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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
Study period:
2013-03-01 to 2013-04-04
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
Strontium metal completely dissolves upon contact and during the reaction with water under a strong evolution of gas and an immediate precipitation of a white crystalline solid, presumably strontium hydroxide (Sr(OH)2). The water solubility test of strontium (OECD TG 105) indicates a high dissolution from strontium metal (6.74 g/L at 20°C, determined as dissolved strontium, separated by filtration from undissolved test item and precipitates), a rapid formation of Sr2+ + 2OH- + H2 (g) and a corresponding increasing solution pH to a pH > 13. Due to the buffering capacity of most environmental systems, it may reasonable be assumed that the formed hydroxide ions are neutralised in the environment by different processes including precipitation. The solubility of strontium is not greatly affected by the presence of most inorganic anions as there is little tendency for strontium to form complexes with inorganic ligands (Krupka et al. 1999. EPA 402-R-99-004B and references therein). Free Sr2+ cations are mobile under most environmental conditions, despite the relatively low solubility of strontium carbonate and strontium sulfate at neutral to high pHs. In solutions with a pH below 4.5, the Sr2+ ion is dominant. Under more neutral conditions (pH 5 to 7.5), SrSO4 forms. Strontium carbonate controls strontium concentrations in solutions only under highly alkaline conditions. Further, dissolved strontium forms only weak aqueous complexes with chloride and nitrate (Salminen et al. 2015 and references therein, Krupka et al. 1999. EPA 402-R-99-004B). Regarding monodentate and bidentate binding to negatively-charged oxygen donor atoms, including natural organic matter, alkaline earth metals, such as strontium, tend to form complexes with ionic character as a result of their low electronegativity. Ionic bonding is usually described as resulting from electrostatic attractive forces between opposite charges, which increase with decreasing separation distance between ions (Carbonaro and Di Toro. 2007. Geochim Cosmochim Acta 71 3958–3968; Carbonaro et al. 2011. Geochim Cosmochim Acta 75: 2499-2511 and references therein). Thus, strontium does not form strong complexes with fulvic or humic acids based on the assumption that strontium would exhibit a similar (low) stability with organic ligands as calcium and that strontium could not effectively compete with calcium for exchange sites because calcium would be present at much greater concentrations (Krupka et al. 1999. EPA 402-R-99-004B). In sum, strontium ions are highly mobile, occur only in one valence state (2+), i.e. are not oxidized or reduced, and do not form strong complexes with most inorganic and organic ligands (Krupka et al. 1999. EPA 402-R-99-004B; Salminen et al. 2015). Thus, it may further be assumed that the behaviour of the dissociated strontium ions in the environment determine the fate of strontium upon dissolution with regard to (bio)degradation, bioaccumulation, partitioning as well as the distribution in environmental compartments (water, air, sediment and soil) and subsequently the ecotoxicological potential.Therefore, the assessment of the ecotoxicity of strontium is based on elemental strontium concentrations. Read-across of ecotoxicity data available for soluble strontium substances is applied since the strontium ions determine the ecotoxicological potential of strontium.
according to
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
adopted on 17th July 1992
Principles of method if other than guideline:
For the treatments 6.25 mg/L and 12.5 mg/L the flow rates were slightly increased (5.8 and 5.9 vessel volumes/day, respectively). However no effects were observed up to and including the highest treatment level.
GLP compliance:
yes (incl. certificate)
signed 2011-11-21
Analytical monitoring:
Details on sampling:
- Concentrations: 6.25 mg/l and 100 mg/l on day 0, 6, 13, 20, 27 and 34 of the test period; control (0 mg/L) at day 34- Sample storage conditions before analysis: ambient conditions
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: A stock solution of the test item at a nominal concentration of 100 mg test item/L was prepared by weighing 13.0012–13.0044 g of the test item into 130 L or 12.0003 - 12.0011 g of the test item into 120 L of dilution medium. The test item was mixed into the test water as homogeneously as possible by intense stirring (stainless steel propeller stirrer). After that, the solution was directly used as stock solution. The stock solution was prepared every week. Corresponding volumes of the stock solution were used to prepare the (lower) test solutions by dilution with reconstituted water. The stock solution was delivered by the flow-through system to mixing vessels. The test solutions were delivered directly from the mixing vessels to the designated test vessels using teflon tubing and a multi-channel peristaltic pump set to deliver the desired volume. - During dosing into the flow-through system (between renewals), the test solutions were stored at room temperature. During storage, the tanks were closed with a lid to reduce airexchange and evaporation.- The flow rates (volume delivered per stroke) were measured volumetrically at least 3 times per week. The volume delivered/measured was divided by the number of strokes. The variation of the nominal stroke-volume was calculated by dividing the measured flow [mL/stroke] by the nominal volume per stroke of the designated pump expressed in percentage of nominal values. Additionally the distribution between the replicates was measured once per week.- The actually measured flow rates in treatment levels C3 (25 mg/L) up to C5 (100 mg/L) and the control were within ±10% of the mean flow rates as described above. For the treatments C1 (6.25 mg/L) and C2 (12.5 mg/L) the flow rates were generally slightly increased (5.8 and 5.9 vessel volumes per day, respectively). Since no effects were observed up to and including the highest treatment level an impact on the outcome or the integrity of this study is excluded. - Controls: dilution medium
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM- Common name: Zebrafish- Strain: Hamilton-Buchanan 1822- Source: Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since August 2011.METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS- glass bowls covered with stainless steel mesh were introduced in the holding tanks of the adult zebrafish- water plants (Microsorium 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- the content of the bowls was poured over a sieve, rinsed with reconstituted water and collected in a glass vessel filled with reconstituted water- immediately afterwards groups of eggs were transferred to glass dishes containing test solution of each treatment- dishes were placed into an incubator set to26°C; approximately 55 minutes after start of the pre-exposure, groups of fertilised eggs were transferred to other pre-exposure vessels using a randomisation procedure until each pre-exposure vessel contained 30 eggs.- approximately 5 hours later all eggs were transferred from the pre-exposure vessels to theexposure vessels under flow-through conditionsPOST-HATCH FEEDING- Start date: day 3 of exposure (on the day the first larva in a test vessel is recorded to be hatched)- Type/source of feed: 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)- Amount given: ad libitum; the food ration was adjusted to the number of living fish per test vessel. - Frequency of feeding: The daily ration was fed in 3–5 equal portions on workdays. On weekends the daily ration was fed in 2–3 portions. Food was withheld from the fish for 24 hours prior to test end.
Test type:
Water media type:
Limit test:
Total exposure duration:
34 d
Remarks on exposure duration:
The test was continued until 30 days after 95% of the control fish had hatched.
Post exposure observation period:
not applicable
Measured in each test vessel at the start and the end of the exposure period:9.0-12.2 °dH (min-max), 3.2 °dH (range), n=24161-218 mg CaCO3/L (min-max), 57 mg CaCO3/L (range), n=24
Test temperature:
Measured in each test vessel once per week during the test and at the start and the end of the exposure period; temperature was recorded in one test vessel twice per hour throughout the test:Temperature (target): 25±2°CAutomatic measurement: 25.0 - 26.8 °C (min-max), 1.8 °C (range), n=1629Manual measurement: 23.4 - 25.5 °C (min-max), 2.1 °C (range), n=84
Measured in each test vessel once per week during the test and at the start and the end of the exposure period:7.2-7.6 (min-max), 0.4 (range), n=72
Dissolved oxygen:
Measured in each test vessel once per week during the test and at the start and the end of the exposure period:- 5.8-8.9 mg/L (min-max), 3.1 mg/L (range), n=84- 71-99 % (min-max), 28 % (range), n=84
not applicable
Nominal and measured concentrations:
- Nominal concentrations: 6.25, 12.5, 25.0, 50.0, 100.0 mg test item/L, plus a control- Analysed concentrations:nominal: 6.25 and 100 mg Sr(NO3)2/L measured at t=0: 4.99 and 92.5 mg Sr(NO3)2/Lmeasured at t=6d: 6.34 and 94.2 mg Sr(NO3)2/Lmeasured at t=13d: 6.41 and 88.1 mg Sr(NO3)2/Lmeasured at t=20d: 5.95 and 91.3 mg Sr(NO3)2/Lmeasured at t=27d: 6.24 and 94.2 mg Sr(NO3)2/Lnominal at t=34 d: 0, 6.25 and 100 mg Sr(NO3)2/Lmeasured at t=34d:
Details on test conditions:
TEST SYSTEM- Test vessel; material, size, headspace, fill volume: Glass aquaria, diameter: 14 cm, height: 7 cm; vessels were fitted with a meshed overflow: outflow at: 5.2 cm, 0.8 L test solution volume.- Aeration: the test vessels were aerated- Type of flow-through (e.g. peristaltic or proportional diluter): permanent (flow-through)- Renewal rate of test solution (frequency/flow rate): flow-through, five vessel volumes per vessel and day- No. of fertilized eggs/embryos per vessel: 30- No. of vessels per concentration (replicates): 2- No. of vessels per control (replicates): 2- Biomass loading rate: loading rate did not exceed 5 g fish/L of solution, and a loading rate of 0.5 g fish/L per 24 hours at any time of the test; the loading rate was calculated by dividing the maximum total fish wet weight per vessel by the target flow rate of five vessel volumes per day; The maximum loading rate was 0.254 g fish/L per 24 hours.TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Reconstituted water (OECD guideline No. 203) mixed with deionised water (1:1; v/v), supplemented with 1% artificial seawater. Local tap water was treated by reverse osmosis and ion-exchanger to prepare deionised water. Therefore a contamination with heavy metals, pesticides and total organic carbon is excluded. The required amount of reconstituted water was prepared within four weeks before use.During storage reconstituted water was aerated.- Conductivity: 722-810 µS/cm (min-max), 88 µS/cm (range/span)- Salinity:- Culture medium different from test medium: reconstituted water was used for both mediaOTHER TEST CONDITIONS- Adjustment of pH: no- Temperature: 26±2°C- Photoperiod: 12/12 hours light/dark cycle- Light intensity: 100–1000 lx; measured: 665–992 lxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :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 fishThe following biological parameters were assessed:- time to start of hatching and end of hatching- number of larvae hatching each day- macroscopic morphological abnormalities- behavioural abnormalitiesDATA EVALUATION Endpoints: NOEC, LOECThe following biological parameters were evaluated statistically in comparison to the control fish where the data allowed such comparisons:- hatching success, mortality (post-hatch success): Fisher exact test with Bonferroni correction- numbers of healthy fish (Data are identical with data for post-hatch success, therefore no separate statistical evaluation)- dry weight of the surviving fish, per treatment means, length of the surviving fish, per treatment means: Williams testThe normal distribution was checked with Shapiro-Wilk's Test.Variance homogeneity was checked with Levene's Test.Due to a lack of concentration-response relationship LCx or ECx values were not calculated.The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.RANGE-FINDING STUDY- no data
Reference substance (positive control):
Key result
34 d
Dose descriptor:
Effect conc.:
>= 41.4 mg/L
Nominal / measured:
Conc. based on:
analytically verified
Basis for effect:
other: Hatching success, mortality (post-hatch success), numbers of healthy fish, length of the surviving fish, dry weight of the surviving fish
34 d
Dose descriptor:
Effect conc.:
> 41.4 mg/L
Nominal / measured:
Conc. based on:
analytically verified
Basis for effect:
other: Hatching success, mortality (post-hatch success), numbers of healthy fish, length of the surviving fish, dry weight of the surviving fish
Details on results:
- The measured concentrations were within 80 to 120% of the nominal concentrations, thus, the biological endpoints were not corrected based on measured concentration in the test solutions.- Mortality/survival at embryo, larval, juvenile, and adult stages: At test end, out of 60 introduced eggs the final number of healthy fish in controls was 48.Across the treatments the number of living, healthy fish ranged from 50 to 54 of 60 introduced eggs.- Days to hatch or time to release of young: hatching from day 3 to day 7 of exposure; no dose-response relationship- Hatching success: 98.3 % (controls), > 96.7 % in all treatments up to the highest concentration; no correlation was observed between theconcentration of the test item and the hatching success- Post-hatch success: 81.4 % (control), 84.7 - 89.7 % (test groups); statistically significant differences of post-hatch success and survival of fish 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 post-hatch success and survival of fish.- Observations on body length and weight of young and/or exposed parents at one or more time periods: Statistically significant differences of weight or 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 dry weight or length of fish.- Number of healthy fish at end of test: 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.- Incidents in the course of the test which might have influenced the results:

The validity criteria required by the study plan and guideline were fulfilled as follows:

- 81.4% post-hatch success

- dissolved oxygen level during routine water quality analyses not below 60% of air saturation: 71% (minimum)

- water temperature: ≤0.7°C between test vessels on the same day; ≤1.05°C per vessel between successive measuring days; max.: 26.8°C, min.: 25.0°C (automatic measurement), range: 1.8°C; max.: 25.5°C, min.: 23.4°C (manual measurement), range: 2.1°C

- the concentrations of the test substance in solution have been maintained within +/- 20% of the mean measured values

- survival of fertilised eggs in controls > 70 %

Validity criteria fulfilled:
for details see "Any other information on results incl. tables"
Under the experimental conditions of this study, a concentration-response relationship was not observed. The NOECs (34 d, hatching success, mortality (post-hatch success), numbers of healthy fish, length of the surviving fish, dry weight of the surviving fish) based on nominal concentrations were >=100 mg strontium nitrate/L corresponding to >= 41.4 mg Sr/L. The corresponding LOECs based on nominal concentrations were higher than 100 mg strontium nitrate/L.

Description of key information

One reliable long-term toxicity study (Klimisch 1, GLP) for a fish species -the zebrafish Danio rerio - was conducted. No effect (mortality) was noted at the highest measured test concentration of niminal 100 mg Strontium nitrate/L. Hence, the NOEC is set to >= 100 mg/L and a LOEC of > 100 mg/L Strontium nitrate/L corresponding to a NOEC of ≥ 41.4 mg Sr/L.

Key value for chemical safety assessment

Additional information