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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The justification for read across is attached in IUCLID Section 13.
Reason / purpose:
read-across source
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
Ba
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
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
> 2.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
Ba(NO3)2
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
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-02-12 to 2014-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
adopted 1992-07-17
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2011-11-21
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.25, 12.5, 25.0, 50.0, and 100 mg test item/L on day 0, 7, 14, 16, 28 and 30 of the test period; control (0 mg/L) at day 0
- Sample storage conditions before analysis: ambient conditions
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Several stock solutions of the test item were prepared by weighing:
- 1003.2 mg of the test item into 10 L of dilution medium, day 0–6;
- 1502.0 mg of the test item into 15 L of dilution medium, day 7–13;
- 2001.1–2006.0 mg of the test item into 20 L of dilution medium, day 14–21;
- 4001.4–4001.6 mg of the test item into 40 L of dilution medium day 23–28.

The test item was mixed into the test medium by intense stirring (magnetic or stainless steel propeller stirrer). After addition of the test item a cloudy white preparation of the stock solution occurss, a whithout stirringcloudy, white precipitate was visible which can be explained by the precipitation of barium sulfate in the test medium. The stock solution was directly used or stored at ambient temperature in the dark until further use.

Immediately before and meanwhile the stock solution was used to prepare the lower concentrated test solutions by dilution with reconstituted water, the stock solution was homogenised by intense stirring (magnetic or stainless steel propeller stirrer).

The stock solution was prepared once to three-times per week. After the stirring period, the stock solution (dispersion) was stored at room temperature in the dark.

The test solutions were prepared by diluting desired volume of stock solution (S1) with test medium. The volume of stock solution and dilution medium used to prepare were measured using graduated glass flasks, graduated glass cylinder or was weighed using a balance. The test solutions were homogenised before use, using a magnetic stirrer, stirred for about 2 minutes or were shaken upside down a several times.
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 the laboratory since August 2011, August/September 2012 and Mai 2013.

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 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 tempered reconstituted water and collected in a glass vessel filled with tempered reconstituted water
- immediately afterwards groups of eggs were transferred to glass dishes containing test solution of each designated exposure vessel
- dishes were placed into an incubator set to26°C (measured: 25.4°C) for 1.17 hour
- 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.

POST-HATCH FEEDING
- Start date: when the first larva per test vessel was recorded to swim up (day 3 of exposure), feeding was started.
- Type/source of feed: dry food (NovoBaby 01 and NovoBaby 02, JBL), live Artemia nauplius larvae and paramaecia (Paramecium caudatum) ad libitum.
- Amount given: food ration was adjusted to the number of living fish per test vessel.
- Frequency of feeding: daily ration was fed in 3–4 equal portions on workdays. On weekends the daily ration was fed in 2–3 portions, except on day 3 and 4 the daily ratio was fed in one portion. The Food was withheld from the fish for 24 hours prior to test end.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Hardness:
During the test (total hardness):
minimum: 9.2 °dH; maximum: 10.2 °dH
minimum: 164 mg CaCO3/L; maximum: 182 mg CaCO3/L
Test temperature:
During the test:
minimum: 23.4 °C; maximum: 26.2 °C
pH:
During the test:
minimum: 7.2; maximum: 7.6
Dissolved oxygen:
During the test:
minimum: 3.43 mg/L; maximum: 8.4 mg/L
minimum: 42.5%; maximum: 102%
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25.0, 50.0, and 100 mg test item/L
Details on test conditions:
TEST SYSTEM
Test vessel, material, size, headspace, fill volume :
- vessels of glass and stainless steel
- dimensions of test vessels:
day 0–7: 150 mL glass vessels were used (inner-diameter: 5.5 cm, high: 8 cm).
day 7–14: 1000 ml glass vessels (inner diameter: 13.6 cm, high: 8 cm), with inner-vessels (plastic vessels, the bottom covered with a 250µm-mesh (diameter: 10.5 cm, high: 5 cm).
day 14 to 21 days: 4000 mL stainless steel vessels (length: 26 cm, depth: 16, high: 15 cm), with inner-vessels (plastic vessels, the bottom covered with a 250µm-mesh (diameter: 10.5 cm, high: 5 cm)).
- until end of exposure: 4000 mL stainless steel vessels (length: 26 cm, depth: 16, high: 15 cm).
- volume of test solution per test vessel: 50–3250 mL (nominal).

- Aeration: from day 0–3 the test solutions were not aerated. At day 4 the test solutions were gently aerated using smal pieces of teflon tubes.

- Renewal rate of test solution (frequency/flow rate):
Day 0: 50 ml of freshly prepared test solution was added to the test vessel.
Day 2: addition of 50 mL of freshly prepared test solution to the test vessel containing 50 ml aged test solution.
Day 7: transfer of larvae and remaining eggs (not hatched larvae) in freshly prepared test solution. The young larvae were slowly poured in the fine meshed inner-vessels (plastic vessels, the bottom covered with a fine mesh) and thereafter gently transferred in the new test solution by dip the inner-vessels into the test solution.
Day 7–21 days: three times per week (usually Monday, Wednesday and Friday). The young larvae were transferred by transpose the inner-vessel in a new set of test vessels containing fresh test solutions, except on day 12, see section 16.
Until end of exposure: three times per week (preferably Monday, Wednesday and Friday). The young larvae were slowly poured in the fine meshed inner-vessels (plastic vessels, the bottom covered with a fine mesh) and thereafter gently transferred in the new test solution by dip the inner-vessels into the test solution.

- 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: theoretical maximum loading was 0.82 g fish/L

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.
- Culture medium different from test medium: reconstituted water was used for both media

OTHER TEST CONDITIONS
- Photoperiod (light/dark): 12/12
- Light intensity (measured): 314–894 lx, measured on day 1 (climate chamber); 735–989 lx, measured on day 5.

EFFECT PARAMETERS MEASURED:
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 assessed:
- time to start of hatching and end of hatching
- number of larvae hatching each day
- macroscopic morphological abnormalities
- behavioural abnormalities
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 40.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
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
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
barium
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
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 40.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
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
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 1.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
barium
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:
- Mortality/survival at embryo, larval, juvenile, and adult stages: an influence of the test item concentrations on the post-hatch success and survival of fish could not be observed. Statistically significant differences of post-hatch success and survival of fish were not be 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.
- Days to hatch: hatching on day 2 to day 4 for all treatments. A concentration-response relationship could not be observed.
- Hatching success: controls: mean hatching success was 100%; treatments: mean hatching success ranged between 98.3%–100%. An influence of the test item concentrations on the hatching success could not be observed. Statistically significant differences on the hatching success 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 hatching success.
- Numbers hatched: in all groups (including the control group) 60/60 fish hatched, except in the 100 mg test item/L group in which 1/60 fish did not hatch
- Number of healthy fish at end of test: all surviving fish (control: 59/60; 6.25 mg test item/L: 50/60; 12.5 mg test item/L: 55/60; 25.0 mg test item/L: 59/60; 50.0 mg test item/L: 56/60; 100mg test item/L: 52/60)
- Type of and number with morphological abnormalities: all surviving fish appeared healthy at the end of exposure, morphological abnormalities were not observed.
- Type of and number with behavioural abnormalities: all surviving fish appeared healthy at the end of exposure, behavioural abnormalities were not observed. A meaningful correlation of abnormal behaviour of fish larvae were not observed with increasing test item concentrations. Some observations were determined in all test item concentrations at a lower level (fish lying on the bottom of the test vessel, larvae showing reduced swimming activity, fish were showing curved spine, fish showed loss of equilibrium).
- Other biological observations:
Weight of the surviving fish at test end: an influence of the test item concentrations on the weight of the surviving fish could not be observed. 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.
Length of the fish at test end: an influence of the test item concentrations on the length of the surviving fish could not be observed. 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):
no data
Reported statistics and error estimates:
Endpoints: NOEC, LOEC
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): Fisher exact binomial-test
- 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 multiple sequential t-test
The normal distribution was checked with Shapiro-Wilk's Test (dry weight and length of the surviving fish).
Variance homogeneity was checked with Bartlett´s (dry weight and length of the surviving fish).
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.

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

- 98.3% post-hatch success

- dissolved oxygen level during routine water quality analyses not below 60% of air saturation: 43% (minimum)

- water temperature: min: 23.4 °C, max.: 26.2°C (manual measurement), difference: 2.8°C; min.: 24.6°C, max.: 26.3°C (online measurement), difference: 1.7°C

Validity criteria fulfilled:
yes
Remarks:
for details see "Any other information on results incl. tables"
Conclusions:
The NOECs (33 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 barium dichloride dihydrate/L. The corresponding LOECs based on nominal concentrations were higher than 100 mg barium dichloride dihydrate/L.

Since the recovery rates (RR) are < 80% the results are given as actual (geometric mean measured) concentrations.

(a) The NOEC >= 61.1 mg BaCl2/L (>= 40.3 mg barium/L) based on total barium concentration.
(b) The NOEC >= 1.91 mg BaCl2/L (>= 1.26 mg barium/L) based on dissolved barium concentration.

Description of key information

A read across approach was followed to cover this endpoint. The key study is an early life stage study in Danio rerio performed with barium chloride (Gilberg, 2014). Because no adverse effects were observed up to and including the highest test concentration, the 33-d NOEC was reported to be >= 100 mg BaCl2.2H2O/L (nominally). When based on mean measured barium concentrations in filtered and unfiltered medium, the 33-d NOEC becomes >= 1.26 mg dissolved Ba/L and >= 40.3 mg Ba total/L. This result was recalculated to barium dinitrate based on the barium content of both substances (i.e. >= 2.39 mg barium trinitrate/L).

Key value for chemical safety assessment

Additional information

A single relevant and reliable study was identified. In this study (Gilberg, 2014), an early life stage toxicity test was performed with zebra fish using the read across substance barium chloride as test substance. No adverse effects were observed up to and including the highest test concentration, which was 100 mg barium dichloride dihydrate/L (nominal concentration). The 33-d NOEC was therefore >= 100 mg test item/L. When based on mean measured barium concentrations in filtered and unfiltered medium, the 33-d NOEC becomes >= 1.26 mg dissolved Ba/L and >= 40.3 mg Ba total/L.