish early life stages using another marine fish species, spotted halibut (Verasper variegatus), to validate the obtained toxicity values. Using the estimated chronic toxicity values, we used a conventional approach to conduct a primary risk assessment of PC in marine environments. During the ELS tests using mummichog, observation in hatched mummichog larvae of morphological abnormality — specifically, distortion of the trunk derived from notochord undulation — in hatched

⁎ Corresponding author at: National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan. E-mail address: [email protected]ffrc.go.jp (K. Mochida).

https://doi.org/10.1016/j.cbpc.2018.09.001 Received 29 June 2018; Received in revised form 29 August 2018; Accepted 4 September 2018 Available online 07 September 2018 1532-0456/ © 2018 Elsevier Inc. All rights reserved.

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Hayashi Pure Chemical Ltd. (Osaka, Japan). Stock solutions of PC were prepared by dilution with dimethylsulfoxide (DMSO; > 99% purity, Wako Pure Chemical Industries, Osaka, Japan). Acute toxicity tests for PC were carried out under semi-static conditions. The tests were carried out with a geometric series of nominal PC concentrations that varied by factors of 2 from 6.3 to 100 μg/L for larval mummichogs, or from 200 to 3200 μg/L for juvenile mummichogs. For the larval mummichog test, each experimental group of 10 larval mummichogs was placed in a small chamber that included a net (14 × 12 cm, 100-μm mesh) with a float that kept the net about 10 cm under the surface of the test solution in the exposure tank. Exposure was conducted in a glass tank (300 × 200 × 250 mm) filled with 10 L of the test solution. For the juvenile mummichog tests, 10 fish were placed directly into the exposure tanks containing the test solutions. The tests lasted 96 h. The exposure solutions were changed daily. During the acute toxicity tests (96 h), mortality was monitored daily and no food was provided. The test apparatus was shaded from exposure to direct light to prevent photodegradation of PC. Water quality parameters for the fish acute toxicity tests of PC were as follows: water temperature, 19 ± 0.55 °C; dissolved oxygen, 6.7 ± 0.89 mg/L; oxygen saturation, > 90%; pH, 8.1 ± 0.01; and salinity, 32 ± 0.05 (mean ± standard deviation).

mummichog larvae indicated that the abnormality was already present during embryonic development. The notochord distortion was also observed in the embryo toxicity test using spotted halibut. It has already been reported that DTCs and several degradation products such as tetramethylthiuram disulfide induce wavy distortions of the notochord in zebrafish (Teraoka et al., 2006; Tilton et al., 2006). The induction of the distorted notochord is thought to be a toxic effect that DTCs have in common. Type II collagen is thought to be involved in embryonic development and is an essential component of the notochord in vertebrates (Cheah et al., 1991; Bieker and Yazdani-Buicky, 1992; Seufert et al., 1994; Yan et al., 1995), and a study using a specific inhibitor for lysyl oxidase (LO) revealed that LO plays a crucial role in frogs in the organization of collagen fibers before notochord formation (Geach and Dale, 2005). LO inhibition-mediated induction of notochord undulation has also been reported in embryos of two fish species exposed to the thiocarbamate insecticide Cartap [S,S′-(2-dimethylaminotrimethylene)bis (thiocarbamate)] (Zhou et al., 2009) and metal pyrithione degradation products, such as 2-mercaptopyridine-N-oxide (HPT) (Anderson et al., 2007) and 2,2′-dipyridyldisulfide [(PS)2] (Mochida et al., 2012a). To investigate the relationship between the inhibition of LO activity and the notochord undulation induced by PC exposure, we also carried out in vitro LO activity inhibition tests using a homogenate of mummichog embryos to see if PC was the source of the LO inhibition activity. We also conducted embryo toxicity tests using mummichog to confirm the reproducibility of the induction of notochord undulation by PC. Furthermore, we carried out preliminary transcriptomic analysis and compared several of the genes related to notochord formation between non-treated embryos and PC-treated embryos to elucidate the molecular mechanism of the induced morphological abnormality.

2.2.2. ELS toxicity test For the ELS test, stock solutions were prepared to adjust the concentration of PC to 375, 750, 1500, and 3000 mg/L. The stock solutions of PC for the ELS test were prepared by dilution with DMSO (plant cell culture tested grade, > 99.5% purity, Sigma-Aldrich, St. Louis, MO, USA). The test and solvent control (DMSO only) solutions were diluted in the exposure tanks (to concentrations noted below) by seawater fed from a header tank (flow rate, 300 mL/min). The stock solutions in Hamilton glass syringes (Sigma-Aldrich) were fed by a micro-syringe pump (IC3230; International Scientific Instruments Supply Co., Ltd., Osaka, Japan) directly into the exposure tanks at a flow rate of 3.0 μL/ min to achieve the target nominal concentrations. For the 60-μg/L exposure group, two syringes with 3000-mg/L stock solution were fed into the exposure tank. The DMSO concentration in the exposure tank for the solvent control was 10 μL/L. For the control, only seawater was fed from a header tank (flow rate, 300 mL/min) to the exposure tank. For ELS tests we used embryos from late blastula to early gastrula stage. Subgroups of 20 embryos (for the seawater only control and the solvent control) or 18 embryos (for PC exposure) were transferred to one of four chambers for exposure to the test solutions. The nominal PC concentrations for the ELS tests varied by a factor of two in a series from 3.8 to 60 μg/L. After hatching, larvae in each subgroup were fed about 8 × 103 of Artemia nauplii once daily. Hatchability (%) was calculated as (number of hatched larvae)/(number of embryos used in the experiment) × 100, and average time-to-hatch was measured for each subgroup. The test duration was 55 d. Mortality was monitored daily, and all dead animals were removed. Survival (%) was calculated as (number of surviving larvae) / (number of hatched larvae) × 100. At the end of each test, all fish were measured for total length and body weight to determine growth, and the number of survivors with or without morphological abnormalities was also counted. The test apparatus was shaded from exposure to direct light to prevent photodegradation of PC. Water quality parameters for the ELS tests for PC were as follows: water temperature, 19 ± 0.10 °C; dissolved oxygen, 7.5 ± 0.11 mg/L; pH, 7.3 ± 0.02; salinity, 30 ± 0.10; and oxygen saturation, > 90%.

2. Materials and methods 2.1. Animals The Arasaki strain of the mummichog (Shimizu, 1997) has been bred in our laboratory for several years. Ten adult mummichogs, both male and female with secondary sexual characteristics and weighing approximately 10 g each, were kept for at least seven months (April to October) in a 60-L aquarium with a flow-through seawater system and natural light illumination. Once daily during mating season, the fish were fed an appropriate commercial diet (C-2000; Kyowa Hakko, Tokyo, Japan). Spawned eggs were carefully collected by using a pipette, and embryos in the early blastula stage were selected under a microscope and used for ELS tests. Larvae at 2 to 3 d post-hatch (total length, 6.4 ± 0.1 mm; body weight, 2 ± 0 mg) or juvenile mummichogs (total length, 37 ± 0.59 mm; body weight, 660 ± 36 mg) (mean ± standard error) were used in the acute toxicity tests and were maintained in indoor 60-L aquaria under a natural photoperiod until initiation of toxicity testing. Embryos were staged according to the criteria reported by Armstrong and Child (1965). Embryos at various stages were used for LO activity measurements. Fertilized eggs of spotted halibut (Verasper variegatus) were generously provided by National Research Institute of Tohoku, Fisheries Research and Education Agency, Miyako, Japan. 2.2. Toxicity tests 2.2.1. Acute toxicity test Toxicity tests were performed as recommended in the test guidelines of the Organization for Economic Cooperation and Development (1992a, 1992b). Test solutions for the acute toxicity tests were prepared by diluting the stock PC solution (500 mg/L) to the concentrations described below with seawater that had been aerated and filtered through sand and activated carbon. PC (molecular weight, 581.62; purity, 95.1%) was purchased from

2.2.3. Embryo toxicity test We carried out embryo toxicity tests for PC using spotted halibut or mummichog under semi-static conditions. Embryos at blastula to early gastrula stages were exposed to PC at 3.7, 11, 33, 100, or 300 μg/L for spotted halibut, or 3.3, 10, 30, 90, or 270 μg/L for mummichog. To make test solutions, 10 μL of a solution of PC in DMSO (Sigma-Aldrich), 62

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Mochida et al., 2012a). A 250-μl aliquot of the lysate was mixed with 750 μL of a 4 × reaction buffer containing 4 U/mL type II horseradish peroxidase (Sigma), 40 mM cadaverine, and 40 mM N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red; AnaSpec Inc., San Jose, CA, USA) in 1.2 M urea–50 mM sodium borate (pH 8.2). One microliter of PC, DMSO (Sigma-Aldrich) as a solvent control, or 2-mercaptopyridine-N-oxide (HPT; molecular weight, 127.16, Tokyo Kasei Kogyo Company, Tokyo, Japan) as a positive control (Anderson et al., 2007) at an appropriate concentration was then added to the mixture to achieve 0.1, 1, and 10 μM for PC or 10 μM for HPT. These concentrations correspond to 58, 580, and 5800 μg/L for PC and 1300 μg/L for HPT. After incubation of the mixture for 90 min at 37 °C, Amplex Red fluorescence was measured at defined intervals with a fluorescence spectrophotometer (F-2000, Hitachi, Ibaraki, Japan) using an excitation of 560 nm and an emission of 590 nm. For each sample, the background fluorescence was subtracted to generate a corrected fluorescence.

1000 times higher than the target concentration, was diluted with 10 mL of seawater sterilized with a sterile filter unit (50 mm diameter, 0.2 μm pore size; Nalgene, Rochester, NY, USA). For the solvent control, 10 μL of DMSO was diluted with 10 mL of sterile seawater. The test solutions, including the solvent control solution, were changed every two days or once daily for spotted halibut or mummichog, respectively, and all dead embryos were removed daily. For spotted halibut, 75 embryos (15 embryos × 5 subgroups) were exposed to PC for 10 d at 12 °C in a 6-well culture plate (BD Falcon, Becton, Dickinson and Company, Tokyo, Japan) containing 10 mL of each test solution. Our preliminary observations revealed that embryonic development of the spotted halibut was temperature-sensitive, and hatchability decreased prominently at temperatures above 12 °C due to abnormal embryonic development (Table S1). At 12 °C, spotted halibut embryos hatch within one week, and hatched larvae start feeding about one week after hatching. The test duration was thus set to 10 d. For mummichog, 30 embryos were exposed to PC for 14 d at 20 °C in a 6-well culture plate (BD Falcon, Becton, Dickinson and Company, Tokyo, Japan) containing 10 mL of each test solution. We documented morphological abnormalities, such as notochord undulation, and survival at the end of the test by microscopic examination with a stereomicroscope. We used the presence or absence of a beating heart to determine whether an embryo had survived. For the seawater-only controls, embryos at the same stages mentioned above were incubated in 10 mL of sterilized seawater. Water quality parameters for the embryo toxicity tests with spotted halibut were as follows: water temperature, 12 °C; dissolved oxygen, 7.9 ± 0.33 mg/L; oxygen saturation, > 90%; pH, 7.8 ± 0.26; and salinity, 30 ± 0.10.

2.5. Data analysis Estimation of 96-h LC50 values, the multiple-comparison test, and calculation of Pearson product-moment correlation coefficient were performed using R software (R Development Core Team, 2007), mainly with the drc (Ritz, 2010) package, multcomp (Dunnett's test) package, and cor.test function, respectively. Differences between groups were considered significant at P < 0.05. 3. Results 3.1. Toxicity

2.2.4. Chemical analysis PC concentration in the test seawater was analyzed with liquid-liquid extraction based on the decomposition of PC to DMDC followed by methyl derivatization to methyl dimethyldithiocarbamate (DMDC-methyl) (Hano et al., 2015b). Seawater samples from test aquaria were collected for chemical analysis 0 h and 24 h after the start of each acute toxicity test, once every 10 d for ELS tests, or 0 and 48 h after the start of the embryo toxicity tests for spotted halibut. For the mummichog embryo toxicity tests, the actual toxicant concentrations were not analyzed. The analytes extracted from test water were measured using an Agilent 6890N gas chromatograph (Agilent Technologies, Tokyo, Japan) by the method described previously (Hano et al., 2015b). The detection limit for PC in test water samples was 0.05 μg/L. We used geometric mean values of toxicant concentrations to estimate the LC50 values, lowest observed effect concentrations (LOECs), and no observed effect concentrations (NOECs).

3.1.1. Acute toxicity Actual measured concentrations of PC averaged about 44% ± 18% of targeted nominal concentrations for acute toxicity tests (Table 1 and S2). Survival in the solvent control group of the acute toxicity test was 100%. The twice-repeated acute toxicity tests had reproducible results (Table 1 and S2), and the 96-h LC50 values of PC for larval and juvenile mummichog averaged from replicate experiments were about 12 and 630 μg/L, respectively (Table 1 and S2). 3.1.2. Chronic toxicity In the ELS tests, actual measured concentrations of PC were about 52–61% of targeted nominal concentrations in all experimental groups (Table 2). Results from all PC exposure groups were compared to those of the solvent control for all endpoints because there were no significant differences between results from the seawater control group and the solvent control group. Although there was no effect on time-to-hatch and hatchability in any of the experimental groups, survival at 55 d was significantly lower in the 60-μg/L exposure groups than in the solvent control groups (Table 2 and S3-1,2,3). A marked toxic effect of PC was also observed on the growth of mummichogs. Both total length and body weight were significantly lower (P < 0.05) in the groups exposed to PC concentrations ≥7.5 μg/L than in the solvent control group, except for 15-μg/L (Table 2 and S3-4). Severe morphological deformities, such as vertebral deformity, were observed for > 8.6% and 11% of the hatched larvae in the 30-μg/L and the 60-μg/L exposure groups, respectively (Table 2 and S5). Similar vertebral deformities were observed in only one out of 57 or one out of 51 individuals in the control and solvent control groups, respectively (Table 2 and S3–5). Collectively, therefore, growth was the most sensitive endpoint, and the LOEC and NOEC were determined to be 3.9 and 2.1 μg/L, respectively, from the actual toxicant concentrations.

2.3. Histologic techniques After exposure to PC, some of the mummichog embryos were fixed in 10% formalin for observation of morphological abnormalities. Cartilage and bone were cleared and stained with alcian blue (Wako Pure Chemical Industries) and alizarin red S (Wako Pure Chemical Industries), respectively, according to the procedures of Dingerkus and Uhler (1977). 2.4. LO assay Ten embryos, from the cell multiplication stage to the end of the gastrula stage, were mechanically homogenized with a Polytron homogenizer (Kinematica, Luzern, Switzerland) in 1 mL 6-M urea–50mM sodium borate (pH 8.2) and extracted overnight with agitation at 4 °C. The supernatant was isolated by centrifugation at 10,000 ×g for 10 min and diluted with 50 mM sodium borate (pH 8.2) to a urea concentration of 1.2 M. Assays were carried out according to published methods (Palamakumbura and Trackman, 2002; Anderson et al., 2007;

3.1.3. Embryo toxicity In the embryo toxicity test for the spotted halibut, actual measured PC concentrations were about 42–94% of targeted nominal concentrations in all experimental groups (Table 3). Results from all PC exposure 63

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Table 1 Acute toxicities of polycarbamate to larval (2–3 days post-hatch) and juvenile mummichogs (Fundulus heteroclitus). Experimenta

Polycarbamate concentration (μg/L) Nominal

1

0 (SC) 6.3 13 25 50 100 0 (SC) 6.3 13 25 50 100 0 (SC) 200 400 800 1600 3200 0 (SC) 200 400 800 1600 3200

2

3

4

Number of fish

Actual

96-h LC50 (μg/L)

0h

24 h

Geometric mean

Exposed

Survived

n.d. 1.2 5.3 16 30 61 n.d. 2.7 6.8 19 33 68 n.d. 109 256 1260 1000 2160 n.d. 264 94.0 914 690 2350

n.d. 0.69 5.5 9.7 20 45 n.d. 0.46 1.4 3.1 9.5 19 n.d. 93.7 171 406 761 1410 n.d. 7.60 197 295 548 1490

n.d. 0.91 5.4 12 24 52 n.d. 2.1 3 8 18 36 n.d. 101 209 715 872 1745 n.d. 45 136 519 615 1871

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

10 10 10 5 0 0 10 10 10 9 2 0 10 8 10 5 0 0 10 8 9 9 0 0

12 (10–14)b

13 (8.2–22)b

710 (670–760)b

540 (220–870)b

SC, solvent control. n.d., not detected. a Tests were carried out under semi-static conditions. Larval fish were used in experiment 1 and 2, juveniles in 3 and 4. b Values in parentheses are the 95% confidence limits.

basis of actual toxicant concentrations, the LOEC and NOEC were determined to be 8.5 and 6.7 μg/L, respectively. The 10-d EC50 values and 95% confidence intervals (in parentheses) for hatchability and abnormality were 13 (4.0–23) μg/L and 8.1 (7.5–8.8) μg/L, respectively. The embryo toxicity test for mummichog revealed that 15% and 59% of the embryos developed notochord undulation in the 90-μg/L (0.15 μM) and 270-μg/L (0.46 μM) exposure groups, respectively (Fig. 2).

groups were compared to those of the solvent control for all endpoints because there were no significant differences between the seawater control group and the solvent control group. There were significant effects on time-to-hatch and hatchability in the 33-μg/L and 100-μg/L exposure groups. Abnormality of the embryo also increased with exposure in a concentration-dependent manner (correlation coefficient = 0.8075, P < 0.05). Severe morphological deformity of the notochord was observed in 35% and 55% of the embryos in the 33-μg/L and the 100-μg/L exposure groups, respectively (Table 3, S4-1,2, and Fig. 1). There was no statistical difference of the 300-μg/L exposure group because most of the embryos died before hatching. In the control and solvent control groups, 5.3% and 2.7% of embryos, respectively, also showed abnormalities, although these values were lower than those in the PC exposure groups (Table 3 and S4-3). Thus, both hatchability and abnormality were the most sensitive endpoints. On the

3.2. Inhibition of LO activity To elucidate the mechanism of notochord undulation induced in the embryos when they were exposed to PC during the ELS test, we performed an in vitro assay to investigate whether PC could inhibit LO activity in mummichog embryos. The inhibition of LO activity

Table 2 Effect of long-term exposure to various concentrations of polycarbamate (PC) on time-to-hatch, hatchability, survival, growth, and morphological abnormalities in mummichog (Fundulus heteroclitus). Group

Polycarbamate concentration (μg/L) Nominal

Control Solvent cont. PC-exposed

0 0 3.8 7.5 15 30 60

No. of used embryosa

Time to hatch (d)

Hatchability (%)

Survival (%) (55 d)

Actual 1

2

3

4

5

Geometric mean

n.d. n.d. 2.4 5.6 25 17 25

n.d. n.d. 2.3 2.4 6.2 10 36

n.d. n.d. 2.6 4.8 7.5 21 40

n.d. n.d. 1.9 4.3 6 20 40

n.d. n.d. 1.5 3.4 8.4 16 35

n.d. n.d. 2.1 ± 0.22 3.9 ± 0.62 9.0 ± 4.1 16 ± 2.2 35 ± 3.1

80 80 72 72 72 72 72

21 24 23 27 22 22 23

± ± ± ± ± ± ±

0.1 1.5 0.7 0.7 0.5 0.6 0.5

75 68 71 69 75 67 58

± ± ± ± ± ± ±

3.5 6.0 5.3 3.6 1.6 6.8 4.8

95 ± 2.9 95 ± 3.5 100 ± 0.0 96 ± 2.3 91 ± 1.6 79 ± 3.2 77 ± 8.2b

n.d., not detected; TL, total length; BW, body weight. a 4 subgroups of 20 for n = 80 and 4 subgroups of 18 for n = 72. Data are expressed as means ± standard error. b Significantly different from the value for the solvent control (P < 0.05). 64

Growth (55 d)

Abnormality (%)

TL (mm)

BW (mg)

(55 d)

15 15 16 14 14 13 12

42 38 37 30 34 25 24

1.7 ± 1.7 1.6 ± 1.6 0.0 ± 0.0 2.1 ± 2.1 2.1 ± 2.1 8.6 ± 5.1 11 ± 11

± ± ± ± ± ± ±

0.4 0.4 0.2 0.3b 0.4 0.3b 0.4b

± ± ± ± ± ± ±

3.2 2.1 1.5 1.4b 2.5 2.0b 2.4b

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Table 3 Effect of exposure to various concentrations of polycarbamate (PC) for 10 days on time-to-hatch, hatchability, survival, growth, and morphological abnormalities in spotted halibut (Verasper variegatus). Group

Polycarbamate concentration (μg/L) Nominal

Control Solvent cont. PC-exposed

a b

0 0 3.7 11 33 100 300

No. of used embryosa

Time to hatch (d)

Hatchability (%)

Abnormality (%) 10 d

75 75 75 75 75 75 75

6.5 6.5 6.4 6.2 7.6 8.2 11

81 ± 4.9 92 ± 2.8 93 ± 3.3 80 ± 11 51 ± 5.1b 19 ± 8.3b 1.3 ± 1.5

5.3 ± 3.7 2.7 ± 1.8 2.7 ± 1.8 4.0 ± 3.0 35 ± 4.4b 55 ± 10b 2.7 ± 3.0

Actual 0h

48 h

Geometric mean

n.d. n.d. 2.4 7.5 12 54 204

n.d. n.d. 2.0 5.9 6 58 387

n.d. n.d. 2.2 6.7 8.5 56 280

± ± ± ± ± ±

0.14 0.0 0.10 0.11 0.22b 0.11b

5 subgroups of 15 for n = 75. Data are expressed as means ± standard error. Significantly different from value for the solvent control (P < 0.05). Fig. 1. Distortion of the notochord induced in embryos of spotted halibut (Verasper variegatus) exposed to polycarbamate (PC). a) Normal hatched larvae, b) embryo with abnormal shape; i.e. distorted trunk, and c) short trunk as observed after 10-d exposure to 33 or 100 μg/L PC. Arrows indicate distorted region of notochord. Scale bars, 2 mm.

a)

SC

90 g/L

270 g/L

Abnormality (%)

b)

70

60 50 40 30 20 10 0

C SC 3.3 10

30 90 270

Concentration ( g/L)

65

Fig. 2. a) Notochord undulation and b) frequency of abnormality in mummichog (Fundulus heteroclitus) induced in the embryo toxicity test for polycarbamate (PC). Thirty embryos were used in each experimental group in b). Under panels in a) show typical examples of mummichog embryos exposed to PC; lower panels show enlarged views of a normal notochord (lower left) and a distorted notochord (lower center, lower right) stained with alcian blue. Arrows indicate the distorted region of the notochord. SC, solvent control. Scale bars, 1 mm or 200 μm in upper and lower panels, respectively, in a).

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4.1. Ecotoxicity data

4.2. Abnormalities induced by exposure to PC

In this study, we documented both chronic and acute toxicity of PC to a marine fish, the mummichog. We have previously demonstrated that 96-h LC50 values for PC to other marine teleost fish, such as juvenile marbled flounder (body weight, 1.6–2.0 g), red sea bream (1.1–1.5 g), and spotted halibut (0.4–2.2 g) are 300 ̶ 360 μg/L, 22 ̶ 24 μg/L, and 240 ̶ 550 μg/L, respectively (Hano et al., 2017). The 96-h LC50 value for juvenile mummichog (body weight, 0.7 g) of about 630 μg/L obtained in the present study suggests that red sea bream is the species most susceptible to PC toxicity among these four fish species. We compared the acute toxicity value for PC to those of other frequently used antifouling biocides in Japan such as copper pyrithione [CuPT, bis-(hydroxy-2(H)-pyridine thionate-O,S)‑copper], pyridine triphenylborane (PTPB), and 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (Sea-Nine 211) (Okamura and Mieno, 2006). The 96-h LC50 values of PC, CuPT, PTPB, and Sea-Nine 211 for mummichog larvae (2–3 d post hatch) are 21–22 nM (present study), 27 nM, 1100–1500 nM, and 16–17 nM, respectively, and those for juvenile red sea bream are 38–41 nM, 29 nM, 900–1200 nM, and 13–23 nM, respectively (Mochida et al., 2006, 2010, 2012b; Hano et al., 2017). These results indicate that the magnitude of toxicity to the two fish species were in the order of PC ≈ CuPT ≈ Sea-Nine 211 ≫ PTPB. We also determined chronic toxicity value of PC for mummichog from the ELS test. The maximum acceptable toxicant concentration (MATC) expressed as the geometric mean of the LOEC and the NOEC was calculated to be 2.9 μg/L (√3.9 × √2.1 μg/L). To estimate the chronic toxicity value of PC for marbled flounder, red sea bream, and spotted halibut, we calculated the ACR to be 217 (630/2.9) using the acute toxicity value for mummichog larvae and the chronic toxicity value for mummichog. Using the ACR and the above-mentioned acute toxicity values (Hano et al., 2017), the chronic toxicity values for marbled flounder, red sea bream, and spotted halibut were estimated to be 1.4–1.7, 0.10–0.11, and 1.1–2.5 μg/L, respectively. The MATC

Vertebral deformities were observed in the mummichog ELS test. In addition, PC exposure induced morphological defects in the notochord in the embryo toxicity test for both spotted halibut and mummichog. The notochord plays an essential role in vertebrate development and is a major skeletal element of the developing embryo (Stemple, 2005). Thus, the vertebral deformity observed in the two fish species may be due to the failure of complete differentiation of the notochord as a result of PC toxicity. In the present study, exposure to PC induced a marked decrease in LO activity in mummichog embryo homogenate. LO catalyzes the crosslinking of collagen and elastin fibers in the extracellular matrix, which is necessary for the structural integrity and function of connective tissues (Geach and Dale, 2005; Mäki et al., 2005). Type II collagen is thought to play a pronounced role in embryonic development and is an essential component of the notochord in vertebrates (Cheah et al., 1991; Bieker and Yazdani-Buicky, 1992; Seufert et al., 1994; Yan et al., 1995). Thus, LO also plays a crucial role in the formation of the notochord. These ideas and our gene expression analysis support the suggestion that the notochord undulation induced in mummichog embryos exposed to PC was due to the inhibition of LO followed by collagen fiber disorganization, but not to a decrease in the supply of collagen. In the embryo toxicity test, PC evoked the morphological abnormality at concentrations of 90 μg/L (0.15 μM) and higher, whereas a much higher PC concentration, e.g. 1 μM, was required to induce significant inhibition of LO activity in the in vitro assay. These results may reflect the bioconcentration factor of PC for mummichog (3.1, Hano et al., 2015b) and the differences in the test duration between the in vivo (14 d) and in vitro (a few minutes) tests. Although we did not analyze LO activity in halibut embryos after exposure to PC, the notochord undulation observed in the embryo toxicity test was possibly caused by inhibition of LO activity due to PC toxicity. In the preliminary transcriptome analysis of the PC-exposed embryos, expression levels of notochord-formation and morphogenesis-

Relative fluorescence units

4. Discussion

obtained from the embryo toxicity test for spotted halibut was 7.5 μg/L (√8.5 × √6.7 μg/L) and is similar to the estimated chronic toxicity value. Kenaga (1982) examined 135 ACRs for 84 chemicals using 9 species of fish and species of aquatic invertebrates and showed that 93% of the ACRs for industrial organic chemicals were 25 or lower. In addition, Hayashi and Kashiwagi (2015) examined 186 publicly accessible ecotoxicity datasets for fish and showed that the median (10th, 90th percentile) ACR was 11.8 (2.0, 118.8). By comparison, the ACR for PC obtained in the present study is exceptionally large. Kenaga (1982) pointed out the possibility that metabolites of a parent chemical could cause a delayed chronic effect at low concentrations and therefore result in a large ACR. Because metabolites of PC such as EBDC and DMDC are also toxic to several fish species (Van Leeuwen et al., 1985a, 1986), it is likely that these metabolites at low concentration chronically affect the early life stages of mummichog during the ELS test. This might be one reason for the exceptionally large ACR for PC. So far, concentrations of DTCs including PC reported by Hano et al. (2015a) is the sole monitoring data in marine environment. The maximum PC concentration in seawater was estimated to be 0.11 μg/L (Hano et al., 2015a). An experimentally acquired chronic toxicity value and three chronic toxicity values estimated from the acute toxicity value and the ACR for marine fish species ranged from 0.1 to 2.9 μg/L. Because this study corresponds to the case where one chronic toxicity value is obtained from either fish or Daphnia, according to OECD (2011), an uncertainty factor of 100 was applied. The predicted no effect concentration (PNEC) was estimated to be 0.001–0.029 μg/L. Because the environmental concentration is above the PNEC, the ecological risk of PC to fish in Hiroshima Bay is relatively high, and the compound should have a high priority for further research on its marine contamination status.

100

*

80

*

60

*

40

20 0

DMSO

0.1

1 PC

10

10 HPT ( M)

Fig. 3. Inhibition of lysyl oxidase activity in mummichog embryos after exposure to polycarbamate (PC), or 2-mercaptopyridine-N-oxide (HPT) as a positive control. Data are presented in relative fluorescence units with the value of dimethyl sulfoxide (DMSO) as a control being 100%. Each bar represents the mean ± standard error (n = 3). Asterisks indicate a significant difference between treatments and DMSO (P < 0.05, Dunnett's test).

increased with increasing concentrations of PC (Fig. 3), and the activities in the groups with PC added to 1 μM (582 μg/L) and 10 μM (5820 μg/L) were significantly lower than that in the group exposed only to DMSO (P < 0.05). In comparison to the inhibition by HPT as a positive control, inhibition by PC was about 75% that of HPT.

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related genes varied (Supplementary text and Table S5). Although the expression level of type II collagen, one of the components of the notochord, in the PC exposure group was similar to those in the control group, decrease in the expression levels of several types of laminin, that participate in the formation of the peri-notochordal basement membrane in zebrafish (Stemple, 2005), and the lysin 6-oxidase gene was observed in the PC-exposed embryos. These phenomena might also be involved in the notochord undulation induced by PC exposure. Quantitative analysis, such as real-time quantitative PCR, is needed for more precise clarification of the adverse effects of PC on the expression of these genes.

Fuji, K., Tanaka, H., 2015a. Primary risk assessment of dimethyldithiocarbamate, a dithiocarbamate fungicide metabolite, based on their probabilistic concentrations in a coastal environment. Chemosphere 131, 225–231. Hano, T., Ohkubo, N., Kono, K., Tanaka, H., 2015b. Bioconcentration and elimination of the dithiocarbamate fungicide polycarbamate in marine teleost fish and polychaete. Bull. Environ. Contam. Toxicol. 95, 340–343. Hano, T., Ohkubo, N., Mochida, K., 2017. A hepatic metabolomics-based diagnostic approach to assess lethal toxicity of dithiocarbamate fungicide polycarbamate in three marine fish species. Ecotoxicol. Environ. Saf. 138, 64–70. Hayashi, I.T., Kashiwagi, N., 2015. Inspection of the validity of using acute-chronic ratio (ACR) by regression analysis. Nihon Risk Kenkyu Gakkaishi 24, 213–220 (in Japanese with English abstract). Kenaga, E.E., 1982. Predictability of chronic toxicity from acute toxicity of chemicals in fish and aquatic invertebrates. Environ. Toxicol. 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5. Conclusion In this study, we clarified the various effects of PC exposure on the early life stages of two marine fish species: mummichog and spotted halibut. The PNEC estimated from the chronic toxicity value and application of the uncertainty factor was close to the estimated PC concentrations in Hiroshima Bay. Therefore, the ecological risk of this compound is considered a matter of concern, and continuous monitoring is needed. We also showed that PC could induce notochord undulation in conjunction with inhibition of LO activity, and also possibly inhibition of expression of a gene or genes related to notochord formation. Further study is needed to clarify the precise molecular mechanisms causing PC exposure-induced notochord undulation and inhibition of LO activity. Acknowledgments We are grateful to Drs. Hideki Nikaido and Daisuke Shimizu (Tohoku National Research Institute, Fisheries Research and Education Agency) for generously providing spotted halibut embryos. We also thank Ms. Chiaki Hiramoto and Miki Shoda (National Research Institute of Fisheries and Environment of Inland Sea) for their kind assistance. This work was supported in part by the Fisheries Agency of Japan, Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpc.2018.09.001. References Anderson, C., Bartlett, S.J., Gansner, J.M., Wilson, D., He, L., Gitlin, J.D., Kelsh, R.N., Dowden, J., 2007. Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis. Mol. BioSyst. 3, 51–59. Armstrong, P.B., Child, J.S., 1965. Stages in the normal development of Fundulus heteroclitus. Biol. Bull. 128, 143–168. Bieker, J.J., Yazdani-Buicky, M., 1992. Distribution of type II collagen mRNA in Xenopus embryos visualized by whole-mount in situ hybridization. J. Histochem. Cytochem. 40, 1117–1120. Cheah, K.S.E., Lau, E.T., Au, P.K.C., Tam, P.P.L., 1991. Expression of the mouse α1(II) collagen gene is not restricted to cartilage during development. Development 111, 945–953. Davies, P.E., White, R.W.G., 1985. The toxicology and metabolism of chlorothalonil in fish. I. Lethal levels for Salmo gairdneri, Galaxias maculatus, G. truttaceus and G. aurtatus and the fate of 14C-TCIN in S. gairdneri. Aquat. Toxicol. 7, 93–105. Dingerkus, G., Uhler, L.D., 1977. Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain. Technol. 52, 229–232. Geach, T.J., Dale, L., 2005. Members of the lysyl oxidase family are expressed during the development of the frog Xenopus laevis. Differentiation 73, 414–424. Hano, T., Ito, K., Mochida, K., Ohkubo, N., Kono, K., Onduka, T., Ito, M., Ichihashi, H.,

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