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Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD 210 Guideline test, conducted under GLP
Justification for type of information:
see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.
according to guideline
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
The stock solution was prepared once per week by dissolving 10.428 g of the test item (nominal; actually weighed amounts: 10.4284 to 10.432 g) in 3 L of dilution water (reconstituted water) using a 3 L graduated flask. This stock solution (S1, 3476 mg/L) was stirred for 1 min. Immediately thereafter, the stock solution was used to prepare the test solutions.
The test solutions were delivered directly from the tanks to the designated test vessels using teflon tubing and a multi-channel peristaltic pump set to deliver the desired volume per day. During dosing into the flow-through system (between renewals), the test solutions were stored at room temperature in the dark. During storage, the tanks were closed with a lid to reduce air-exchange and evaporation.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Danio rerio Hamilton-Buchanan 1822 (Zebrafish). Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since May 2004.
In the culture tanks, the fish were kept in reconstituted water (OECD guideline 203, 1992) supplemented with 1% of artificial seawater (Tropic Marin, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28 ‰ ± 2 ‰). Fish were held in glass aquaria with internal ceramic filters at 26°C ± 2°C with a 12 h/12 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. (Sanders Brine Shrimp Co., Morgan, UT 84050, U.S.A.) and TetraMin (Tetra Werke, Melle, Germany).

On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding tanks of the adult zebra fish. Water plants (Microsorum pteropus) were placed on the mesh, allowing the fish to spawn. The spawning bowls were removed from the holding tanks on the day of test start shortly after onset of illumination, which triggers fertilization. The content of the bowls was poured over a sieve (mesh size 0.5 mm), rinsed with reconstituted water and collected in a glass vessel filled with reconstituted water. Immediately afterwards groups of 10 - 20 eggs were transferred to glass dishes containing test solution of each designated exposure vessel (acclimated at test temperature), until all dishes contained approximately 130 eggs. The dishes were placed into an incubator set to 25°C for two hours. Unfertilized and damaged eggs were removed and the number of eggs per dish was reduced to 30. Subsequently the eggs were transferred to the exposure vessels.
Test type:
Water media type:
Limit test:
Total exposure duration:
34 d
min: 164 mg/L as CaCO3
max: 193 mg/L as CaCO3
Test temperature:
23.8 - 25.6 degrees Celcius
min: 7.0
max: 7.5
Dissolved oxygen:
min: 5.2 mg/L (=64% of air saturation value)
max: 9.0 mg/L (108% of air saturation value)
Nominal and measured concentrations:
Nominal test concentrations: 316, 100, 31.6, 10, and 3.2 mg test item/L plus a control
Details on test conditions:
Reconstituted water according to OECD guideline No. 203 was used to dilute the test item and to keep the fish before and during the period of the test. Local tapwater was treated by reverse osmosis and ion-exchanger to prepare deionised water. Therefore a contamination with heavy metals, pesticides and TOC is excluded.

- Test medium: Reconstituted water (OECD guideline No. 203) mixed with deionised water (1:1; v/v), supplemented with 1% artificial seawater
- Biological material: Newly fertilised eggs were exposed to the test item dissolved in water in a flow-through system for 30 days post-hatch. The method of administration is in accordance with the requirements of the test guideline
- Loading: did not exceed a loading of 5 g fish/L of solution, or a loading rate of 0.5 g fish/L per 24 hours at any time of the test.
- Endpoints: ECx, NOEC, LOEC
- Biological parameters: –Hatching success: for eggs and embryos (particularly in the early stages) mortality was related to a marked loss of translucency and change in colouration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance
– Mortality (post-hatch success): immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque colouration of central nervous system and/or lack of reaction to mechanical stimulus.
– Body abnormalities
– Behavioural abnormalities
– Length of the surviving fish
– Dry weight of the surviving fish
- Test duration: 34 days
- Temperature: Max.: 25.5°C, Min.: 24.8°C (online measurement)
- Photoperiod: 12/12 hours light/dark cycle
- Light intensity: 281 to 600 lx
- Test units: Glass vessels, 0.5 L volume
- Dimensions of test vessels: Diameter: 14 cm, Height: 6 cm; vessels were fitted with a meshed overflow: center of outflow: 3.5 cm, diameter of outflow: 1 cm. The vessels were covered with a transparent lid to reduce air-exchange and evaporation.
- Volume of test solution per test vessel: 0.3 L (nominal).
- No. of replicates per treatment: 2
- Renewal of test solution during exposure: Permanent (flow-through)
- Flow-through test equipment (hardware): Peristaltic pump: Type Ismatec IPC 24, tubing type: TYGON ST, Ismatec, Wertheim Mohnfeld; Teflon tubing
- Flow rates: measured volumetrically at least 3 times per week. The volume measured per per time unit (2 minutes per measurement), multiplied by the number of minutes per day, results in the daily volume. Dividing the daily volume by the nominal vessel volume (0.3 L) results in the daily vessel-volume exchange rate (+/- five velles volumes per vessel and day).
- Aeration: test vessels were aerated
- Feeding: On the day after the first larva per test vessel was recorded to swim up (day 3 of exposure), feeding was started by adding food to the vessels. The test organisms were fed dry food (NovoBaby 01 and 02, Tetra), live Artemia nauplius larvae and parametia (Paramecium caudatum) ad libitum; the daily ration was fed in 3 - 4 equal portions on workdays. On weekends the daily ration was fed in 2 - 3 portions. Uneaten food was removed at least once daily. The food ration was adjusted to the number of living fish per test vessel. Food was withheld from the fish for 24 hours prior to test end.
- Dead eggs, embryos or fish were removed from the test vessels daily upon observation throughout the test.

The actual measured flow rates per treatment were within ± 20% of the mean flow rates as described above. This is higher than recommended in the test guideline, but since in this study the test solutions were not prepared by use of the flow-through system (e.g. by automatically dosing stock solution into dilution water) but by direct delivery of test solution into the test vessels, this slightly higher variation of the flow rate has no influence on the actual test concentration
Reference substance (positive control):
34 d
Dose descriptor:
Effect conc.:
>= 316 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: hatching success, mortality (post-hatch success), numbers of healthy fish, length of surviving fish, dry weight of the surviving fish
Details on results:
Validity criteria:
- 97% post-hatch success in the controls (required: 70% post-hatch success)
- Dissolved oxygen level during routine water quality analyses was 64% (or higher) of air saturation (required minimum: 60%)
- Water temperature differed ≤ 0.5 degrees Celcius) between test vessels on the same day or ≤ 1.4 °C per vessel between successive measuring days (allowed: maximum difference of 1.5 degrees Celcius between test vessels or between successive days)
- The water temperature should be within a range of 2 degrees Celcius of the desired test temperature: maximum was 26.0 degrees Celcius, minimum was 24.0 degrees Celcius (online measurement) (difference of 2 degrees Celcius); For manual measurement, the maximum was 25.6° degrees Celcius and the minimum was 23.8 degrees Celcius (difference of 2 degrees Celcius)

- In controls the hatching success was 100%. In all treatments up to the highest concentration the hatching success was above 90%.
- At test end, out of 60 introduced eggs, the final number of healthy fish in controls was 58. In the treatments the number of living, healthy fish ranged from 45 at 10 mg/L to 54 at 316 mg/L, respectively.
- All surviving fish appeared healthy at the end of exposure, i.e. behavioural or morphological abnormalities were not observed. Therefore, a separate statistical evaluation was not performed on the number of healthy fish.
- Statistically significant differences of weight were not observed between treated and control groups at p ≤ 0.05. No correlation was observed between the concentration of the test item and the dry weight of fish.
- Statistically significant differences of length were not observed between treated and control groups at p ≤ 0.05. No correlation was observed between the concentration of the test item and the length of fish.
Results with reference substance (positive control):
The following biological parameters were recorded during and/or at the end of the test:
- cumulative mortality
- numbers of healthy fish
- time to start of hatching and end of hatching
- number of larvae hatching each day
- number of deformed larvae
- number of organisms exhibiting abnormal behaviour
- length and weight of surviving fish

The following biological parameters were evaluated from a biological point of view:
- time to start of hatching and end of hatching
- number of larvae hatching each day
- macroscopic morphological abnormalities
- behavioural abnormalities
Reported statistics and error estimates:
The following biological parameters were evaluated statistically in comparison to the control fish where the data allowed such comparisons:
- hatching success, mortality (post-hatch success), numbers of healthy fish : Fisher's Exact Binomial Test with Bonferroni Correction
- dry weight of the surviving fish, per treatment means: Williams' test
- length of the surviving fish, per treatment means: Williams' test

Due to a lacking concentration-response relationship LCx or ECx values were not calculated. Variance homogeneity was checked with Levene´s Test. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf, Germany) was used for these calculations.
An OECD No.210 test was conducted, using sodium sulfite as test substance. Newly fertilised eggs of zebrafish were exposed to a series of 7 test concentrations plus a control without test item under flow-through conditions. The test was continued until 30 days after 50% of of the control fish had hatched. Lethal and sub-lethal effects on eggs, larvae and juvenile fish were assessed in comparison with control values to determine the ECx (concentration that is expected to cause an effect in x% of the animals), the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC). No significant effects were found for any of the tested concentrations. Based on this study, an unbounded NOEC of >= 316 mg test item/L is put forward. These data can be used for the effects assessment of sodium sulfite and other (bi-)sulfite compounds (read-across principle).

Description of key information

Effects of sodium sulfite on the early life stages of zebrafish (Danio rerio) were not observed in a GLP-OECD 210 early life stage toxicity test and a 34-d NOEC ≥ 316 mg test substance/L (i.e., ≥ 200.5 mg SO32 -/L) was derived. Furthermore, potassium as essential element has a very low potential for chronic toxicity to fish. Based on read-across to thiosulfate/sulfite/disulfite substances and soluble potassium substances and taking into account ubiquitousness and essentiality of potassium and sulfur, the potential of potassium thiosulfate for chronic toxicity to fish is expected to be low.

Key value for chemical safety assessment

Additional information

Studies on the long-term toxicity of potassium thiosulfate to fish are not available, and thus, read-across to thiosulfate and sulfite/disulfite substances is applied. Upon contact with water, salts of sulfur oxyacids including potassium thiosulfate dissociate into sulfur oxyacid anions and the respective counterions. Only the properties of the thiosulfate anion are considered a relevant determinant of environmental toxicity since potassium cations are essential and have a very low potential for chronic toxicity to freshwater and marine fish. Please refer to the respective endpoint summary for potassium (K) for further details.Thiosulfate anions are unstable under environmentally relevant conditions, will disproportionate to sulfite and will further be oxidized to sulfate or reduced to sulfide.


Thiosulfate and sulfite/disulfite substances –Long-term toxicity to fish

In a reliable chronic toxicity study according to OECD Guideline 210 on the effects of sodium sulfite (Na2SO3) on the early-life stages (ELS) of Danio rerio, five different substance concentrations were evaluated but no adverse effects on number of healthy fish, hatching, post–hatch mortality, health, length and dry weight of surviving fish (Danio rerio) were observed. Consequently, a chronic 34-d NOEC of ≥ 316 mg Na2SO3/L, corresponding to ≥ 200.5 mg SO32-, was derived.