Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Summary

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Abstract

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L⁻¹ in different concentrations of ERM at pH 7 and 25 °C in the dark.

We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Introduction

Since the establishment of the Organisation for Economic Co-operation and Development (OECD) test guidelines for testing chemicals in 1979, 38 test guidelines have been published in Section 2 of the guidelines, Effects on Biotic Systems¹. All of the aquatic organisms tested have been from freshwater habitats, namely freshwater plants; algae; invertebrates such as daphnia and chironomids; and fishes such as medaka, zebrafish, and rainbow trout. Compared to saltwater environments, freshwater environments are more directly affected by human economic and industrial activities. Therefore, freshwater environments have been prioritized for testing because they are at higher risk from pollution.

In coastal areas, including estuaries, salinities vary between brackish water and seawater conditions, and these areas are often polluted by industrial activity². Coastal areas and their associated wetlands are characterized by high ecological biodiversity and productivity. Coastal ecosystems must therefore be protected from chemical pollution. However, there has been limited ecotoxicological research in brackish water and seawater habitats.

Sakaizumi³ studied the toxic interactions between methyl mercury and salinity in Japanese medaka eggs and found that increasing the osmotic pressure of the test solution enhanced the toxicity of the methyl mercury. Sumitani et al.⁴ used medaka eggs to investigate the toxicity of landfill leachate; they found that the osmotic equivalency of leachate to the eggs was the key to inducing abnormalities during embryogenesis. In addition, Kashiwada⁵ reported that plastic nanoparticles (39.4 nm in diameter) easily permeated through the medaka egg chorion under brackish conditions (15x embryo rearing medium (ERM)).

A typical small fish model, the Japanese medaka (Oryzias latipes) has been used in basic biology and ecotoxicology⁶. Japanese medaka can live in conditions ranging from freshwater to seawater because of their highly developed chloride cells⁷. They are therefore likely to be useful for testing in conditions with a wide range of salinities.

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Protocol

The Japanese medaka used in this study were treated humanely in accordance with the institutional guidelines of Toyo University, with due consideration for the alleviation of distress and discomfort.

1. Silver Nanocolloids (SNCs)

1. Purchase purified SNCs (20 mg/L⁻¹, 99.99% purity, particle mean diameter about 28.4 ± 8.5 nm suspended in distilled water).
2. Validate the purity and concentration of the silver by inductively coupled plasma mass spectrometry (ICP-MS) analyses according to operating manual⁸. The pretreatment method for ICP-MS analyses is described in section 7.

2. Preparation of SNC Solutions (Mixtures of Silver Colloids and Ag⁺) with Different Salinities

1. Prepare 60× ERM consisting of 60 g NaCl, 1.8 g KCl, 2.4 g CaCl₂·2H₂O, and 9.78 g MgSO₄·7H₂O in 1 L of ultrapure water; adjust the pH to 7.0 with 1.25% NaHCO₃ in ultrapure water.
2. Stir the ERM solution at 25 °C overnight.
3. Mix SNCs with diluted ERM. Prepare 40 ml of each SNC-ERM mixed solution. The final concentration is 10 mg/L⁻¹ of SNCs in different concentrations of ERM (1x, 5x, 10x, 15x, 20x, or 30x).
4. Adjust pH of the SNC-ERM mixed solution to 7.0 with 0.625% NaHCO₃ in ultrapure water. pH adjustment is very important in preparing the SNC solution, because Ag⁺ release is facilitated by acidic conditions⁹.
5. Use AgNO₃ as a reference compound for SNCs.
   1. Mix AgNO₃ with diluted ERM. Prepare 40 ml of AgNO₃-ERM mixed solution at an AgNO₃ concentration of 15.7 mg/L^-1 (10 mg/L⁻¹ silver) in different concentrations of ERM (1x, 5x, 10x, 15x, 20x, or 30x).
      Note: To examine silver colloid toxicity, AgNO₃ solution, which is a source of soluble silver, is used as a reference compound for SNCs, which are a mixture of silver colloids and soluble silver.

3. Medaka Culture and Egg Harvesting

1. Obtain the medaka (O. latipes) (orange-red strain) (60 males and 60 females).
2. Culture medaka as groups (20 males and 20 females as one group) in 1x ERM in 3 L tanks by using a medaka flow-through culturing system.
   1. Culture at the following conditions:
      pH range of the culture medium: 6.2 to 6.5  
      light:dark cycle: 16:8 hr
      temperature of the culture medium: 24 ± 0.5 °C
      osmotic pressure of the culture medium: 257 mOsm
3. Feed medaka on Artemia salina nauplii at 10:00 (once a day) and feed an artificial dry fish diet at 09:00, 11:00, 13:00, 15:00, and 17:00 (five times a day).
   1. Obtain A. salina nauplii.
   2. Prepare 5 L of a 3.0% salt solution in a plastic beaker.
   3. Add 30 g of brine shrimp eggs to the salt solution in the beaker.
   4. Incubate the eggs at 25 °C for 48 hr with bubbling (4 L/min⁻¹) using an aeration pump.
   5. After 48 hr, stop the bubbling.
   6. Allow the solution to stand for 5 to 10 min to separate the hatched A. salina nauplii (lower part of the solution) from the unhatched eggs and eggshells (upper part of the solution).
   7. Remove the upper layer of the solution by decantation.
   8. Filter the lower portion of the solution through a sieve with openings of 283 µm, and collect the nauplii that pass through on a net with openings of 198 µm.
   9. Feed the nauplii to the medaka within 6 hr.
4. After the female medaka have spawned, remove the external egg clusters gently from the females' bodies or collect the eggs from the bottom of the fish tank by using a small net (net size 5 cm x 5 cm, hole size 0.2 mm x 0.2 mm).
5. Rinse the egg cluster with flowing tap water for 5 sec.
6. Add all of the rinsed egg clusters to 30x ERM solution.
7. Remove the clusters from the solution after 1 min and place the egg clusters between dry paper towels and roll gently.
8. Put the eggs back into the 30x ERM.
9. Select fertilized eggs under a dissecting microscope.
10. Place selected 810 eggs in 1x ERM in six-well plastic plates by using forceps.
11. Incubate the eggs at 25 ± 0.1 °C in an incubator until developmental stage 21. (Developmental stages of the medaka embryos were defined from the work of Iwamatsu¹⁰.)
12. Pick out incubated eggs at developmental stage 21 under a dissecting microscope.
13. Rinse selected eggs with 1x ERM.
14. Subject the rinsed eggs to exposure experiments (section 4).

4. Toxicity Testing of SNCs or AgNO₃ at Different ERM Salinities

1. Rinse medaka eggs (stage 21) three times with test solution [SNCs (10 mg/L⁻¹) or AgNO₃ (15.7 mg/L⁻¹ as 10 mg/L⁻¹ silver) at each concentration of ERM (1x, 5x, 10x, 15x, 20x, or 30x) at pH 7]. As controls, use eggs in 1× to 30× ERM at pH 7.
2. Add 15 rinsed eggs to 5 ml of each test solution in six-well plastic plates. (Perform the exposure experiments three times for SNC or AgNO₃ toxicity testing using each test solution.)
3. Wrap the plates in aluminum foil.
4. Incubate the wrapped plates at 25 °C in the dark until hatching or for 14 days.
5. Observe the exposed eggs every 24 hr for biological changes and dead eggs (Figures 1 and 2).
6. Exchange the test solutions every 24 hr.
7. Perform observations as follows.
   1. On day 6 of exposure, count the heart rate (per 15 sec) of medaka embryos under a dissecting microscope by using a stopwatch (Figure 3a).
   2. On day 6 of exposure, measure the eye size (diameter) of medaka embryos under a dissecting microscope by using a micrometer (Figure 3b).
   3. On hatching day, measure the full body lengths of larvae under a dissecting microscope by using a micrometer (Figure 3c).
   4. Count the total number of exposed eggs that hatch over the 14 days (Figure 3d).

5. Isolation of Soluble Silver from SNC Solution, and Silver Analysis

1. Isolate soluble silver from each SNC solution (a mixture of silver colloids and soluble silver) by filtering through a 3 kDa membrane filter at 14,000 x g and 4 °C for 10 min. Use a 3 kDa membrane filter to isolate soluble silver from the SNCs, because the reported mean diameter of aggregated SNCs in 1x ERM is 67.8 nm¹¹ and that of Ag⁺ is 0.162 nm¹²; the 3 kDa membrane excludes particles with diameters of 2 nm or more¹³.
2. Measure the silver concentration in 50 µl of filtered solution (= the soluble silver concentration) by ICP-MS analysis (Figure 3e) according to the ICP-MS operating manual⁸. The pretreatment method for the ICP-MS analyses is described in section 7.

6. Measurement of Silver Bioaccumulation in Medaka Embryos

1. Expose medaka eggs (stage 21) to SNCs or AgNO₃ as described in section 4.
2. On day 6 of exposure, remove chorion from the egg (i.e., dechorion) by using medaka hatching enzyme according to the protocol described in the Medaka Book¹⁴.
3. Measure the silver concentration of the dechorioned eggs by ICP-MS analysis according to the ICP-MS operating manual⁸ (Figure 3f). The pretreatment method for the ICP-MS analyses is described in section 7.

7. Measurement of Silver Concentration by ICP-MS Analysis

1. Add samples [50 µl of silver solution (for validation of the silver concentration; section 1); three dechorionated embryos (section 5); or 50 µl of filtered solution (section 5)] to a 50 ml Teflon beaker.
2. Add 2.0 ml of ultrapure nitric acid to the 50 ml beaker.
3. Heat the mixture on a hot plate at 110 °C until just before it dries out (about 3 hr).
4. To dissolve the organic matter completely, add 2.0 ml of ultrapure nitric acid and 0.5 ml of hydrogen peroxide to the beaker.
5. Heat the mixture again on the hot plate until just before it dries out (about 3 hr).
6. Dissolve the residue in 4 ml of 1.0% ultrapure nitric acid solution.
7. Transfer 4 ml of solution to a centrifuge tube.
8. Repeat 7.6 to 7.7 twice (a total of three times). The final volume is 12.0 ml.
9. Measure the silver concentration of the sample (dissolved in 1.0% ultrapure nitric acid) by using ICP-MS analysis according to the operating manual⁸.
   1. Use an internal and an external standard solution (See Materials List) to quantify the silver concentration. The internal and external standard solution is accredited by American Association for Laboratory Accreditation (A2LA). Detection limits of silver were 0.0018 ng/ml⁻¹ (solution) and 0.016 ng mg-weight⁻¹ (embryo body).

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Results

The effect of salinity on SNC toxicity was very obvious: the induction of deformity or death was salinity dependent (Figures 1 and 2). We measured phenotypic biomarkers (heart rate, eye size, full body length, and hatching rate) in SNC (10 mg/L⁻¹)-exposed embryos. These phenotypic biomarkers revealed salinity-dependent SNC toxicity.

Heart rates ranged from 29.6 to 32.2 beats/15 ...

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Discussion

Medaka is a freshwater fish that is highly tolerant to seawater; it is not well known that the original natural habitat of this fish was saltwater off the Japanese coast⁶. Hence, medaka fish have well-developed chloride cells⁷. This unique property provides scientists with a new way to test the toxicity of chemicals in the environment as a function of salinity (freshwater to seawater) by using only a single species of fish.

To obtain medaka eggs at stage 21, eggs must be ...

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Materials

Name	Company	Catalog Number	Comments
Silver nanocolloids	Utopia Silver Supplements
NaCl	Nacalai Tesque, Inc.	31319-45	For making ERM
KCl	Nacalai Tesque, Inc.	28513-85	For making ERM
CaCl2·2H2O	Nacalai Tesque, Inc.	06730-15	For making ERM
MgSO4·7H2O	Nacalai Tesque, Inc.	21002-85	For making ERM
NaHCO3	Nacalai Tesque, Inc.	31212-25	For making ERM
AgNO3	Nacalai Tesque, Inc.	31018-72
pH meter	HORIBA, Ltd.	F-51S
Balance	Mettler-Toledo International Inc.	MS204S
medaka (Oryzias latipes) orange-red strain	National Institute for Environmental Studies
medaka flow-through culturing system	Meito Suien Co.	MEITOsystem
Artemia salina nauplii eggs	Japan pet design Co. Ltd	4975677033759
aeration pump	Japan pet design Co. Ltd	non-noise w300
Otohime larval β-1	Marubeni Nissin Feed Co. Ltd	Otohime larval β-1	Artificial dry fish diet
dissecting microscope	Leica microsystems	M165FC
micrometer	Fujikogaku, Ltd.	10450023
incubator	Nksystem	TG-180-5LB
shaker	ELMI Ltd.	Aizkraukles 21-136
6-well plastic plates	Greiner CELLSTAR	M8562-100EA
aluminum foil	AS ONE Co.	6-713-02
stopwatch	DRETEC Co. Ltd.	SW-111YE
3 kDa membrane filter	EMD Millipore Corporation	0.5 ml centrifugal-type filter
50 ml Teflon beaker	AS ONE Co.	33431097
Custom claritas standard	SPEXertificate	ZSTC-538	For internal standard
Custom claritas standard	SPEXertificate	ZSTC-622	For external standard
ultrapure nitric acid	Kanto Chemical Co.	28163-5B
hydrogen peroxide	Kanto Chemical Co.	18084-1B	for atomic absorption spectrometry
ICP-MS	Thermo Scientific	Thermo Scientific X Series 2
hot plate	Tiger Co.	CRC-A300